MOLECULAR BIOLOGY OF THE CELL - ascb.org

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MBoC
MOLECULAR BIOLOGY OF THE CELL
ASCB AWARD ESSAYS, BIG DATA AND THE FBI,
AND THE 2015 PAPER OF THE YEAR
MBoC
Published by the American Society for Cell Biology
2015 ASCB Award Essays,
Selected Perspective, and
MBoC Paper of the Year
Contents
EDITORIAL
Great science inspires us to tackle the issue of data reproducibility
D. G. Drubin
1–2
ASCB AWARD ESSAYS
Advice to a young scientist (by someone who doesn’t know how to give it)
V. Denic
What does it take to get the job done?
M. Serpe
How nontraditional model systems can save us
A. S. Gladfelter
A case for more curiosity-driven basic research
A. Amon
Surviving as an underrepresented minority scientist in a majority environment
E. D. Jarvis
An unconventional route to becoming a cell biologist
E. Fuchs
3–5
6–8
9–11
12–13
14–18
19–21
PERSPECTIVE
Biosecurity in the age of Big Data: a conversation with the FBI
K. G. Kozminski
22–25
MBoC PAPER OF THE YEAR
Subcellular optogenetic inhibition of G proteins generates signaling gradients
and cell migration
P. R. O’Neill and N. Gautam
27–36
MBoC
ASCB Award Essays, BIg Data and the FBI, and the 2015 Paper of the Year
A cell activated by spatially uniform chemoattractant responds by increasing the level of a
signaling lipid all over the cell, shown as translocation of green fluorescence from the cytosol to
the plasma membrane. After this transient response subsides, optical activation (white box) is
applied to one side of the cell to recruit an inhibitor (red) of an intracellular signaling protein
(heterotrimeric G protein) that is known to be activated downstream of the receptor. This causes
a subsequent increase in the level of the signaling lipid and initiation of migration, both directed
toward the opposite side of the cell. See the 2015 MBoC Paper of the Year (O’Neill and Gautam,
Mol. Biol. Cell 25, 2305–2314; reprinted on p. 27). The MBoC Paper of the Year is selected by
the Editorial Board from among papers published in the journal each year that have a postdoc
or student as the first author. (Image: Patrick O’Neill, Washington University School of Medicine,
St. Louis)
The Philosophy of Molecular Biology of the Cell
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MBoC
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David G. Drubin
University of California, Berkeley
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CEA, Hopital Saint Louis
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University of Colorado, Boulder
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ETH Zurich
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MBoC | EDITORIAL
adequate independent replicates and report: 1) how many independent replicates were performed and 2) the variance in the results.
Appropriate statistical practices are important but are not a panacea. This is because there is a difference between precision and accuracy. An experimental setup with a systematic design flaw can
produce data that are precise but inaccurate. Second, it is vital that
the variables in each procedure be altered systematically to deterDavid G. Drubin
mine which parameters are critical for making results reproducible.
Department of Molecular and Cell Biology, University of California,
Having done these two things, it is next essential that the study be
Berkeley, Berkeley, CA 94720
communicated in sufficient detail to allow others to reproduce the
key findings. Finally, one of the best ways to insure that a result is
correct is to get the same answer using at least two independent
approaches. In their classic study, Jamieson and Palade (1967) used
This special MBoC edition celebrates the American Society for Cell
both cell fractionation and radioautography to discover the intracelBiology’s 2015 award winners by featuring essays describing their
lular trafficking route.
inspiring scientific journeys and sharing their impressive wisdom.
Journals are part of this reproducibilThese articles remind us that, while much
ity
issue in another way. Many scientists
attention has recently been focused on
feel that it is necessary to publish in highconcerns about research practices and
profile journals to secure funding, emdata reproducibility, we live in an era of unployment, and career advancement.
precedented achievements in biomedical
Attempting to publish in such journals,
research discovery, such as immune checkhowever, may make authors feel the
point therapy for cancer, which arose dineed to oversimplify their results and
rectly out of basic research (Sharma and
omit inconvenient data, both of which
Allison, 2015).
compromise the integrity of the reported
As the successful careers of these ASCB
results. Moreover, higher-profile journals
awardees serve to remind us, it is importend to have severe constraints on article
tant that all scientists promote research
length, compounding these problems.
practices and standards that result in highVital to making research reproducible is
quality, reproducible research. If the scienbeing able to report ALL of the nuances
tific community cannot convince the pubof the experimental procedures and relic that we have control of this issue, we
sults, including “inconvenient facts” that
risk reduced funding and imposition of
might not fit perfectly with the major
guidelines developed and enforced by
findings of a study. One back-to-basics
government legislators.
David G. Drubin
solution to this problem is to publish reBecause peer-reviewed publications
Editor-in-Chief
search articles in professional society
are both the product of research and the
journals like MBoC that are concerned
vehicles for communicating scientific disonly with results being new and true and not with their popular
coveries, journals have a critical role to play, promoting the practices
appeal or flashiness. MBoC and some other professional society
that make research reproducible. It is appropriate that scientist-run
journals do not have artificial limits on the number of figures or
journals like MBoC take a lead in this effort. MBoC has always emthe length of the text. Such limits impair the ability to provide
braced a “back-to-basics” approach to promote research integrity.
sufficient detail so others can reproduce published work.
In other words, major innovations are not needed to promote reproA promising way to address reproducibility issues is through deducibility, just an emphasis on sound fundamentals.
velopment
of field-specific, community standards. Achieving reproReproducibility must begin with those individuals performing the
ducibility
can
be challenging, because scientific research is difficult
initial study. First and foremost, it is vital that investigators perform
and protocols are often complex (Aschwanden and King, 2015).
However, this is not an excuse for publication of results that cannot
DOI:10.1091/mbc.E15-09-0643. Mol Biol Cell 26, 3679–3680.
be reproduced. Rather, it is an important acknowledgment of the
David G. Drubin is Editor-in-Chief of Molecular Biology of the Cell.
reality that there are a lot of variables in performing experiments
Address correspondence to: David G. Drubin ([email protected]).
and in collecting and analyzing data. Seemingly insignificant
© 2015 Drubin. This article is distributed by The American Society for Cell Biology
under license from the author(s). Two months after publication it is available to
changes in execution or analysis can have profound impacts on rethe public under an Attribution–Noncommercial–Share Alike 3.0 Unported Cresults. Because the ways in which complex phenomena are observed,
ative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
classified, and reported are often research area specific, one-size®
®
“ASCB ,” “The American Society for Cell Biology ,” and “Molecular Biology of
fits-all solutions for the reproducibility issue are unattainable. The
the Cell®” are registered trademarks of The American Society for Cell Biology.
Great science inspires us
to tackle the issue of data
reproducibility
2015 ASCB Award Essays, Selected Perspective, and MBoC Paper of the Year
1
autophagy field has developed its own standards by consensus
(Klionsky et al., 2012), providing a powerful example for others to
follow.
Many of these approaches to the data-reproducibility problem
are discussed in a report by the ASCB Data Reproducibility Task
Force (American Society for Cell Biology, 2015). In the near future,
MBoC will be developing strategies to implement the task force’s
recommendations in ways that do not place excessive administrative burdens on authors.
In closing, I offer congratulations to the 2015 ASCB award winners! You inspire us all with your creativity and passion, exemplify
sound science practices, and remind us that great scientific achievements result when all of these elements are combined.
2 | D. G. Drubin
REFERENCES
American Society for Cell Biology (2015). How can scientists enhance rigor
in conducting basic research and reporting research results? A white
paper from the American Society for Cell Biology. www.ascb.org/files/
How-can-scientist-enhance-rigor.pdf.
Aschwanden C, King R (2015). Science isn’t broken. FiveThirtyEight. http://
fivethirtyeight.com/features/science-isnt-broken.
Jamieson JD, Palade GE (1967). Intracellular transport of secretory proteins
in the pancreatic exocrine cell. II. Transport to condensing vacuoles and
zymogen granules. J Cell Biol 34, 597–615.
Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A,
Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, et al.
(2012). Guidelines for the use and interpretation of assays for monitoring
autophagy. Autophagy 8, 445–544.
Sharma P, Allison JP (2015). The future of immune checkpoint therapy.
Science 348, 56–61.
Molecular Biology of the Cell
MBoC | ASCB AWARD ESSAY
Advice to a young scientist (by someone who
doesn’t know how to give it)
Vladimir Denic
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
ABSTRACT While trying to extract original and general advice from the details of my career,
I realized this might not be possible. My path, like those of so many others, had too many
idiosyncratic twists and turns that had to work out just the way they did to be mined for
generally useful strategies. So I abandon the conceit of advice and simply give you my story.
There are many like it, but this one is mine. Take what you wish from it.
AND YET IT COLLAPSES
that Mr. Patterson refused to confirm or
In Belgrade, where I grew up, I was a merefute my explanation. Instead, he chaldiocre science student, unlikely to sponlenged me to devise an experiment that
taneously improve. I have to believe this
could falsify my working model. I returned
was because the subject was taught by
the challenge: “Doesn’t this way of thinkrote memorization, but regardless, I was
ing call into question all the other stuff in
more interested in the indolent pursuits
the textbook?” Smiling mischievously, he
of disaffected youth in latter-day Yugoslaretorted, “What do you think?” I didn’t
via, like stealing car radios (easier than
have an answer, but what I should have
you might think) and pilfering supermarsaid is “I think, therefore I am … a workket baguettes (harder). In an attempt to
ing model.”
alter my steady course toward juvenile
Learning that I, rather than the authoridelinquency, my mom sent me to live with
ties (textbooks, Mr. Patterson himself)
my dad, then in the throes of his second
could be both originator and verifier of hymarriage, in the mythic land of affluent
potheses was one of the most empowerhigh school kids I had been watching on
ing revelations of my life, a quiet and meltelevision: the United States. For the next
ancholic form of resistance against my
year, despite being in rural Pennsylvania, I
Vladimir Denic
parents’ divorce, against the authoritarian
lived a new life that seemed as glamorous
system back home, and, I realized as I got
and as far from post-Tito Belgrade as the
older, against the dying day itself. As summer began, Steven Spielone Brandon and Brenda Walsh were living in Beverly Hills.
berg fed my growing interest in science by genetically resurrecting
One day, in Mr. Patterson’s chemistry lab, I finally took notice of
the dinosaurs. Soon thereafter, I was sent back to Belgrade, just in
science. The task: explain why a soda can containing a dollop of
time for the war in Bosnia. I dodged the draft by immigrating to
boiling water collapsed when inverted in a beaker of ice water.
New Zealand, where I attended college. Perhaps inspired by the
What was remarkable to me was not that the can collapsed, but
velociraptors—clever girls—I majored in biochemistry.
Vladimir Denic is the recipient of the 2015 Early Career Life Scientist Award from
the American Society for Cell Biology.
Address correspondence to: Vladimir Denic ([email protected]).
Abbreviations used: ER, endoplasmic reticulum; VLCFAs, very-long-chain fatty
acids.
© 2015 Denic. This article is distributed by The American Society for Cell Biology
the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
“DEFINITELY. IT TAKES ANOTHER 3 HOURS BY PLANE
FROM SYDNEY”
Near the end of college, I started reading recent papers in major
journals. One study that caught my eye described how unfolded
proteins in the endoplasmic reticulum (ER) send a signal to the nucleus to activate genes encoding ER chaperones (Cox and Walter,
1996). This feedback loop required the ER transmembrane protein
kinase Ire1 and a new transcription factor, Hac1. Instead of activating Hac1 by yet another kinase cascade, Ire1 splices an intron from
3
the HAC1 mRNA to relieve a block in Hac1 synthesis by ribosomes.
The work was done at UCSF (closer to Beverly Hills) in the lab of
Peter Walter. This was not the first molecular biology paper that I
read, but it was the first that made me dream. I cold-called Peter
from a phone booth in Auckland and asked him to let me work in
his lab. He initially demurred, suggesting half-jokingly (or, knowing
Peter, not jokingly at all) that a competing lab that had recently
relocated to Australia was sending me as an infiltrator. Ultimately,
he assented, but only after I convinced him that New Zealand and
Australia are different countries.
When I left New Zealand after my third year of college, I planned
to return at the end of the summer break, but I never did. Instead, I
worked as an intern in Peter’s lab for little over a year before joining
the graduate program at UCSF. Living in San Francisco over the next
10 years, I came of age both personally and scientifically.
THE LONELINESS OF THE LONG-DISTANCE GRADUAL
STUDENT
I pursued my PhD in Jonathan Weissman’s lab, a scientific paradise
that I managed to turn into a personal scientific hell—but I’m getting ahead of myself. I was attracted to the Weissman lab partly
because it was new and relatively small, so Jonathan was often available to hang out in the lab and discuss science. However, discussing
science with Jonathan meant always being a few steps behind. My
brilliant solution was to insist that I work, essentially in isolation, on
a problem that was at best tangential to Jonathan’s main research
interests. I had also somehow gotten the idea that a mentee should
be petulant and jokingly dismissive of his mentor’s scientific ideas.
Despite my recalcitrance, Jonathan offered me several projects that
were guaranteed to work, but I turned them down in favor of pursuing my own ideas.
Cut to four years, several “clever” genetic screens, and zero
publications later. Jeffery Cox, one of the students who revealed
Ire1 and Hac1’s unique relationship in Peter’s lab, once said (to
someone else), “If you can’t clone the gene you love, love the gene
you clone.” What he didn’t say is what to do if you don’t know what
love is.
In my case, that meant not knowing how to explore the other
worlds of cell biology that lay in the direction my cloned genes were
trying to take me. In part, my resistance was based on fear that pursuing the obvious questions would require me to master biochemistry, which at the time I considered to be both less elegant and more
laborious than genetics. By my sixth year of graduate school, the
dream of crushing my own can of science was slipping away. But
then something unremarkable happened: existing projects in the
lab needed an extra pair of hands to get finished. My hands, idled
by disillusionment, were available. I got some results. Results became figures. Figures became papers.
Year 7. Some of the aforementioned results had suggested that
the uncharacterized gene YJL097w was involved in sphingolipid
metabolism. In a previous clever (but fruitless) genetic screen, I had
cloned two other genes involved in sphingolipid metabolism. As
that project collapsed into irrelevance, I had occupied myself by
accumulating an absurdly disproportionate familiarity with the
sphingolipid literature. On the basis of that knowledge (heretofore
useless to me), I intuited that YJL097w might be the missing biosynthetic enzyme for very-long-chain fatty acids (VLCFAs), the building
blocks of sphingolipids.
Contemporaneously, a couple of publications from another lab
had argued that the plant homologue of YJL097w was a protein
phosphatase involved in the cell cycle. I was unconvinced by these
data and felt that all of the phenotypes associated with mutations in
4 | V. Denic
the plant homologue could be explained by a defect in VLCFA synthesis. Thus, finally, I hit my stride: from my first tenuous baby steps
in Mr. Patterson’s chemistry lab, to a few Bambi-on-ice moments
while finishing other people’s projects, to making what was by far
the coolest science prediction I had ever made, which—cherry on
top!—was at odds with the accepted view. The exhilarating thought
of testing (and possibly even confirming) this hypothesis motivated
the next 6 months of labor—at the end of which a peak on a chromatogram showed me that purified Yjl097w had made a dehydrated VLCFA product. My working model had worked! We submitted our paper to a major journal, where it was rejected on the
grounds that it lacked general interest.
Still intoxicated by my discovery that Yjl097w was the missing
dehydratase, I decided that the general reader would be generally
interested in total VLCFA synthesis in vitro using Yjl097w and three
other enzymes. Unfortunately, all of these enzymes were integral
membrane proteins sensitive to detergent. Groping for a path forward, I was inspired by a paper written by Görlich and Rapoport on
an unrelated topic (Görlich and Rapoport, 1993). In their approach,
one places several pure membrane proteins in detergent, mixes
them with detergent-solubilized synthetic phospholipids, and then
removes the detergent (with something called “biobeads”) to yield
proteoliposomes containing the desired proteins. Despite the strategy’s straightforward logic, the remarkably detailed methods section suggested that there might still be some magic involved (for
example, only lot number 810017 of Big CHAP worked), so Jonathan put me in touch with a former UCSF student, Manu Hegde,
who was making proteoliposomes regularly in his own lab at the
National Institutes of Health. Manu and I spent hours on the phone,
like teenagers (“Did you know how much humidity in Bethesda affects my biobeads?” “Tell me about it. No, seriously, tell me ALL
about it.”), and a few weeks later, I was making proteoliposomes
that were making VLCFAs. We submitted our work to another major
journal, where it was rejected on the grounds that it didn’t demonstrate anything new.
Meanwhile, I had figured out how two different versions of yeast
VLCFA enzymes synthesize VLCFA products of different lengths. In
a “natural experiment,” I noticed that evolution had changed the
distance between the active site on the cytosolic end of the synthase (where carbon building blocks feed the growing end of the
fatty acid–chain substrate) and a lysine near the luminal end of a
transmembrane alpha helix. Remarkably, I could make new VLCFA
products of predictable lengths by “sliding” the position of the lysine, like molecular calipers, up or down the helix.
Several months later, Jonathan and I compiled the data for the
molecular caliper story and sent it to the journal that issued our first
rejection. (This felt a bit like trying to convince your ex-girlfriend to
take you back because you spent a year in the gym.) A few weeks
after the submission, as I waited in line in my favorite San Francisco
bakery, Jonathan called to tell me that the paper had been accepted
without revisions. The moment was ecstatic, but also sentimental,
because it meant that our mentor–mentee relationship was finally
coming to an end. It was the culmination of nine years of Jonathan’s
patience with me, during which he cheered me on, just as loudly
every time I fell down as when I finally won the race.
GO EAST(?), YOUNG MAN
I spent my last six months in the Weissman lab helping another project in the lab get finished, lining up a postdoc in Japan, and hedging my career bets by applying for a job to a few departments that
expressed interest in me after the caliper work was published. In the
end, I bailed on Japan and started my lab at Harvard University. At
the time that I was contemplating taking the Harvard job, the word
on the street was not good (“They eat their young”). Why did I still
choose to go there? First, I really enjoyed my interview interactions
with several senior members of the department (I know what you are
thinking: senior, not junior; red flag), who convinced me that they
could be decent Jonathan substitutes for this phase of my career.
Second, I believed that bad reputations are often the disproportionately long shadows of atypical events (I know what you are thinking:
shadows grow long when it is too late in the day for change to occur). And admittedly, my own hubris came into play: even if the
place was bad for junior faculty, I thought I would be somehow different (I don’t even wanna know what you’re thinkin’).
After a year at Harvard, however, progress was not swift. Only
two students had rotated with me, and they both joined other labs
(run by senior faculty). Self-doubt and fear spread through my veins
like poison. As an antidote, I considered an offer from another department with a better reputation for cultivating junior faculty. Why
did I stay, in the end? An old saying: “wherever you go, there you
are.” So, rather than entertaining Borgian fantasies about my senior
lab “competitors,” I tried to improve my own contributions to the
process of attracting talented students. I got myself on the student
radar by spearheading a journal club for first-year students and faculty, modeled on one I had enjoyed at UCSF, and started pitching
projects with the unabashed verve of a used car salesman.
Over the next five years, our group figured out how tail-anchored
proteins are inserted into the ER membrane by the GET pathway.
Before I left the Weissman lab, I had developed a cell-free system
for studying this pathway, which my group stripped down to its purified components. These were exhilarating times, because we were
racing against several fantastic labs to answer the same mechanistic
questions. Even though I was a newcomer to the membrane protein
insertion field, I was encouraged by more senior figures—especially
Manu Hegde, who taught me that scientific competition and criticism are not mutually exclusive with scientific openness.
As the lab established itself, we started parallel work on autophagy. My interest in this field arose during grad school, when I read
a paper from Yoshinori Ohsumi’s lab (where, incidentally, I had planned
to do a postdoc). Autophagy is a half century–old puzzle in cell biology: How do cells wrap targets with a membrane to make a vesicle
that then delivers targets to the lysosome? Many imaging methods
have been used to track the formation of this membrane, but few
biochemical approaches had been attempted. After a couple of years,
we built a cell-free system that allowed us to initiate autophagosome
membrane formation in situ using a purified autophagy target. Other
protein-targeting fields have been transformed following the development and rapid adoption of cell-free systems. Our work adds selective autophagy to this list and will hopefully accelerate the elucidation
of key mechanisms underlying this process
A TALE TOLD BY AN IDIOT, FULL OF SOUND AND FURY,
SIGNIFYING NOTHING
Here is where I intended to summarize my tale, with the implicit
purpose of inspiring younger scientists to do as I did. But what
would that advice be? “Here’s what you need to do, kids: Fail repeatedly for years, alone, but then get serendipitously lucky and
pick a winning horse years in advance of a final payoff. Then sit back
and wait by the phone for a job offer from Harvard, which despite
everything you’ve heard will give you exactly the kind of support
you need to succeed as junior faculty. You’re welcome [mic drop].”
But one person’s rose-tinted view of their own idiosyncratic story
does not constitute “advice,” especially not in an endeavor where
we value reproducibility; I’m not sure I could reproduce my own
good fortune, much less expect someone else to reproduce it from
the same set of initial conditions.
The only thing I know for sure is that the support I was repeatedly
given at every stage of my career was critical to what success I did
have throughout my career. That support enabled me to stay with it
through failures and to do something productive at those times
when I needed, more than anything else, to produce something.
Not everyone who had the support with which I was privileged
would have reached the same result, but I know that I wouldn’t have
succeeded without it. And so, for that, I am truly grateful.
ACKNOWLEDGMENTS
I thank Chris Patil for helping me write this essay and for helping me
finish my college education in San Francisco. I dedicate this essay to
my mother for giving me actually useful advice my whole life.
REFERENCES
Cox JS, Walter P (1996). A novel mechanism for regulating activity of a
transcription factor that controls the unfolded protein response. Cell 87,
391–404.
Görlich D, Rapoport TA (1993). Protein translocation into proteoliposomes
reconstituted from purified components of the endoplasmic reticulum
membrane. Cell 75, 615–630.
No advice to a young scientist
| 5
Mihaela Serpe
Unit on Cellular Communication, Program in Cellular Regulation and Metabolism, National Institute of Child Health
and Human Development, National Institutes of Health, Bethesda, MD 20892
ABSTRACT I am extremely honored to be the recipient of the 2015 Women in Cell Biology
Junior Award. When I reflect on my journey in science, many great people and memorable
experiences come to mind. Some of these encounters were truly career-defining moments.
Others provided priceless lessons. In this essay, I recount some of the moments and experiences that influenced my scientific trajectory with the hope that they may inspire others.
THE BIG QUESTION
multidisciplinary approaches for solving
It was a tense day in late fall of 2011. I was
problems. This scientific strength was
going through my first site visit at the Nashaped by my diverse background and
tional Institutes of Health (NIH), a compreexperiences and is fueled by a need to
hensive review that would define my scienunderstand the molecular mechanisms
tific career. My three-year-old lab had
underlying biological phenomena. I am
published a significant paper on mechadrawn to long-standing mysteries in the
nisms that shape bone morphogenetic
field, and my first impulse is to imagine
protein (BMP) morphogen gradients and
what kind of molecule/function(s) could
control early patterning (Peluso et al.,
fill the missing link. What does it take to
2011), and we had discovered Drosophila
get the job done? In my search for anNeto, an obligatory auxiliary subunit for
swers, I reach across disciplines and
glutamate receptors. The question filled
communicate with diverse experts, from
the room: “The BMP/transforming growth
cell biologists to neuroscientists and
factor β (TGF-β) field has been tackled for
computational biologists. I gather comquite some time by many laboratories. You
prehensive knowledge of the system and
propose to work on these pathways. What
the phenomena to understand the “job”
are you hoping to bring that is new to the
Mihaela Serpe
and to describe it in molecular terms.
field and how?” It was a very fair and simThen I use biochemistry and structure–
ple question coming from one of the most
function insights to envision possibilities and formulate a testable
accomplished and clear thinkers in cellular and developmental biolhypothesis.
ogy today, Eric Wieschaus. In a few words he crystallized the key
“What does it take to get the job done?” not only describes
issue any junior principal investigator should think hard about: What
the thinking process in my lab but also captures our mode of
do I bring that is new to science?
operation. We will do everything possible to answer the next
In my lab, I address fundamental issues of cellular communicaquestion, whether it means perfecting our skills, inventing tools,
tion using a genetic system and a unique set of powerful,
bringing new technologies to the lab, or establishing relevant
collaborations.
Mihaela Serpe is the recipient of the 2015 ASCB Women in Cell Biology Junior
Award for Excellence in Research.
Address correspondence to: Mihaela Serpe ([email protected]).
Abbreviations used: BMP, bone morphogenetic protein; iGluRs, glutamate-gated
ion channels; NIH, National Institutes of Health; NMJ, neuromuscular junction;
pMad, phosphorylated Smad; TGF-β, transforming growth factor β.
© 2015 Serpe. This article is distributed by The American Society for Cell Biology
the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
6 | M. Serpe
FINDING ONE’S PASSION
It takes scholarly work and courage to break new ground, but first
one must find his/her passion. I knew early on that I wanted to be
a scientist. At first I thought I would be a mathematician. But, because I was a girl, I was persuaded to look elsewhere. I chose
chemistry and genetics and decided to study biochemistry. This
was one of the best decisions in my life and was entirely mine. I was
17 years old, and I literally took three days to think hard and
consider everything that I was passionate about: logical thinking,
genetics, molecules, living creatures, and solving puzzles. Within a
year I moved to Bucharest to study biochemistry.
My first encounter with cell biology was self-driven. The biochemistry curriculum at the University of Bucharest was very heavy on
chemistry, especially in the first years. I was searching for ways to put
things in perspective. A friend recommended a cell biology textbook
adapted from Molecular Biology of the Cell by Bruce Alberts and
colleagues (Alberts et al., 2014). I bought it and started to read. I
ended up immersed in it, losing track of time. After graduation, I was
drawn toward the Institute for Cellular Biology and Pathology in
Bucharest, the place with the best cell biology research in Romania.
Many scientists have turning points in their careers, when they find
out what they really want to do in science. Mine was when I met
George Emil Palade, who came to give a seminar at the institute. He
made a comment that intrigued me: he considered his work on protein regulation by phosphorylation even more important than the
discovery of ribosomes. From the perspective of a freshly graduated
scientist, the body of work that brought Palade the Nobel Prize was
simply monumental. How could anything be more important than
that? What was so significant about protein phosphorylation, and
why was this a key finding in biology? I was enthralled. This was my
introduction to cellular signaling. This moment triggered a fascination
with signaling and macromolecular complexes that I have to this day.
THE JOURNEY
To work on signaling, I joined Dan Kosman’s laboratory at the State
University of New York at Buffalo, which focused on cellular mechanisms to acquire and metabolize iron. The first winter in Buffalo was
unforgettable: braving my way through record snowfall, I was taking
terrific courses, such enzyme kinetics taught by Cecile Pickart and
molecular biology taught by Ed Niles. For my PhD thesis, I studied
how a simple eukaryotic cell, the budding yeast, senses and responds to the levels of copper and iron in its environment. It was a
time of intense discoveries in the field. Dan Kosman encouraged us
to go to meetings and grasp the latest news. I learned a lot from
seeing how he discussed our findings and interacted with his competitors. He is a rigorous scientist and a master strategist.
For postdoctoral training, I joined Mike O’Connor’s laboratory at
the University of Minnesota to work on signaling by BMP/TGF-β factors during Drosophila development. Up to that point, I did not
know anything about development, but it seemed to be the best
place to probe for the biological relevance of signaling. It was a
daunting yet exhilarating experience to dive into a model system
with such prominent history in genetics and development. I went
back to take courses, including Drosophila genetics at the Cold
Spring Harbor Laboratory and developmental biology taught by
Ann Rougvie at the University of Minnesota. It was a steep learning
curve, but Mike O’Connor’s laboratory and the Developmental Biology Center offered an interactive and stimulating environment. The
lab was extremely productive and full of fantastic people. New discoveries were coming in big leaps rather than in a linear progression. We all had different individual projects, and it seemed that
each story tackled another field. I was always mesmerized by Mike
O’Connor’s ease at entering a new field and making an important
discovery. The lab was in the middle of a vibrant fly community, including Tom Hays, Jeff Simon, Tom Newfeld, and Hiroshi Nakato.
Fly stocks and good ideas were moving freely. I learned to do microinjections in Xenopus embryos in Jamie Lohr’s lab. And I tested the
axon guidance defects of a Caenorhabditis elegans mutant in Lishia
Chen’s lab. During these years, I described several molecular
mechanisms that shape morphogen gradients during patterning
(Serpe et al., 2005, 2008; Umulis et al., 2006). I formed a long-term
collaboration with David Umulis (now at Purdue University), who introduced me to computational biology. And I characterized a TGF-β
pathway required for motor neuron axon guidance and formation of
neural circuitry (Serpe and O’Connor, 2006). This last bit was the
beginning of a new chapter, neural development, which was about
to captivate me for the rest of my career.
THE UNIT ON CELLULAR COMMUNICATION
The big surprise in starting a new lab is the loneliness that comes with
its beginning. Fresh from a lab buzzing with people and experiments,
you are faced with an empty space. And it is up to you to bring it to
life and make things happen. The way I looked at it was that it another new ground to break, which takes scholarly work and courage.
This time I bought the book At the Helm by Kathy Barker (Barker,
2010), and I took management courses. Courage was asking for help.
My corner of the NIH is full of excellent scientists who challenge
themselves and push the boundaries. In this environment, I found
many answers and important role models. I learned from Alan
Hinnebusch to set high standards and from Mary Dasso to become
an effective mentor and woman scientist. I learned to distill questions in neurobiology from the late Howard Nash, who initiated and
mentored the Drosophila neurobiology interest group. We regularly brainstorm on neurons and circuits in joint lab meetings with
Chi-Hon Lee and Ed Giniger.
The fly neuromuscular junction (NMJ) has been a powerful genetic system to study synapse development. The easily accessible
synaptic structures were well described, the subunits that form the
glutamate-gated ion channels (iGluRs) were known and relatively
well characterized, and dynamic studies have captured growing
synapses (DiAntonio, 2006; Thomas and Sigrist, 2012). But a nagging problem was holding up the field: the mechanisms controlling the synaptic recruitment of iGluRs remained unknown for decades. Studies on iGluR C-tails, which provide rich regulation for
these receptors in other systems, brought marginal progress in
flies. We found an interesting transmembrane molecule, Neto
(Neuropillin and Tolloid-like protein), with a spectacular phenotype: netonull embryos are completely paralyzed and cannot hatch
into the larval stages. This is reminiscent of the phenotype seen
with the loss of iGluRs and lack of functional NMJ. In fact, we found
that Neto and iGluRs form complexes and depend on each other
for trafficking and clustering at synaptic sites (Kim et al., 2012).
Neto is an obligatory auxiliary protein for the fly NMJ iGluRs. The
discovery of Neto provides an entry point to address key questions
in iGluR cell biology and to start dissecting the individual steps of
iGluR assembly, surface delivery, trafficking, and stabilization at
synaptic locations; function; and postsynaptic composition. We
have already found that Neto engages in intracellular and extracellular interactions that recruit postsynaptic components and stabilize iGluRs at synaptic locations (Kim et al., 2015; Ramos et al.,
2015). In collaboration with Mark Mayer, we have achieved the
long-sought functional reconstitution of NMJ iGluRs in heterologous systems and showed that Neto modulates the functioning of
iGluRs but not their assembly or surface delivery (Han et al., 2015).
Neto also appears to be at the center of transsynaptic complexes that monitor the synapse activity status and relay this information to the presynaptic BMP signaling pathway (Sulkowski et al.,
2014). At the fly NMJ, BMP signaling triggers accumulation of phosphorylated Smad (pMad) in motor neuron nuclei and at synaptic
terminals. Nuclear pMad controls gene expression and promotes
NMJ growth; the role of synaptic pMad eluded the field for more
than a decade. We found that synaptic pMad is part of a completely
new BMP pathway that is genetically distinguishable from all known
| 7
BMP signaling cascades. This novel pathway does not contribute to
the NMJ growth and instead appears to set up a positive-feedback
loop that modulates synapse maturation as a function of synapse
activity. Thus, BMPs may monitor synapse activity and coordinate it
with synapse growth and maturation.
THE PEOPLE
When I reflect on how this all came together, many great people
and mentors come to mind. First are the people in my lab, a team of
talented, budding scientists whom I have been privileged to guide
in exciting endeavors. Their success is critical to me from the moment they join my lab to, I suspect, long after they leave. Second are
my colleagues—and here I hit the jackpot. Ours can be a stressful
profession, particularly at the beginning of a new lab. But if you have
colleagues like I do, incredibly accomplished scientists with big
hearts and a deep commitment to mentoring their juniors, you
could survive even when the building is falling on you. I am especially grateful to Alan Hinnebusch, Mary Dasso, and Chi-Hon Lee for
their continuous guidance and support. They believed in me from
the beginning, showed confidence in my decisions, and helped me
keep the course. I owe a great deal to our collaborators, who taught
us exciting new things and enriched our science. Bing Zhang completed the first physiology recordings for our mutants, and then
helped set up our own rig. Without Mark Mayer, we could not have
done the iGluR functional reconstitution and structural studies. We
learned to harness the power of superresolution imaging in Jennifer
Lippincott-Schwartz’s laboratory.
In science, as in life, one could never return the help and support received along the way. All we can hope is to do the same in
the future. When my trainees go on the job market, I paraphrase for
them the advice I received: In this profession there will be many
worries, good and bad. What BMPs do in a particular cell is a “good
worry.” Try to find the place where your worries will be mostly
good. And figure out fast “What does it take to get the job done?”
8 | M. Serpe
REFERENCES
Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P
(2014). Molecular Biology of the Cell, 6th ed., Garland Science.
Barker K (2010). At the Helm: Leading Your Laboratory, 2nd ed., Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory.
DiAntonio A (2006). Glutamate receptors at the Drosophila neuromuscular
junction. Int Rev Neurobiol 75, 165–179.
Han TH, Dharkar P, Mayer ML, Serpe M (2015). Functional reconstitution of
Drosophila melanogaster NMJ glutamate receptors. Proc Natl Acad Sci
USA 112, 6182–6187.
Kim YJ, Bao H, Bonanno L, Zhang B, Serpe M (2012). Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction.
Genes Dev 26, 974–987.
Kim YJ, Igiesuorobo O, Ramos CI, Bao H, Zhang B, Serpe M (2015).
Prodomain removal enables Neto to stabilize glutamate receptors at the
Drosophila neuromuscular junction. PLoS Genet 11, e1004988.
Peluso CE, Umulis D, Kim YJ, O’Connor MB, Serpe M, Shaping BMP (2011).
Morphogen gradients through enzyme-substrate interactions. Dev Cell
21, 375–383.
Ramos CI, Igiesuorobo O, Wang Q, Serpe M (2015). Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila
neuromuscular junction. PLoS Genet 11, e1005191.
Serpe M, O’Connor MB (2006). The metalloprotease tolloid-related and
its TGF-β-like substrate Dawdle regulate Drosophila motoneuron axon
guidance. Development 133, 4969–4979.
Serpe M, Ralston A, Blair SS, O’Connor MB (2005). Matching catalytic activity to developmental function: tolloid-related processes Sog in order
to help specify the posterior crossvein in the Drosophila wing. Development 132, 2645–2656.
Serpe M, Umulis D, Ralston A, Chen J, Olson DJ, Avanesov A, Othmer H,
O’Connor MB, Blair SS (2008). The BMP-binding protein Crossveinless 2
is a short-range, concentration-dependent, biphasic modulator of BMP
signaling in Drosophila. Dev Cell 14, 940–953.
Sulkowski M, Kim YJ, Serpe M (2014). Postsynaptic glutamate receptors
regulate local BMP signaling at the Drosophila neuromuscular junction.
Development 141, 436–447.
Thomas U, Sigrist SJ (2012). Glutamate receptors in synaptic assembly and
plasticity: case studies on fly NMJs. Adv Exp Med Biol 970, 3–28.
Umulis DM, Serpe M, O’Connor MB, Othmer HG (2006). Robust, bistable
patterning of the dorsal surface of the Drosophila embryo. Proc Natl
Acad Sci USA 103, 11613–11618.
How nontraditional model systems can save us
Amy S. Gladfelter
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755; Marine Biological Laboratory,
Woods Hole, MA 02543
ABSTRACT In this essay I would like to highlight how work in nontraditional model systems
is an imperative for our society to prepare for problems we do not even know exist. I present
examples of how discovery in nontraditional systems has been critical for fundamental advancement in cell biology. I also discuss how as a collective we might harvest both new questions and new solutions to old problems from the underexplored reservoir of diversity in the
biosphere. With advancements in genomics, proteomics, and genome editing, it is now technically feasible for even a single research group to introduce a new model system. I aim here
to inspire people to think beyond their familiar model systems and to press funding agencies
to support the establishment of new model systems.
My career as a biologist began in the orange groves and lake waters of central
Florida. An unstructured childhood was
spent learning to observe and wonder.
Without realizing it at the time, my training began with the mantra, “Study nature,
not books,” the familiar entreaty of Louis
Agassiz, a founder of what would become
the Marine Biological Lab (MBL) in Woods
Hole, Massachusetts. In that humid air,
listening to cicadas click, I subconsciously
practiced asking basic questions about
the structure of the natural world. I suspect many of us began our careers this
way, even though we ended up thinking
about systems of molecules from behind
the black curtains of the microscope
room, immersed in the frosty air of the
cold room, or bathed in the glow of a
computer screen. Before the grown-up
challenges of funding, publishing, and
progressing in a career, we easily marveled at the complexity and surprises of
the living world. How can we recapture
the joy that comes from curiosity-driven
inquiry? This being an essay for the midcareer award, it seems appropriate to
blend material for a midlife with a plan for
how we as a cell biology community can
identify big new questions. So instead of
a fancy car, a new microscope, or a dangerous (professional) liaison, try developing a new model system as an outlet for
your midlife crisis!
Photo Credit: Rob Strong
Amy S. Gladfelter
Amy S. Gladfelter is the recipient of the 2015 ASCB Women in Cell Biology
Mid-Career Award for Excellence in Research.
Address correspondence to: Amy S. Gladfelter (Amy.Gladfelter@Dartmouth
.edu).
Abbreviations used: MBL, Marine Biological Lab; NIH, National Institutes of
Health; NSF, National Science Foundation.
© 2015 Gladfelter. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
NONTRADITIONAL SYSTEMS
PRODUCE PUZZLES AND
PARADOXES
I begin with my own experiences in working with a nontraditional model fungus
called Ashbya gossypii. As a postdoc in Peter Philippsen’s group
in UniBasel, Switzerland, I started work with Ashbya, which is relatively closely related on the genome scale to the budding yeast
Saccharomyces cerevisiae but has some strikingly different cell
biology (Dietrich et al., 2004). As a postdoc, I began studying the
perplexing ability of nuclei in Ashbya’s multinucleate cells to
divide asynchronously despite being in a common cytoplasm
(Gladfelter et al., 2006). This was a paradox, because decades of
classic work demonstrated that cytosolic factors (CDK/cyclins)
control the cell cycle and should synchronize nuclei in a syncytium
(Johnson and Rao, 1970). This observation has fueled many experiments in my lab for the past decade, and we have been led to
new models for compartmentalizing cytosol, have gained insight
9
into how ploidy can vary in syncytia, and
have been continually surprised and inspired (Anderson et al., 2013, 2015; Lee
et al., 2013, 2015). For example, one set of
experiments led us to realize functional
uses for polyQ-tract proteins in localizing
the mRNAs encoding cyclins (Lee et al.,
2013). This discovery was an early example of a now growing list of functional uses
for protein aggregation outside pathologies. Importantly, as a junior faculty
member, I had enough of an autonomous
niche that my students and I could ask big
questions without concerns about being
scooped by bigger labs.
FIGURE 1: Images of nontraditional model systems discussed in this essay. (A) Ashbya (fungus),
image provided by Hanspeter Helfer. (B) Tardigrade (water bear), image provided by Bob
Goldstein (University of North Carolina, Chapel Hill). (C) Aiptasia (sea anemone), image provided
by John Pringle (Stanford University). (D) Sepioteuthis sepioidea (reef squid), image provided by
Roger Hanlon (Marine Biological Laboratory).
WHAT ARE THE CHALLENGES OF WORKING IN A
NONTRADITIONAL SYSTEM?
While there is a tremendous amount of stimulation and freedom
that comes from work on a less-studied system, there are also challenges. For example, there is not a large community of users, so I
sought out the yeast, filamentous fungus, and cell biology communities—all of whom have been open to Ashbya as an alternative
system. It also means most reagents are not simply an email away
but instead typically have to be developed in the lab. This is slow,
but again, if you are not in a race to publish, it is less of a concern.
A molecular geneticist extraordinaire, such as Patricia Occhipinti, a
long-term technician in my lab, makes it possible to generate any
kind of new strain. Investing in a person like this as a lab constant
is critical for establishing a new system. Also, if a system is somewhat related to an established model system, some tools may be
transportable or at least adaptable, and so we have borrowed
many tricks from the yeast genetics world. Finally, one has to convince funding agencies of the value of the problems that can be
addressed by this system. (More on this later.) If the system is conducive to studying a fundamental problem, brings contrasting
mechanisms into our current thinking about a key process, and is
tractable, it should be possible to make a compelling case for support. There are truly unknown problems in biology waiting in the
wings.
SUCCESS STORIES OF ESTABLISHING NONTRADITIONAL
MODEL SYSTEMS
In the most successful new system launches, there is either new
biology or a new handle on an old problem that make the systems
especially suited for investment. The entry point to a new experimental system tends to be either through old descriptive literature
that captures some fascinating phenomena or through a genomic
approach that reveals a surprising manifestation of, or absence of,
genes thought to be critical for a process. For example, Bungo
Akiyoshi, who recently established his own lab at Oxford, comes
from a PhD working on kinetochores in yeast with Sue Biggins.
Akiyoshi noticed a complete absence of typical kinetochore proteins in the genome of Trypanosoma brucei, a protozoan parasite
(Akiyoshi and Gull, 2013, 2014; Figure 1). He went on to identify a
novel mechanism of chromosome segregation for eukaryotes,
and, because of the unique identity of the proteins, they are likely
to be drug-able targets for treatment of African sleeping sickness
disease, which is caused by infection with T. brucei. In seeking out
basic biology in an understudied system, Akiyoshi’s lab is finding
new mechanisms to old cell biological problems that could lead to
a potential disease cure.
10 | A. S. Gladfelter
Adoptions of alternative systems can also stem from a lab with
a principal investigator familiar with established systems and willing to take risks. Wallace Marshall at the University of California,
San Francisco, for example, long a student of flagella in Chlamydomonas, has recently established work in Stentor, a ciliate among
the largest single-celled organisms known. Marshall’s lab has established genetic techniques, proteomics, and transcriptomics to
study cell polarity, cytoplasmic flow, and regeneration in these
simple and intriguing pond dwellers (Slabodnick et al., 2013, 2014;
Slabodnick and Marshall, 2014). Similarly, Bob Goldstein at the
University of North Carolina, Chapel Hill, an established leader in
Caenorhabditis elegans developmental cell biology, invested in
establishing another microscopic animal—the Tardigrade or aptly
named “water bear” (Figure 1). The water bear is fascinating in
terms of evolutionary development but also in its ability to withstand prolonged desiccation (Gabriel and Goldstein, 2007; Gabriel
et al., 2007; Tenlen et al., 2013). The water bear has already journeyed into space for experiments with the aim of understanding
how these tiny animals that normally reside in moss are so resilient.
Both water bears and Stentor are proving to be tractable, rich
sources of new biology and may provide important insight into
stress responses and regeneration.
There are also many “forgotten” systems that were once widely
studied but fell out of favor in the recombinant DNA revolution at
the end of the 20th century. Many of these creatures happen to
reside in the sea or other aquatic habitats and boast deep literatures of physiology, behavior, and, in some cases, cell biology
(Figure 1). The squid is a great example, showing us microtubule
motors and the action potential, yet the squid research community
has shrunk dramatically, in part because squid cannot be readily
cultured, thus requiring a steady supply from a marine lab such as
the MBL in Woods Hole. Yet there is still tremendous biology being discovered in squid and other cephalopods. These organisms
widely use RNA editing to generate variation in protein sequences
posttranscriptionally, as is being studied intensively by Josh Rosenthal at the University of Puerto Rico and the MBL (Alon et al.,
2015). RNA editing is dependent on temperature and, likely, other
environmental conditions (Garrett and Rosenthal, 2012). Investment in cephalopodomics is promising to bring these traditional
systems back in vogue. Finally, a hero of yeast geneticists, John
Pringle, almost completely switched model systems to a simple
sea anemone about 10 years ago. He has rejuvenated his program
with new questions relevant to coral ecology and symbiosis
(Lehnert et al., 2012, 2014). The sea is vast, and we have tapped
into an extremely small amount of the biodiversity, especially at
the cell and molecular level.
HOW CAN WE MAKE IT MORE FEASIBLE FOR LABS
TO ESTABLISH NEW SYSTEMS?
Millions of years of evolution has done the hard work of creating
vast biodiversity on the planet, so how can we start feasting intellectually on this biology at the level of molecules, cells, and tissues?
There is no question that, in this century, we will face problems arising out of climate change, habitat destruction, and expanding and
aging human populations that are highly mobile, allowing rapid
spread of infectious diseases. How can we best prepare to solve the
challenges that will arise from these monumental changes on the
planet? I would argue that the track record of basic science solving
problems relevant to human health and society is sparkling. Why
should we stop here when the technology is in our reach to readily
tame many systems?
The question is who can pay for the risk of attempting to establish a new system. For Ashbya, Peter Philippsen had the drive and
ingenuity to fund sequencing the Ashbya genome more than
15 years ago. He was willing to take the risk and had the creativity to
invest in a completely new system after decades of work in budding
yeast. He had enough funding flexibility at the time, being in a
European institution with industry support, to see it through to complete establishment. What could be done to make it more feasible
for even small and new labs to find a parallel alternative model? It
could be argued that this is for the realm of the National Science
Foundation (NSF), as the work will likely initially result in new science
but may not immediately be translated to human health. However,
the National Institute of General Medical Sciences at the National
Institutes of Health (NIH) has a stellar history in funding basic science
that leads to Nobel Prizes and cures for diseases with time. What if
everyone who had an R01 and the interest could apply for a “model
organism development” supplement to perform foundational
experiments to establish a new system in parallel with their conventional system?
The advantages and joys of work in an unchartered system revolve around the intellectual challenges, the breathing room of
broad problems, which is especially useful for junior scientists, and
the potential to discover truly new solutions in biology and for society. Each summer I move part of my lab to the MBL in Woods Hole,
and one future for this hallowed place may well be to facilitate the
development of new systems—if we can convince funding agencies
and our deans to allow us this freedom. My children, ages seven and
nine, eagerly attend the Children’s School of Science in Woods Hole
while we are there, and each day they head to the ponds, the surf,
and the fields to observe nature. For their generation, who are currently learning to gaze at diverse creatures, I hope they have a menagerie of choices of systems in which to study biology.
ACKNOWLEDGMENTS
I apologize to any nonconventional model systems that I did not
have space to discuss. I acknowledge my intellectual family of formal
advisors, mentors, students, lab managers, and postdocs for making this such a rewarding profession. I am also grateful to the U.S.
government for generous support of our work through the NIH and
the NSF. Finally, I would not be in this position without the encouragement of my parents and sister or support of my husband and
children.
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Akiyoshi B, Gull K (2013). Evolutionary cell biology of chromosome segregation: insights from trypanosomes. Open Biol 3, 130023.
Akiyoshi B, Gull K (2014). Discovery of unconventional kinetochores in
kinetoplastids. Cell 156, 1247–1258.
Alon S, Garrett SC, Levanon EY, Olson S, Graveley BR, Rosenthal JJ,
Eisenberg E (2015). The majority of transcripts in the squid nervous
system are extensively recoded by A-to-I RNA editing. eLife 4, 05198.
Anderson CA, Eser U, Korndorf T, Borsuk ME, Skotheim JM, Gladfelter AS
(2013). Nuclear repulsion enables division autonomy in a single cytoplasm. Curr Biol 23, 1999–2010.
Anderson CA, Roberts S, Zhang H, Kelly CM, Kendall A, Lee C,
Gerstenberger J, Koenig AB, Kabeche R, Gladfelter AS (2015). Ploidy
variation in multinucleate cells changes under stress. Mol Biol Cell 26,
1129–1140.
Dietrich FS, Voegeli S, Brachat S, Lerch A, Gates K, Steiner S, Mohr C,
Pöhlmann R, Luedi P, Choi S, et al. (2004). The Ashbya gossypii genome
as a tool for mapping the ancient Saccharomyces cerevisiae genome.
Science 304, 304–307.
Gabriel WN, Goldstein B (2007). Segmental expression of Pax3/7 and
engrailed homologs in Tardigrade development. Dev Genes Evol 217,
421–433.
Gabriel WN, McNuff R, Patel SK, Gregory TR, Jeck WR, Jones CD,
Goldstein B (2007). The Tardigrade Hypsibius dujardini, a new model for
studying the evolution of development. Dev Biol 312, 545–559.
Garrett SC, Rosenthal JJ (2012). A role for A-to-I RNA editing in temperature adaptation. Physiology 27, 362–369.
Gladfelter AS, Hungerbuehler AK, Philippsen P (2006). Asynchronous
nuclear division cycles in multinucleated cells. J Cell Biol 172, 347–362.
Johnson RT, Rao PN (1970). Mammalian cell fusion: induction of premature
chromosome condensation in interphase nuclei. Nature 226, 717–722.
Lee C, Occhipinti P, Gladfelter AS (2015). PolyQ-dependent RNA-protein
assemblies control symmetry breaking. J Cell Biol 208, 533–544.
Lee C, Zhang H, Baker AE, Occhipinti P, Borsuk ME, Gladfelter AS (2013).
Protein aggregation behavior regulates cyclin transcript localization and
cell-cycle control. Dev Cell 25, 572–584.
Lehnert EM, Burriesci MS, Pringle JR (2012). Developing the anemone
Aiptasia as a tractable model for cnidarian-dinoflagellate symbiosis: the
transcriptome of aposymbiotic A. pallida. BMC Genom 13, 271.
Lehnert EM, Mouchka ME, Burriesci MS, Gallo ND, Schwarz JA, Pringle JR
(2014). Extensive differences in gene expression between symbiotic and
aposymbiotic cnidarians. G3 (Bethesda) 4, 277–295.
Slabodnick M, Prevo B, Gross P, Sheung J, Marshall W (2013). Visualizing
cytoplasmic flow during single-cell wound healing in Stentor coeruleus.
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(2014). The kinase regulator mob1 acts as a patterning protein for
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Tenlen JR, McCaskill S, Goldstein B (2013). RNA interference can be used to
disrupt gene function in tardigrades. Dev Genes Evol 223, 171–181.
Nontraditional model systems
| 11
A case for more curiosity-driven basic research
Angelika Amon
Koch Institute for Integrative Cancer Research, Department of Biology, Howard Hughes Medical Institute,
Massachusetts Institute of Technology, Cambridge, MA 02139
ABSTRACT Having been selected to be among the exquisitely talented scientists who won
the Sandra K. Masur Senior Leadership Award is a tremendous honor. I would like to take this
opportunity to make the case for a conviction of mine that I think many will consider outdated. I am convinced that we need more curiosity-driven basic research aimed at understanding the principles governing life. The reasons are simple: 1) we need to learn more
about the world around us; and 2) a robust and diverse basic research enterprise will bring
ideas and approaches essential for developing new medicines and improving the lives of
humankind.
are lists, some better than others. We now
When I was a graduate student, curiosity-driven
know how many coding genes define a
basic research ruled. Studying mating-type
given species and how many protein kiswitching in budding yeast, for example, was
nases, GTPases, and so forth there are in
exciting because it was an interesting problem:
the various genomes we sequenced. This
How can you make two different cells from a sinknowledge, however, does not even
gle cell in the absence of any external cues? We
scratch the surface of understanding their
did not have to justify why it is important to study
function. When I browse the Saccharomywhat many would now consider a baroque quesces cerevisiae genome database (my section. Scientists and funding agencies alike agreed
ond-favorite website), I am still amazed
that this was an exciting biological problem that
how many genes there are that have not
needed to be solved. I am certain that all scieneven been given a name.
tists of my generation can come up with similar
To me the most important achieveexamples.
ment the new genome-sequencing and
Since the time I was a graduate student, the
genome-editing technologies brought us
field of biological research has experienced a
is that nearly every organism can be a
revolution. We can now determine the genetic
model organism now. We can study and
makeup of every species in a week or so and have
Angelika Amon
manipulate the processes that most fascian unprecedented ability to manipulate any genate us in the organisms in which they ocnome. This revolution has led to a sense that we
cur, with the exception, of course, of humans. Thus, I believe that
understand the principles governing life and that it is now time to
the golden era of basic biological research is not behind us but in
apply this knowledge to cure diseases and make the world a better
front of us, and we need more people who will take advantage of
place. While applying knowledge to improve lives and treat disthe tools that have been developed in the past three decades. I
eases is certainly a worthwhile endeavor, it is important to realize
am therefore hoping that many young people will chose a career
that we are far from having a mechanistic understanding of even the
in basic research and find an exciting question to study. The more
basic principles of biology. What the genomic revolution brought us
of us there are, the more knowledge we will acquire, and the
higher the likelihood we will discover something amazing and
important. There is so much interesting biology out there that
Angelika Amon is the recipient of the 2015 ASCB Women in Cell Biology Sandra
we should strive to understand. Some of my favorite unanswered
K. Masur Senior Leadership Award.
questions are: What are the biological principles underlying symAddress correspondence to: Angelika Amon ([email protected]).
biosis and how did it evolve? Why is sleep essential? Why do
© 2015 Amon. This article is distributed by The American Society for Cell Biology
plants, despite an enormous regenerative potential, never die of
the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Crecancer? Why do brown bears, despite inactivity, obesity, and high
levels of cholesterol, exhibit no signs of atherosclerosis? How do
sharks continuously produce teeth?
12 | A. Amon
One could, of course, argue that the knowledge we have accumulated over the past 50 years provides a reasonable framework,
and it is now time to leave basic science and model organisms behind and focus on what matters—curing diseases, developing
methods to produce energy, cleaning up the oceans, preventing
global warming, building biological computers, designing organisms, or engineering whatever the current buzz is about. Like David
Botstein, who eloquently discussed the importance of basic research in these pages in 2012 (Botstein, 2012), I believe that the
notion that we already know enough is wrong and the current application-centric view of biology is misguided. Experience has taught
us over and over that we cannot predict where the next important
breakthrough will be emerge. Many of the discoveries that we consider groundbreaking and that have brought us new medicines or
improved our lives in other ways are the result of curiosity-driven
basic research. My favorite example is the discovery of penicillin.
Alexander Fleming, through the careful study of his (contaminated)
bacterial plates, enabled humankind to escape natural selection.
More recent success stories such as new cures for hepatitis C, the
human papillomavirus vaccine, the HIV-containment regimens, or
treatments for BCR-ABL induced chronic myelogenous leukemia
have also only been possible because of decades of basic research
in model organisms that taught us the principles of life and enabled
us to acquire the methodologies critical to develop these treatments. Although work from my own lab on the causes and consequences of chromosome mis-segregation in budding yeast has not
led to the development of new treatments, it has taught us a lot
about how an imbalanced karyotype, a hallmark of cancer, affects
the physiology of cancer cells and creates vulnerabilities in cancer
cells that could represent new therapeutic targets.
These are but a few examples for why it is important that we
scientists must dedicate ourselves to the pursuit of basic knowledge
and why we as a society must make funding basic research a priority.
Achieving the latter requires that we scientists tell the public about
the importance of what we are doing and explain the potential implications of basic research for human health. At the same time, it
will be important to manage expectations. We must explain that not
every research project will lead to the development of new medicines and that we cannot predict where the next big breakthroughs
will materialize. We must further make it clear that this means we
have to fund a broad range of basic research at a healthy level. Perhaps a website that collects examples of how basic research has led
to breakthroughs in medicine could serve as a showcase for such
success stories, bringing the importance of what we do to the
public.
While conducting research to improve the lives of others is certainly a worthy motivation, it is not the main reason why I get up very
early every morning to go to the lab. To me, gaining an understanding of a basic principle in the purest Faustian terms is what I find
most rewarding and exciting. Designing and conducting experiments, pondering the results, and developing hypotheses as to how
something may work is most exciting, the idea that I, or nowadays
the people in my lab, may be (hopefully) the first to discover a new
aspect of biology is the best feeling. It is these rare eureka moments, when you first realize how a process works or when you discover something that opens up a new research direction, that make
up for all the woes and frustrations that come with being an experimental scientist in an expensive discipline.
For me, having a career in curiosity-driven basic research has
been immensely rewarding. It is my hope that basic research remains one of the pillars of the American scientific enterprise, attracting the brightest young minds for generations to come. We as a
community can help to make this a reality by telling people what we
do and highlighting the importance of our work to their lives.
ACKNOWLEDGMENTS
I am grateful to my friend and colleague Frank Solomon for his
thoughts and discussions.
REFERENCE
Botstein D (2012). Why we need more basic biology research, not less. Mol
Biol Cell 23, 4160–4161.
Why we need more basic research
| 13
Surviving as an underrepresented minority
scientist in a majority environment
Erich D. Jarvis
Department of Neurobiology, Duke University Medical Center, Durham, NC 27710; Howard Hughes Medical Institute,
Chevy Chase, MD 20815
ABSTRACT I believe the evidence will show that the science we conduct and discoveries we
make are influenced by our cultural experience, whether they be positive, negative, or neutral. I grew up as a person of color in the United States of America, faced with challenges that
many had as members of an underrepresented minority group. I write here about some of the
lessons I have learned that have allowed me to survive as an underrepresented minority
scientist in a majority environment.
THE DIRECT INFLUENCE OF E. E.
JUST ON MY CAREER
heard gunshots several nights a week.
Most of those gunshots were not meant
I am honored to be the recipient of the
for target practice, including the one that
2015 Ernest Everett Just Award from the
killed my father. One of my mentors at
American Society for Cell Biology and to
Rockefeller at the time, Peter MacLeish,
write this associated essay. Just had an iman African-American assistant professor in
pact on my scientific life well after his
the lab of Nobel laureate Torsten Wiesel
death in 1941. I was a beginning graduate
and professor next door at Weill Cornell
student at the Rockefeller University in
Medical College, sat me down in his office
New York City at the end of the 1980s,
to talk about my life. At the end of our
struggling to get a grip on the drama that
conversation, MacLeish gave me his copy
was unfolding in my life. I had graduated
of a book published five years earlier
from Hunter College in New York City with
(1983) titled The Black Apollo of Science:
a double major in biology and mathematThe Life of Ernest Everett Just by Kenneth
ics, published several papers from my unR. Manning. MacLeish said, “I want you to
dergraduate research, was accepted into
read the book, and then come back and
top-tier graduate programs, and then my
talk to me.”
grandfather, who helped support me, died
Erich D. Jarvis
I read the book and identified with
of a heart attack, his brothers died soon
Just’s experience. Like Just, my mother
after, my homeless father was shot and
was single, raising multiple (four) children. I had been ill on and off
killed, my first child was on the way, and although technically being
at a young age (6–8 years old), intermittently taken out of school,
a middle-class person of color with a house I inherited from my
and partly as a result was delayed in my education, being below
grandfather, I lived in a Bronx ghetto neighborhood where we often
grade level in reading and writing. One physician even told my
mother that I could become mentally retarded due to a blow my
father had made to my back. He did it in a state of anger for me goDOI:10.1091/mbc.E15-06-0451. Mol Biol Cell 26, 3692–3696.
ing into his pants pockets and eating some of his drugs (which he
Erich D. Jarvis is the 2015 recipient of the E. E. Just Award from the American
had synthesized as a student of chemistry) and also for my brother
Society for Cell Biology.
and me throwing toys and clothes out the window in our Harlem
Address correspondence to: Erich D. Jarvis ([email protected])
apartment. Most importantly, Just was an African American who was
© 2015 Jarvis. This article is distributed by The American Society for Cell Biology
struggling to survive as a scientist in a Caucasian majority environthe public under an Attribution–Noncommercial–Share Alike 3.0 Unported Crement (I say environment instead of world, because the majority of
the world was and still is not Caucasian). Just had been considered
for a faculty position at Rockefeller in the 1930s for his genius in cell
14 | E. D. Jarvis
biology but was rejected in part due to a scientific dispute with a
former mentor and collaborator, Jacques Loeb at Rockefeller, and in
part due to his race. I came back to MacLeish, had that discussion
with the resolve that if Just could make it through his hardships, so
could I. And further, Just’s hardships were orders of magnitude more
difficult than mine, due in part to greater discrimination and lower
expectations for him.
It was at that time I began to appreciate how much of a profound
impact ethnicity, culture, and gender can have on an individual’s
career. Before then, I was surrounded by persons of diverse backgrounds, many of whom looked like me. However, now I was at a
mostly Caucasian institution, clearly one of the world’s best in biomedical science, faced with culture shock of wondering why did
most of the students have a shared experience different from me?
Although they had their struggles, why were mine different and, in
many cases, tougher? In this essay, I discuss some of the answers to
these questions and lessons learned that helped me survive as an
underrepresented minority scientist, all of which I hope will be helpful for all scientists and all people as they navigate their careers.
THEY KILLED HIM BECAUSE OF THE COLOR
OF HIS SKIN
I was born in New York City in 1965, at a high point of the Civil Rights
movement, including the signing of the Voting Rights Act. My maternal grandparents and paternal great-grandparents were from
North Carolina and Virginia, having moved north to New York either
in the late 1800s or during the Great Depression of the 1930s, most
being descendants of slaves. I remember the day when I was watching a black-and-white television at my maternal grandparents’ house
in Queens, New York, where we lived after my mother divorced in
the early 1970s, about a news story of the anniversary of Martin Luther King Jr.’s death. Being between 6 and 7 years old and an inquisitive child, I asked my mother, “Why did they kill him?” She
seemed to have a hard time explaining it me, and finally came out
with it was because the color of his skin, he was black, a Negro, and
wanted to bring peace to all. I remember looking at my skin, feeling
afraid, and wondering, “Are they going to kill me one day?” From
that time onward as a child, I remember wanting my skin to become
white, my hair straighter, and my nose and lips thinner. My family
was diverse, and I envied those who looked more European than
me. I internalized a feeling of “less than.” It now makes me wonder
how young African-American children feel today, when they see on
the news stories about young black men killed by police with the
reason partly linked to ethnicity.
BEING TRAINED TO PERSEVERE IN THE FACE
OF DAUNTING OBSTACLES
Following my father’s conversion to a Japanese sect of Buddhism
(Soka Gakkai), my mother converted as well and taught us (her
children) its philosophies. Although her mother, a Baptist Christian,
secretly told us kids that she thought it was the devil’s religion, many
of the views of Buddhism made sense to me. I applied them to my
life, including that I am responsible for my own destiny, must pursue
my most ambitious dreams, dream the impossible, and that, no matter what, all obstacles can be overcome as long as you work at it.
Although I stopped practicing the faith-based part of the religion
in my teens, I still followed the philosophical views, which I found I
needed to get through the “less than” internalized feelings. I first
applied them to pursue a career in dancing. I was accepted into the
High School of Performing Arts, was on scholarships at the Joffrey
Ballet and Alvin Ailey Dance schools in New York, and performed
with the Westchester Ballet Company. But at the end my senior year,
I was at a junction between choosing opportunities for a career in
dance or something else I fell in love with, science. I chose science,
following my mother’s training of doing something that has a positive impact on society. I thought I could accomplish that better as
a scientist than as a dancer. So again, I applied my training in
Buddhism and now in the arts, into the sciences as an undergraduate student at Hunter College of the City University of New York. I
found that the transition between dance and science was natural, as
both required discipline, creativity, hard work, and, often, acceptance of failure before something works.
Hunter was and still is an ethnically diverse school, although I did
not realize that at the time, because it looked like the rest of New
York City’s melting pot. There I was taken under the wing of Rivka
Rudner, a molecular microbiologist studying ribosomal genes, which
synthesize proteins. I invested many hours in the lab, sometimes
staying overnight to finish my experiments, and pursued projects
that eventually led me to publish six research papers with her, including two as first author and one as senior corresponding author
(three of which were published by the time I graduated). We developed molecular tools to map the chromosomal organization of the
protein synthesis genes in bacteria and determined how their organization affected their genome evolution and function. Making such
discoveries, working with collaborators, and publishing these papers gave me the confidence that I could be a scientist.
At this juncture, I was considering the choices of applying to
medical school, graduate school, or both to pursue a MD/PhD. This
was a struggle many from economically challenged backgrounds
face, where there is a drive in the family to have my “son/daughter
the doctor.” Although I prepared for and performed well on entrance exams for both kinds of degrees, I decided to focus on the
basic science PhD route because this was where my passion was,
and it felt more closely connected to my artistic side.
THE ONLY WAY I FOUND I WAS ABLE TO OVERCOME
THE FEELING OF LESS THAN WAS BY BEING SUCCESSFUL
I applied to and was accepted into many of the top graduate
programs in the country in addition to Rockefeller, including
MIT, Harvard, Yale, Princeton, Johns Hopkins, Berkeley, and the
University of California, San Francisco. During my interviews, some
of the faculty members I interviewed with cautiously informed me
that they were surprised that an African-American kid from Harlem
had achieved what I did and were wondering how I did it. I did not
have an answer and was wondering, “What did I do?” I never
thought that I could not. More ethnically sensitive questions and
statements came from students. At one place, in a group conversation, a student told me that I should be careful of going to a specific
neighborhood, because there are “blacks and Puerto Ricans there
and it is dangerous.” I stared back at the person speechless, with a
half-smile, wondering to whom does he think he is talking? That is
the kind of neighborhood I come from; my wife at the time was
Puerto Rican. Were she and I dangerous? Another place had two of
their students, one African American and the other Caucasian, contact me on separate occasions to say that if I did not accept their
offer for graduate school, there would be no more African-American
students in their PhD program. I wondered whether I needed their
program, or they needed me. Why was this happening?
I accepted the offer to join the graduate student program at
Rockefeller but found a different experience from my previous
schools. At Hunter, as at many institutions with a diverse population,
there was a hold-your-hand support system practiced by the faculty.
At Rockefeller, it was sink or swim, as it is at many Research 1 institutions. I was swimming and sinking in my first four years as a graduate
Underrepresented scientist survival
| 15
student. I had never heard of parents being able to purchase a car
for their children after they graduate college; instead, I was helping
my parents keep out of financial troubles. There was an unspoken
feeling among a very small number of students that I was there to fill
a quota, and although tiny, it was enough to contribute to me feeling that way too. At the same time, my father was killed, elders in my
family were dying of natural but still more long-term preventable
causes, I was taking care of a family, I had failed my first prelim examination (which I passed the second time), and I was struggling to
get my experiments to work. I began to again internalize feelings of
less than. I had felt that I did not belong in this sink-or-swim world,
despite that fact that I had by then seven publications with my undergraduate advisor. But I did not give up. I worked hard, trying
different ways of overcoming these obstacles, and in my last two
years of graduate school, my earlier life experience training kicked
in. Once the small amount of negative influence around me moved
on, I found my groove and quickly progressed in my research, publishing three papers from my graduate research (including one as
first author).
I decided to break the rules and stay on as a postdoctoral fellow
in the same lab where I did my PhD, that of Fernando Nottebohm at
Rockefeller, because I felt I was just beginning to really swim. My
litmus test for making decisions was asking: “What are people going
to remember me for after I die?” Whether I followed standard rules
or made a significant impact in science? From the three years of my
postdoctoral research, I published three papers on the molecular
biology of vocal learning, eventually 10 altogether, in high-impact
journals, including Nature. I had finally overcome the feeling of less
than. I found that the only way I could overcome the feeling of less
than was to be successful in what I set out do to.
THE COLOR OF MY SKIN AND MY GENDER IS EITHER A
DISADVANTAGE OR AN ADVANTAGE, BUT RARELY
NEUTRAL
My postdoctoral years were the mid-1990s, a period during which
funding in science had been looking grim. Very good people were
applying for academic jobs and not getting them. Politicians were
being blamed for decreasing funding to science and making it
harder for us to make new discoveries and contribute to society. I
made a vow to myself that I was going to figure out a way to succeed and survive by doing the best science I could do. I worked very
hard, sometimes too hard. I applied for a small number of professorship research positions and received offers from all.
Once again, similar to my experience as a graduate student, faculty members at these institutions appeared to be surprised that a
person of color, an African American, born in the United States, had
accomplished what he did. Suddenly, was I heavily recruited, like an
up-and-coming basketball star. I felt that the color of my skin and
the drive to increase diversity among the academic ranks combined
with my scientific successes suddenly made me a commodity. This
was the first time in my life, at 32, when I felt the color of my skin was
an advantage. Then I was told about a Duke University tuition benefit program in which, if I were a faculty member for more than five
years, Duke University would pay for 100% of up to two of my children’s tuition at Duke or up to 75% of Duke’s rate at any other university in the world. Finding this out floored me. It washed away
generations of oppression for me in an instant. African Americans
were not allowed to be students at Duke before the early 1960s,
soon before I was born, and still had a hard time being accepted.
My parents and grandparents would not have been able to get into
Duke, regardless of their talents. And now, my children (I had two)
would later be able to get a high-quality, expensive college tuition
16 | E. D. Jarvis
paid for, anywhere in the world. It hit me that this was an affirmative
action program for Caucasians that had been around for generations. I cried, and accepted Duke’s offer. I was now in one of the
world’s leading neuroscience programs with the resources I needed
to accomplish the science I wanted to achieve and make sure my
children had an opportunity for a high-quality education.
From this experience, I learned that the color of my skin or my
gender or that of anyone else is either an advantage or a disadvantage, but rarely neutral. For most of my life it had been a disadvantage, but for once and at that moment, it was an advantage. I wanted
it to be neutral, but this was beyond my immediate control. It also
made me realize that the affirmative action programs I had benefited from, such as the National Institute of General Medical Sciences
Minority Biomedical Research and Minority Access to Research Careers programs as an undergraduate and graduate student were affirmative action programs that offset a disadvantage that many underrepresented students and others do not realize that they have.
I HAVE TWO JOBS: BE THE BEST SCIENTIST I CAN BE
AND HELP CURE SOCIETY’S RACIAL DISEASE
After I arrived at Duke in 1998, I was inducted into many initiatives
to help diversify the scientific workforce, including the push for
women in science. I wondered, as a man, what did I know about
women? But there was an assumption that a person of an underrepresented minority background knew more of what was needed
for any minority to succeed, including women in science, relative to
white males. There may be some truth to this, but certainly not an
absolute truth. I began to realize that as a young professor at Duke
University, and within the scientific community generally, I was being
unintentionally asked to take on two jobs: 1) be the best scientist I
could be, as expected of everyone else; and 2) help cure society’s
racial disease, unlike everyone else. After two years, I made the conscious decision that I could not do both jobs well at the same time.
I decided on job 1, to pursue being the best scientist I could be, and
only taking on those few tasks for job 2 in which I felt I could make
the biggest impact.
However, I felt that job 1 could indirectly help cure society’s disease. There were many people before me who had taken on activist
roles in bringing down obstacles and opening up opportunities for
others like me to become scientists. I felt what we needed more of
now was underrepresented minority scientists leading by example,
so that others would not say they are there because of a quota, that
they are less than, and that they have an advantage because of the
color of their skin. This is what I set out to do as a scientist and professor at Duke University. To do so, I had to learn how to say no to
many requests, balanced with yes for those that had the biggest
impacts. Before doing so, I would often consult with others to get a
second opinion. I also would also encourage others who were not
underrepresented minorities to help with job 2, as I felt the cure to
this disease required participation and education of everyone and
not just underrepresented minorities.
OVERCOMING CULTURE SHOCK AND LEARNING HOW
TO REACH OUT
When I first arrived at Duke, I had a similar but more stark experience than at Rockefeller, where I felt I was in the middle of Europe
in terms of ethnicity. This was despite the fact that, relative to the
rest of the country, there was a high proportion of African Americans
in the surrounding population. I recall interviewing a female Hispanic student our department brought in as a candidate for graduate school; during the interview, she opened a booklet that included
the Duke student demographics. African Americans were in the low
single digits, and Hispanics even lower. She actually cried in my office, wondering whether she had any chance of getting in, and if she
did, would she really be accepted culturally? Pertaining to faculty,
years later in 2006, the dean called a meeting of African-American
faculty members of the medical school to discuss our views of the
now infamous Duke lacrosse case, in which an African-American female stripper accused team members of rape (later turned out to be
false). At the meeting, I learned that I was only the second basic
science African-American faculty member in the medical school, out
of more than 200, in more than 28 years. The hiring of clinical faculty
fared better in absolute numbers, although not proportionally, as
there were more than 1800 clinical faculty members. Basically, some
others and I, including students, were feeling culture shock.
The ethnic diversity at Duke and some other Research 1 schools
has changed quite a bit, particularly, at the student level, since I became a professor in the late 1990s. The change was ushered in by
purposeful efforts made by leadership, including the Duke President’s Council on Black Affairs chaired by President Nannerl Keohane, the Dean’s Council on Diversity led by Nancy Andrews, admissions offices of the medical schools and graduate schools led by
Brenda Armstrong and Jacklynn Looney, respectively, and now the
Office of Biomedical Graduate Diversity started by Dona Chickaraishi; this last office is now led by one of the students I mentored at
Duke and the second African-American student to graduate from our
neurobiology department, Sherilynn Black. What I have learned from
these experiences about surviving as an underrepresented minority
in the sciences is that many such students do not take proactive steps
to reach out, seek help, and get answers to concerns they have. They
wait for someone to come to them. But they must learn how to reach
out in order to survive. For institutions, creating a culture of inclusion
and a space for these students to air their thoughts is a great help.
ACCEPT ALL APPROACHES AND BY ANY MEANS
NECESSARY
I have seen from firsthand experience how cultural background
plays a strong role in the way we go about conducting our science.
Growing up as a child in the 1960s and 1970s, my perception was
that you were either a “Martin Luther King family” or a “Malcolm X
family.” The Martin Luther King family adopted the belief of loving
and accepting everyone to bring about world peace; the Malcolm
X family adopted the belief of achieving equality by any means necessary. We were a Martin Luther King family. In this regard, in my
laboratory, I brought together people of diverse ethnicities and cultures with diverse ways of thinking, and found that this diversity led
to more rapid advances in our science relative to a mono-ethnic or
mono-gender group. But I have stolen from Malcolm X’s thinking as
well and have taken the approach to addressing scientific questions
by any means necessary, as long as it does not harm anyone. In this
manner, I have incorporated molecular biology, anatomy, physiology, evolutionary biology, genetics, and computational biology into
our research program to address questions on the brain mechanisms and evolution of vocal learning and spoken language. An
analogy is the marriage between physics and biology that led to the
discovery of the structure and genetic code of DNA. If I had not
been trained to think in this way as a child from a diverse background, it might have been harder for me to learn how to do so as
an adult.
ACCEPTING ALL OF ME
Growing up in the United States, we are often trained to think in a
black-and-white world. Further, black was considered bad, white
good, and any drop of blackness meant that you were considered
black. Although the definition of mulatto existed, it or admixture
was not a recognizable category in the check box of many forms.
You had to pick and choose, which became the title of a popular
song by one my cousins, a Native American singer, Pura-Fe. My
perception of growing up and how I was treated was mainly (∼80%)
African, with a mixture of (∼10% each) Native American and European. However, my family had debates about exactly what we were.
I decided at a young age to investigate the discrepancies, which I
still continue to do to this day. In contrast to perception, based on
the oral history of my elders and many others (and even in contrast
to some of their own perceptions), my ancestors have been admixed over and over again between these three ethnic groups for
several hundred years. My oral history calculations are 48% African,
37% European, and 14% Native American, each of multiple ethnic
groups from these ancestral populations. But my genetic ancestry
from ancestry.com, 23andMe, and whole-genome sequencing
shows 49% African, 49% European, and less than 1% Native
American. There are various reasons for the oral and genetic discrepancies, including that either the oral tradition is wrong or there
is not sufficient Native American DNA diversity in the databases.
One thing that genetics taught me is that my African and European
ancestries are heavily admixed from seven different ethnic groups
each, and this is not because they were already admixed before
arrival in the Americas—most of the admixtures occurred after
arrival in the Americas.
The biggest lesson I learned from taking a cultural and scientific
perspective in trying to figure out who I am, was that I had to learn
to accept all of me in order to help propel me along my scientific
journey more successfully. Neither I nor anyone else is really living in
a black-and-white world. Our current president, Barack Obama, is
considered the first “African-American” president of the United
States. But like me, he is as much European as he is African. So what
happened to the European part in the minds of Americans? It is
buried, not thought about, because we still live with this social disease of racism. If you can transcend that thought, accept all of who
you are, all of who we are, then I think you have a much greater
ability to communicate and interact with the broader world and advance in your science or whatever path in life you choose.
In closing, the challenges that Ernest Everett Just faced externally and internally and the approach he took to try to overcome
them has influenced how I handled trying to overcome the challenges I faced. Some of my challenges were easier, and some different, due in part to others making it more possible for me to succeed
than was possible in Just’s time. I suspect and hope that my brief
story here will help the next generation to overcome their challenges, which will invariably have overlap and differences. One such
difference I predict is that the countries and thus the environments
in which science is highly valued will have less of a black-and-white
view of the world, in part due to a greater understanding of human
and thus species genetics, and due in part to greater numbers of
admixed peoples according to current racial categories. I hope that
my story can be of comparable help to underrepresented minorities
and the majority. Finally, I note that not all formulas work for all people. My PhD advisor, Fernando Nottebohm, said he would tell his
son to “understand 100% of what I say, but only believe in 50% of
it.” This meant to me that not all formulas work for all, but there is
always room for improvement and change, and sometimes we get
things wrong, need to recognize when we do, correct them, and
then move on.
For further reading on my life story in the sciences written by others see publications by Rimer (1989), Dreifus (2003), Adler (2006),
Blakeslee (2005), NOVA (2005), and Berstein (2015a, 2015b). For
Underrepresented scientist survival
| 17
reading about some of the past and recent scientific discoveries I
have contributed to that I feel are broadly important see publications by Jarvis (2004), Jarvis et al. (2005, 2014), Petkov and Jarvis
(2012), Pfenning et al. (2014), Whitney et al. (2014), and Zhang et al.
(2014a,b).
ACKNOWLEDGMENTS
I acknowledge the support of my mother, father, stepfather,
grandparents, other family members, and friends, as well as my
undergraduate advisor Rivka Rudner and graduate advisor
Fernando Nottebohm, who helped me get to where I am now. I
also acknowledge the opportunities and funding provided to me
by the National Institutes of Health (particularly the National
Institute of General Medical Sciences), the National Science
Foundation, the Howard Hughes Medical Institute, the Society
for Neuroscience Scholars Program, Hunter College, The Rockefeller University, Duke University, and many others, without
which it would not have been possible for me to progress as a
scientist.
REFERENCES
Adler J (2006). Song and dance man. Smithsonian November. www
.smithsonianmag.com/making-a-difference/song-and-dance
-man-135440722.
Berstein R (2015a). Following the birdsong of science. Science Careers
January 19. http://sciencecareers.sciencemag.org/career_magazine/
previous_issues/articles/2015_01_19/caredit.a1500015.
Berstein R (2015b). Science by any means necessary. Science 347, 686. www
.sciencemag.org/content/347/6222/686.short.
Blakeslee S (2005). Minds of their own: birds gain respect. New York Times
February 1. www.nytimes.com/2005/02/01/science/minds-of-their-own
-birds-gain-respect.html.
18 | E. D. Jarvis
Dreifus C (2003). A conversation with: Erich Jarvis: a biologist that explores
the minds of birds that learn to sing. New York Times, January 7.
www.nytimes.com/2003/01/07/science/conversation-with-erich-jarvis
-biologist-explores-minds-birds-that-learn-sing.html.
Jarvis ED (2004). Learned birdsong and the neurobiology of human
language. Ann NY Acad Sci 1016, 746–777.
Jarvis ED, Güntürkün O, Bruce L, Csillag A, Karten H, Kuenzel W, Medina
L, Paxinos G, Perkel DJ, Shimizu T, et al. (2005). Avian brains and a
new understanding of vertebrate brain evolution. Nat Rev Neurosci 6,
151–159.
Jarvis ED, Mirarab S, Aberer AJ, Li B, Houde P, Li C, Ho SYW, Faircloth BC,
Nabholz B, Howard JT, et al. (2014). Whole genome analyses resolve
the early branches to the Tree of Life of modern birds. Science 346,
1320–1331.
Manning KR (1983). The Black Apollo of Science: The Life of Ernest Everett
Just, New York: Oxford University Press.
NOVA (2005). ScienceNOW profile of Jarvis. www.pbs.org/wgbh/nova/
nature/erich-jarvis.html.
Petkov CI, Jarvis ED (2012). Birds, primates, and spoken language origins:
behavioral phenotypes and neurobiological substrates. Front Evol
Neurosci 4, 121–24.
Pfenning A, Hara E, Whitney O, Rivas MV, Wang R, Roulhac PL, Howard JT,
Wirthlin M, Lovell PV, Ganapathy G, et al. (2014). Convergent transcriptional specializations in the brains of humans and song learning birds.
Science 346, 1256846.
Rimer S (1989). Random death claims a man who struggled to regain life.
New York Times May 27. www.nytimes.com/1989/05/27/nyregion/
random-death-claims-a-man-who-struggled-to-regain-life.html.
Whitney O, Pfenning AR, Howard JT, Blatti CA, Liu F, Ward JM, Wang R,
Audet JN, Kellis M, Mukherjee S, et al. (2014). Core and region enriched
gene expression networks of behaviorally regulated genes and the singing genome. Science 346, 1256780.
Zhang G, Jarvis ED, Gilbert MTP (2014a). A flock of genomes. Science 346,
1308.
Zhang G, Li C, Li Q, Li B, Larkin DM, Lee C, Storz JF, Antunes A, Greenwold
MJ, Meredith RW, et al. (2014b). Comparative genomics reveals insights
into avian genome evolution and adaptation. Science 346, 1311–1320.
An unconventional route to becoming
a cell biologist
Elaine Fuchs
Howard Hughes Medical Institute, Rockefeller University, New York, NY 10065
ABSTRACT I am honored to be the E. B. Wilson Award recipient for 2015. As we know, it was
E. B. Wilson who popularized the concept of a “stem cell” in his book The Cell in Development and Inheritance (1896, London: Macmillan & Co.). Given that stem cell research is my
field and that E. B. Wilson is so revered within the cell biology community, I am a bit humbled
by how long it took me to truly grasp his vision and imaginative thinking. I appreciate it
deeply now, and on this meaningful occasion, I will sketch my rather circuitous road to cell
biology.
I could hardly wait until I was in junior
I grew up in a suburb of Chicago. My father
high school, when I could enter science
was a geochemist, and for everyone whose
fairs. You would think that my scienceparents worked at Argonne National Labominded family might help me choose and
ratories, Downers Grove was the place to
develop a research project. True to their
live. My father’s sister was a radiobiologist
mentoring ethos, they left these decisions
and my uncle was a nuclear chemist, both
to me. My first project was on crayfish beat Argonne; they lived in the house next
havior. I recorded the response of the craydoor. Across the street from their house
fish I had caught to “various external stimwas the Schmidtke’s Popcorn Farm—a
uli.” At the end of this assault, I dissected
great door to knock on at Halloween. The
the crayfish and, using “comparative anatcornfields were also super for playing hideomy,” attempted to identify all the parts.
and-seek, particularly when you happened
The second project was no gentler. I foto be shorter than those Illinois cornstalks.
cused on tadpole metamorphosis and the
I remember when the first road in the
effects of thyroid hormone in accelerating
area was paved. It made biking and rollerdevelopment at low concentrations and
skating an absolute delight. Fields of butdeath at elevated concentrations. Someterflies were everywhere, and with develElaine Fuchs
how, I ended up going all the way to the
opment came swamps and ponds filled
state fair, where it became clear that I had
with pollywogs and local creeks with crayserious competition. That experience, however, whetted my appefish. It was natural to become a biologist. When coupled with a famtite to gain more lab experience and to learn to read the literature
ily of scientists and a mother active in the Girl Scouts, all the remore carefully.
sources were there to make it a perfect path to becoming a
My experience with high school biology prompted me to gravscientist.
itate toward chemistry, physics, and math. When it came to college, my father told me that if there was a $2000/year (translated
in 2015 to be $30,000/year) reason why I should go anywhere
besides the University of Chicago (where Argonne scientists reElaine Fuchs is the recipient of the 2015 E. B. Wilson Medal awarded by the
American Society for Cell Biology.
ceived a 50% tuition cut for their children) or the University of IlAddress correspondence to: Elaine Fuchs ([email protected]).
linois (then $200/year tuition), we could “discuss” it further. HavAbbreviations used: EBS, epidermolysis bullosa simplex; EH, epidermolytic
ing a sister, father, aunt, and uncle who went to the University of
hyperkeratosis; Ifs, intermediate filaments.
Chicago, I chose the University of Illinois and saved my Dad a
© 2015 Fuchs. This article is distributed by The American Society for Cell Biology
bundle of money. At Illinois, I thought I might revisit biology, but
the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Cremy choices for a major were “biology for teachers” or “honors
biology.” The first did not interest me; the second seemed
®
®
“ASCB ,” “The American Society for Cell Biology ,” and “Molecular Biology of
intimidating.
19
I enrolled as a chemistry major. Four years went by, during which
time I never took a biology class. I enjoyed quantum mechanics,
physics, and differential equations, and problem solving became
one of my strengths. In the midst of the Vietnam War era, however,
Illinois was a hotbed of activity. I was inspired to apply to the Peace
Corps, with a backup plan to pursue science that would be more
biomedically relevant than quantum mechanics. I was accepted to
go to Uganda with the Peace Corps, but with Idi Amin in office, my
path to science was clear. Fortunately, the schools I applied to accepted me, even though, in lieu of GRE scores, I had submitted a
three-page essay on why I did not think another exam was going to
prove anything. I chose Princeton’s biochemistry program. This
turned out to be a great, if naïve choice, as only after accepting their
offer did I take a biochemistry class to find out what I was getting
into. I chose to carry out my PhD with a terrific teacher of intermediary metabolism, Charles Gilvarg, who worked on bacterial cell walls.
My thesis project was to tackle how spores break down one cell wall
and build another as they transition from quiescence to vegetative
growth.
By my fourth year of graduate school, I was trained as a chemist
and biochemist and was becoming increasingly hooked on biomedical science. I listened to a seminar given by Howard Green, who
had developed a method to culture cells from healthy human skin
under conditions in which they could be maintained and propagated for hundreds of generations without losing their ability to
make tissue. At the time, Howard referred to them as epidermal
keratinocytes, but in retrospect, these were the first stem cells ever
to be successfully cultured. I was profoundly taken by the system,
and Howard’s strength in cell biology inspired me. It was the perfect
match for pursuing my postdoctoral research. The time happened
to be at the cusp of DNA recombinant technology.
At MIT, I learned how to culture these cells. I wanted to determine their program of gene expression and how this changed when
epidermal progenitors embark on their terminal differentiation program. While the problem in essence was not so different from that
of my graduate work at Princeton, I had miraculously managed to
receive my PhD without ever having isolated protein, RNA, or DNA.
Working in a quintessential cell biology lab and tackling a molecular
biology question necessitated venturing outside the confines of the
Green lab and beyond the boundaries of my expertise. Fortunately,
this was easy at MIT. Richard Hynes, Bob Horvitz, Bob Weinberg,
and Graham Walker were all assistant professors, and their labs were
very helpful, as were those of David Baltimore and Phil Sharp, a
mere walk across the street. On the floor of my building, Steve
Farmer, Avri Ben Ze’ev, Gideon Dreyfuss, and Ihor Lemischka were
in Sheldon Penman’s lab just down the hall, and they were equally
interested in mRNA biology, providing daily fuel for discussions.
Uttam Rhajbandary’s and Gobind Khorana’s labs were also on the
same floor, making it easy to learn how to make oligo(dT)-Sepharose to purify my mRNAs. Vernon Ingram’s lab was also on the same
floor, so learning to make rabbit reticulocyte lysates to translate my
mRNAs was also possible. Howard bought a cryostat, so I could section human skin and separate the layers. And as he was already
working with clinicians at Harvard to apply his ability to create sheets
of epidermal cells for the treatment of burn patients, I had access to
the leftover scraps of human tissue that were also being used in
these operations.
The three years of my postdoc were accompanied by three
Fuchs and Green papers. The first showed that epidermal keratinocytes spend most of their time expressing a group of keratin proteins with distinct sequences. The second showed that these keratins were each encoded by distinct mRNAs. The third showed that,
20 | E. Fuchs
as epidermal keratinocytes commit to terminally differentiate, they
switch off expression of basal keratins (K5 and K14) and switch on
the expression of suprabasal keratins (K1 and K10). That paper also
revealed that different stratified tissues express the same basal keratins but distinct sets of suprabasal keratins. I am still very proud of
these accomplishments, and my MIT experience made me thirst to
discover more about the epidermis and its stem cells.
My first and only real job interview came during my second year
of postdoc, at a time when I was not looking for a job. I viewed the
opportunity, initiated by my graduate advisor, as a free trip home to
visit my parents and my trial run to prepare me for future searching.
I was thrilled when this interview materialized into an offer to join the
faculty, for which the University of Chicago extended my start time
to allow me to complete my three years with Howard.
Times have clearly changed, and it is painful to see talented
young scientists struggle so much more today. That said, I have
never looked ahead very far, and having a lack of expectations or
worry is likely to be as helpful today as it was then. I am sure it is
easier said than done, but this has also been the same for my science. I have always enjoyed the experiments and the joy of discovery. There was no means to an end other than to contemplate what
the data meant in a broader scope.
I arrived at the University of Chicago with a well-charted route. My
aim was to make a cDNA library and clone and characterize the sequences and genes for the differentially expressed keratins I had
identified when I was at MIT. It was three months into my being at
Chicago when my chair lined up some interviews for me to hire a
technician. I was so immersed in my science that I did not want to
take time to hire anyone. I hired the first technician I interviewed.
Fortunately, it worked out. However, I turned graduate students
away the first year, preferring to carry out the experiments with my
technician and get results. After publishing two more papers—one
on the existence of two types of keratins that were differentially expressed as pairs and the other on signals that impacted the differential expression of these keratin pairs, I decided to accept a student,
who analyzed the human keratin genes. My first postdoc was a fellow
grad student with me at Princeton; she studied signaling and keratin
gene expression. My second postdoc was initiated by my father, who
chatted with him at the elevator when I was moving into my apartment. He set up DNA sequencing and secondary-structure prediction methods, and the lab stayed small, focused, and productive.
I was fascinated by keratins, how they assembled into a network
of intermediate filaments (Ifs). When thalassemias and sickle cell
anemia turned out to be due to defects in globin genes, I began to
wonder whether there might be human skin disorders with defective
keratin genes. I had no formal training in genetics, and there were
no hints of what diseases to focus on. Thus, rather than using positional cloning to identify a gene mutation associated with a particular disease, we took a reverse approach: we first identified the key
residues for keratin filament assembly. After discovering that mutations at these sites acted dominant negatively, we engineered transgenic mice harboring our mutant keratin genes and then diagnosed
the mouse pathology. Our diagnoses, first for our K14 mutations
and then for our K10 mutations, turned out to be correct: on sequencing the keratins from humans with epidermolysis bullosa simplex (EBS), we found K14 or K5 mutations; similarly, we found K1 or
K10 mutations in affected, but not in unaffected, members of families with epidermolytic hyperkeratosis (EH). Both are autosomaldominant disorders in which patients have skin blistering or degeneration upon mechanical stress. Without a proper keratin network,
the basal (EBS) or suprabasal (EH) cells could not withstand pressure.
Ironically, family sizes of all but the mildest forms of these disorders
were small, meaning that the disorders were not amenable to positional cloning. But the beauty of this approach is that once we had
made the connection to the diseases, we understood their underlying biology. In addition, the IF genes are a superfamily of more than
100 genes differentially expressed in nearly all tissues of the body.
Once we had established EBS as the first IF gene disorder, the pathology and biology set a paradigm for a number of diseases of
other tissues that turned out to be due to defects in other IF genes.
Fortunately, I had students, Bob Vassar (professor, Northwestern
University) and Tony Letai (associate professor, Harvard Medical
School), and a postdoc, Pierre Coulombe (chair, Biochemistry and
Molecular Biology, Johns Hopkins University), who jumped into this
fearless venture with me. We had to go off campus to learn transgenic technology. I had never worked with mice before. When Bob
returned to campus with transgenic expertise, we hired and trained
Linda Degenstein, whose love for animal science was unparalleled.
Pierre’s prior training in electron microscopy was instrumental in
multiple ways. Additionally, I was not a dermatologist and had no
access to human patients. Fortunately Amy Paller, MD, at Northwestern volunteered to work with us.
The success of this project attests to an important recipe: 1) Pursue a question you are passionate about. 2) In carrying out rigorous,
well-controlled experiments, each new finding should build upon
the previous ones. 3) If you have learned to be comfortable with
being uncomfortable, then you will not be afraid to chart new territory when the questions you are excited to answer take an unanticipated turn. 4) Science does not operate in a vacuum. Interact well
with your lab mates and take an interest in their science as well as
your own. And wherever you embark upon a pathway in which the
lab’s expertise is limited, do not hesitate to reach out broadly to
other labs and universities.
I have followed this recipe now for more than three decades,
and it seems to work pretty well. A lab works only when its students and postdocs are interactive, naturally inquisitive, and freely
share their ideas and findings. I have been blessed to have a number of such individuals in my lab over the years. When push comes
to shove, I am always inclined first to shave from the “brilliant”
category and settle for smart, nice people who are passionate and
interactive about science and original and unconventional in their
thinking.
So what questions have I been most passionate about? I have
always been fascinated with how tissues form during development,
how they are maintained in the adult, and how tissue biology goes
awry in human disorders, particularly cancers. I first began to think
about this problem during my days at Princeton. I also developed a
dogma back then that I still hold: to understand malignancies, one
must understand what is normal before one can appreciate what is
abnormal. I think this is why I have spent so much of my life focusing
on normal tissue morphogenesis, despite my passion for being at
the interface with medicine. And because skin has so many amazingly interesting complexities, and because it is a great system to
transition seamlessly between a culture dish and an animal, I have
never found a reason to choose any other tissue over the one I chose
many years back.
I will not dwell on the various facets of skin biology we have tackled over the years. Our initial work on keratins was to obtain markers
for progenitors and their differentiating lineages. This interest
broadened to understanding how proliferative progenitors form cytoskeletal networks and how the cytoskeleton makes dynamic rearrangements during tissue morphogenesis.
From the beginning, the lab has also been fascinated by how
tissue remodeling occurs in response to environmental signals.
Indeed, signals from the microenvironment trigger changes in
chromatin dynamics and gene expression within tissue stem cells.
Ultimately, this leads to changes in proteins and factors that impact on cell polarity, spindle orientation, asymmetric versus symmetric fate specifications, and ultimately, the balance between
proliferation and differentiation.
The overarching theme of my lab over these decades is clear,
namely, to understand the signals that unspecified progenitors receive that instruct them to generate a stratified epidermis, make
hair follicles, or make sweat and sebaceous glands. And if we can
understand how this happens, then how are stem cells born, and
how do they replace dying cells or regenerate tissue after injury?
And, finally, how does this process change during malignant progression or in other aberrant skin conditions?
In tackling tissue morphogenesis, I have had to forgo knowing
the details of each tree and instead focus on the forest. There are
many times when I stand back and can only admire those who are
able to dissect beautiful cellular mechanisms with remarkable precision. But I crossed that bridge some years ago in tackling a problem that mandates an appreciation of nearly all the topics covered
in Bruce Alberts’ textbook Molecular Biology of the Cell. I am now
settled comfortably with the uncomfortable, and the problem of
tissue morphogenesis in normal biology and disease continues to
keep me more excited about each year’s research than I was the
previous year. Perhaps the difference between my days as a student, postdoc, and assistant professor and now is that my joy and
excitement is as strong for those I mentor and have mentored as it
is for myself.
E. B. Wilson Medal
| 21
MBoC | PERSPECTIVE
Biosecurity in the age of Big Data: a conversation
with the FBI
Keith G. Kozminski
Department of Biology, University of Virginia, Charlottesville, VA 22904; Department of Cell Biology, University of
Virginia, Charlottesville, VA 22908
ABSTRACT New scientific frontiers and emerging technologies within the life sciences pose
many global challenges to society. Big Data is a premier example, especially with respect to
individual, national, and international security. Here a Special Agent of the Federal Bureau of
Investigation discusses the security implications of Big Data and the need for security in the
life sciences.
INTRODUCTION
“The FBI is reading our poster!” Granted, this is not a typical refrain
heard at the annual meetings of the American Society for Cell Biology, but it is heard frequently at other research meetings, for example, in the field of synthetic biology. I admit that it was head
turning when I first heard these words spoken a few years ago at the
International Genetically Engineered Machines (iGEM) Jamboree,
which is an annual, global, intercollegiate synthetic biology competition. In format, the iGEM Jamboree is much like the annual meeting
of any major scientific society. But why, in the aisles, were there
suited people with badges? Perhaps a new age has dawned upon
the research community. The contents of this special issue of Molecular Biology of the Cell, with an emphasis on Big Data, certainly
suggest that this is true. Nonetheless, overt governmental examination of research beyond the standard purview of granting agencies
and its program officers can only raise questions. To answer some of
these questions, I invited, on behalf of Molecular Biology of the Cell,
Supervisory Special Agent (SSA) Edward You, who heads the Biological Countermeasures Unit (BCU) at Federal Bureau of Investigation (FBI) Headquarters in Washington, D.C., and frequently addresses the synthetic biology community, to have a conversation on
Address correspondence to: Keith G. Kozminski ([email protected]).
Abbreviations used: AAAS, American Association for the Advancement of
Science; BCU, Biological Countermeasures Unit; BWC, Biological Weapons Convention; CDC, Centers for Disease Control and Prevention; DoD, Department of
Defense; FBI, Federal Bureau of Investigation; iGEM, International Genetically
Engineered Machines; NIH, National Institutes of Health; SSA, Supervisory Special Agent; WMD, weapons of mass destruction.
© 2015 Kozminski. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
22 | K. G. Kozminski
Monitoring Editor
David G. Drubin
University of California,
Berkeley
Received: Jul 30, 2015
Accepted: Aug 5, 2015
biosecurity, especially with respect to Big Data (Figure 1). This conversation was recorded on July 17, 2015, and is presented here,
abridged and edited for clarity and considerations of space.
ONE FOOT IN NATIONAL SECURITY; ONE FOOT
IN THE LIFE SCIENCES
MBoC: Agent You, before you talk about Big Data, please tell our
readers about your scientific background and path to the FBI.
SSA You: I got my bachelor’s degree in the biological sciences
from the University of California at Irvine, then a master’s degree in
biochemistry and molecular biology at the University of Southern
California. All that has served me well; it does show that there is life
without a PhD.
Before joining the Bureau, I came from the laboratory setting. I
had six years of graduate research in human gene therapy, with a
focus on retrovirology, and three years in the biotech sector at
Amgen, where I did oncology research. Then I decided to go into
public service and apply to the FBI.
MBoC: What are your responsibilities at the FBI? What is your
mission today?
SSA You: I sit at headquarters at the Weapons of Mass Destruction (WMD) Directorate in the Biological Countermeasures Unit.
The WMD Directorate is one of the newest divisions of the FBI. It
was born out of the events of September 11, 2001. On the heels of
that terrorist event, we had the anthrax mailings. It was a serious
wake-up call for the U.S. government and the FBI in particular.
Since then, as a law enforcement service, our priority has become
one of prevention rather than being reactive, just going in and investigating a crime or incident. Now our number one priority is to
prevent in particular a 9/11 from happening again. Safeguarding
science is the theme of my mission. Part of that is reaching out proactively to different members of the scientific community, ranging
from the private sector, biotech and the pharmaceutical industry;
FIGURE 1: FBI SSA Edward You. In addition to heading the FBI’s
BCU, he is a Working Group member of the National Security Council
Interagency Policy Committee on Countering Biological Threats and
an ex officio member of the NIH National Science Advisory Board for
Biosecurity. He also serves on two National Academies committees:
the Institute of Medicine’s Forum on Microbial Threats and the
Committee on Science, Technology, and Law’s Forum on Synthetic
Biology. SSA You also serves on the Strategic Advisory Board for the
Synthetic Biology and Engineering Research Center and as an
instructor for the United Nations Interregional Crime and Justice
Research Institute. He can be reached at (202) 324-0236 or Edward
[email protected].
to universities; to the iGEM; and even to the amateur community,
the sprawling Do-It-Yourself bio community, showing how members
of law enforcement and the life science community have a shared
responsibility of safeguarding the development and very beneficial
applications of the life sciences. I find myself in a unique position,
where I have one foot in national security and another in the life
sciences. I seek very hard to ensure that we are able to support
both at the same time.
BIG DATA WORRIES AT THE FBI
MBoC: You mentioned synthetic biology and have been involved in
that community. However, it seems more recently that the FBI has
been showing more overt concern toward the security of Big Data in
the life sciences. Why does the FBI have concern?
SSA You: If you take my consideration of how to protect the life
sciences in a proactive manner, it is our responsibility to identify
emerging areas. Six years ago the emerging area was synthetic biology. That is why you have seen all this activity and outreach occurring, especially at iGEM.
The reason why Big Data has become very significant is that it is
the next evolutionary step that synthetic biology will take, meaning
that all applications and technologies coming out of this field will be
completely dependent upon data—all the various omics.
A very good example is precision personalized medicine, where
you are seeing tremendous investments in drug development, particularly in cancer research and metabolic disease, where very large
data sets are leveraged. If you are looking at an individual’s genome,
it is just one snap shot. What are needed are data over time, during
exposure to the environment, for example. From the human standpoint, maybe this is looking at your lifestyle—daily diet or exercise.
It all goes into helping determine potential health vulnerabilities
and appropriate therapies. If you set that as a stage and then look
at potential policy aspects, there is a lot of activity looking at privacy,
but not a whole lot looking specifically at security.
So, back in April 2014, I partnered with the American Association
for the Advancement of Science (AAAS) and the United Nations Interregional Crime and Justice Research Institute (UNICRI). We
kicked off a meeting where the theme was national and transnational security implications of Big Data in the life sciences. We really
wanted to tackle some of the security implications in the area of Big
Data, where biology has almost a complete overlap with the digital
world. At this meeting I had representatives from Microsoft, Intel,
IBM, Google, and Amazon, the entities leveraging this Big Data
bio-innovation future, and challenged them at the outset to identify
potential security issues. We did find some significant issues and
published some reports that are now publically available.
MBoC: You had an incredible lineup of expertise contributing to
the AAAS report National and Transnational Security Implications of
Big Data in the Life Sciences (Berger and Roderick, 2014). Was there
any specific event that motivated the FBI to launch this reflection on
biosecurity or was this entirely a proactive endeavor?
SSA You: The anthrax mailing in 2001 was a huge seminal event.
Security discussions in the past tried to overlay security structures
that were used in the nuclear or chemical realm. Completely locking
down certain areas of expertise or materiel is completely antithetical
to how the life sciences operate. If our mission is one of preventing
the misuse, exploitation, or abuse of the life sciences, how do we
approach security without becoming a hindrance to the life science
enterprise?
Over the last two years, we have had the issues with regard to
the Centers for Disease Control and Prevention (CDC) and Department of Defense (DoD). A lot of discussion also came when the
J. Craig Venter Institute synthesized that bacterial genome. There
were a lot of calls and discussions about the scientific community
needing more ethics training and the need to develop a greater
culture of responsibility. From a law enforcement perspective those
are necessary but not sufficient. What has been lacking is the scientific community being provided security awareness—something
that augments how they approach the life sciences. Individuals,
no matter where they are in the world or when they enter the life
sciences, always start with the premise, “Do No Harm,” taking a
page from the Hippocratic Oath. Unfortunately there are groups
and individuals who do not subscribe to the same ethics and norms
and agreements to integrity that we all take for granted and are almost innate for us. How do we graduate from “Do No Harm” to
“Not On My Watch”? It means you take an active role in being
sentinels for what you are doing and preventing the abuse, misuse,
and exploitation of the life sciences. If you are not fully aware what
the security vulnerabilities are, then that becomes a true vulnerability for all of us.
We also have a Biological Weapons Convention (BWC). It is
amazing to me that we have an international treaty to which we are
all beholden, yet there are very few programs, if any, in which incoming biology students are exposed to it or the fact that the BWC exists because biology had been absolutely used and exploited for
Biosecurity in the age of Big Data
| 23
offensive purposes, even by the United States. If we do not teach
that little bit of history and other security aspects, then it becomes
really a challenge in the future on how to better protect biology. It
is not about ethics; it is not about responsibility; it really is about
having a healthy appreciation of some of the security concerns.
MBoC: How real is the threat? The aforementioned AAAS report
read, “very little, if any, information exists about the theft, manipulation, or exploitation of Big Data in the life sciences.”
SSA You: That is the key question. One of the goals of generating this report was to galvanize people to start thinking about security because quite honestly I do not think we really appreciate how
deep or how wide the security vulnerabilities are in leveraging these
large data sets or Big Data in general.
Referring to my prior comments about precision medicine, it all
hinges on genetic information and longitudinal data over time. You
are only as good as the size of the data set. You need a large data
set because when you do an analysis you need statistical significance to know whether your results are right. As you think about
that, let me walk you back to some of the most significant cyber-intrusions this past year. In August 2014, there was a Community
Health Systems hack with 4.5 million patient records accessed; a few
months after that was the large Anthem Blue Cross hack with
80 million individuals impacted; and then a month after that, the
Premera Blue Cross hack in which 11 million patient records were
hit. This is when it keyed off for me. In the Premera Blue Cross hack,
clinical data were accessed too. Across the government, with these
particular intrusions, the focus has been only on the potential loss of
personal identifying information, the risk of fraud, and identity theft.
I do not want to give that short shrift, because we are talking about
tens, hundreds of millions of dollars in potential loss. However, if you
think about the critical data—a beautiful longitudinal data set, containing an individual’s demographics, disease state, drugs administered, and treatment received—someone now has a treasure trove
of clinical trial information. Unfortunately, all of those hacks were allegedly attributed to a hacking group based out of China. It has
become not just fraud anymore. There is a much broader security
vulnerability, the potential loss of our ability to stay globally competitive in the new drug market. Now somebody out there has the
brass ring—this gigantic data set, where the only limitation is deriving the analytical tools to make all that data useful.
There are a couple of issues now. One is to identify how much we
have given up. We have to get beyond the paradigm of just looking
at the financial loss. In the area of Big Data with specific applications
to the life sciences, information taken could potentially be used for
exploitation or extortion. A second is that, with the analytical tools
that are coming online today, it will be almost impossible to deidentify information in the future. This was a key takeaway from the
meeting with the AAAS-UNICRI last year. If you have any short genetic sequence of an individual, you can effectively deanonomize it
in fewer than three steps with publically available tools.
MBoC: Privacy does not exist anymore?
SSA You: Correct. If you are part of an institutional review board,
you are in big trouble in maintaining compliance and keeping up
with protecting human subject information. That is just one regulatory hurdle that will be coming up.
MBoC: Where is the greatest security threat to Big Data through
hacking? Is it through the lone wolf, companies engaged in industrial espionage, or is it from state-sponsored activities?
SSA You: My answer is “yes.” The vulnerabilities are across the
spectrum.
MBoC: Are the threats to Big Data greater for private Big Data,
for example Pharma, or for academic Big Data?
24 | K. G. Kozminski
SSA You: It is all of the above. Our AAAS meeting came to the
crux of it: whoever has the largest and most diverse data set is going
to win. That means we really need to start thinking in a more holistic
manner what security means with a data set.
MBoC: Does the FBI define Big Data in terms of volumes of data
or analytical functions? Is the threat against the volumes of data or
the ability to analyze data?
SSA You: It is both. I do not want to go too far into definitions
because one of the issues is how to define Big Data.
From a life sciences standpoint, we need to be going into this
with our eyes wide open. How do we do anything? A thorough assessment of potential security vulnerabilities is a first step. Second,
identify how to mitigate them up front. Finally, ask whether we have
to come up with novel ways to address security in this bio-future.
The power of the life sciences is open source, open sharing, but in it
there is the added dimension of an individual’s very intimate information. So there may be a call to redefine how we address security
in the future. It may not be building up secure walls, whether they
are physical or virtual, that protect data like our financial data. In this
world of the life sciences, which is inherently open, we are going to
have to rethink security.
MBoC: How should life scientists, faculty members at universities, respond to the worries of the FBI in terms of biosecurity? What
do you see people doing to improve the situation?
SSA You: To me, the strategy is that once we build trusted partnerships with the scientific community, first with the FBI reaching out
and providing the security awareness and education, something really profound happens. We have seen it happen in synthetic biology.
You see the scientific community doing their own assessments of
their technologies, self-identifying potential security vulnerabilities
and then providing notification to the FBI—to my unit or other partners at the FBI. So the tables have turned. The scientific community
educates the FBI on emerging vulnerabilities. They do us a favor,
helping us to be better informed to better protect the life sciences,
universities, and communities. Even better, the community will then
develop security solutions based on their expertise, which is the
best of both worlds. How powerful would that be when the experts,
who are developing these powerful tools and applications of the
future, immediately, on the front end, start developing and implementing security measures within these applications? That is where
we want to be; that is where the future has got to be. So there is
absolutely a very necessary and important partnership between law
enforcement and the scientific community. It is just not a one-way
street.
Take, for example, the scientific papers regarding CRISPR/Cas9
and gene drives and most recently the genetically modified yeast
producing opioids. Scientists drafted the scientific manuscript and a
companion editorial piece calling out the potential security vulnerabilities. That is powerful; that is a home run. We have successfully
empowered the scientific community to understand security and
then to take some proactive actions of their own.
MBoC: It seems one of the concerns of your unit, the BCU, is
dual use of data. Does the BCU have formal relationships or work
with the National Institutes of Health (NIH) National Science Advisory Board for Biosecurity or the CDC?
SSA You: Thank you for that question. It goes to the background
of the WMD Directorate. One of the cornerstone aspects of our
program is the really important position called the WMD Coordinator. These are men and women, Special Agents, trained in chemical,
biological, radiological, and nuclear matters. We have at least one
stationed in each of our 56 field offices across the United States. The
WMD Coordinator’s role, as the name implies, is to coordinate and
lead the notification protocols with state and local law enforcement,
public health, partner with other federal entities, and then build relationships with universities, companies, and institutions within their
jurisdiction. So if anything did occur, a local university, for example,
would then know they have a local federal representative that can
respond. If there is ever a biological incident or actual bio-crime,
then those WMD Coordinators become critical in the response.
They actually have been a big part of the action over the years with
the DoD and CDC events, the discovery of smallpox at NIH at a
Food and Drug Administration laboratory, the two high-profile ricin
mailings almost two years ago, and the incident at Georgetown University where a student was manufacturing ricin in his dorm room. In
all of these different incidents, those WMD Coordinators were called
in and were part of the response. No matter where in the government, the Coordinator is there to help and assist with either preventive training or, if anything did occur, the response.
MBoC: How do people find the WMD Coordinators should they
ever need one?
SSA You: They can just call the FBI field office in their jurisdiction
and ask to be referred to the WMD Coordinator. Should any suspicious or criminal activity be observed that puts personnel, institutions, or materials at risk, contact your local FBI WMD Coordinator
to help with any assessments. Think of them as being a resource to
the scientific community. If you call them, it is not immediately the
opening of an investigation. They are someone specifically within
the FBI who is familiar with the life sciences community and with
whom you can just touch base to see if something passes the sniff
test.
MBoC: Although many readers of Molecular Biology of the Cell
are gaining a greater awareness of Big Data, their own research
does not take them into the realm of Big Data. For those readers,
does the FBI have biosecurity concerns that lie with small data or is
the focus really on Big Data?
SSA You: The focus on Big Data is because it is an emerging
area. If you see all of our activities, the overall theme is safeguarding
science, whether you are working in large data analytics, with select
agents, yeast, or Escherichia coli. We are not honing in on a specific
subgroup or subtopic of the life sciences. It is really preventing the
misuse of the life sciences in general.
MBoC: The FBI’s primary role is safeguarding the homeland, the
United States. Many of our readers are not Americans. Is there a
separate, special, or additional message for people doing life science research, especially Big Data research, outside the boundaries
of the United States?
SSA You: Safeguarding science is universally applicable. I hope
for a future when biologists are working as WMD Coordinators in
other law enforcement agencies around the world. We need that.
The 21st century will see the same leaps and bounds with the life
sciences that we saw in the 20th century with the Internet and personal computing. If there is going be a global impact from the life
sciences, there is absolutely a call to action for biologists wherever
they are in the world to be guardians of science. However, we need
to come to a realization first that there will be issues. We have to
start discussing these things now before it is too late, before any
attempts at security will be too little, too late. We are much better
served tackling issues sooner. I hate to say it, but, if we are not careful and there is a complete overlap of the life sciences and the digital world, we might see ourselves with our security as we are facing
cyber-security right now, and we do not want to be in that position.
FROM BENCH TO BADGE—ARE YOU HIRING?
MBoC: It is clear there is a lot of work ahead, not just for the scientific community, but for the FBI as well. What are the career opportunities for cell biologists in the FBI, whether they have Big Data
experience or not?
SSA You: We are most definitely hiring. You can be a Special
Agent like me, or there are support positions such as the scientists
who work in our laboratory division. These are individuals who develop the tools for forensic analysis. A key piece of our mission is
looking at intelligence; that is an analyst position. There will absolutely be a need for folks with a biology background. You do not
need to have law enforcement experience. I did not.
I will be completely candid, upfront—our hiring is a very competitive process. Prior to 9/11, the FBI’s focus was on hiring individuals with law enforcement or military experience, lawyers, or accountants because the primary mission was tackling organized crime. In
this day, when our number one priority is prevention, there is an
absolute critical need for hiring individuals with background in computer science, foreign languages, and especially the natural sciences. If you have a chemistry or biology background, you are in the
running. The minimal criteria are a bachelor degree and at least
three years of real-world experience. More than anything else, if you
can articulate and show how you excelled in your specific field, then
you are a good candidate. Your field does not necessarily have to be
Big Data. You need to be passionate about what you do because in
doing so, you inherently excel. The key is to set yourself in a position
where you can really excel so when we begin talking to your coworkers and managers about who you really are, you have put them in a
position where they can say you are an integral part of the team and
made significant contributions. That will be a good selling point for
a future career in the FBI.
THE TAKE-AWAY
SSA You: Partnerships between the FBI and the scientific community
to build security awareness are essential. Big Data in the life sciences is taking the biosecurity discussion beyond pathogens and
toxins. Historically, the conversation almost always fell on pathogens
and almost exclusively on select agents. We have to widen the aperture of what we mean by biosecurity in the future.
ACKNOWLEDGMENTS
I thank John Fleischman, American Society for Cell Biology Senior
Science Writer, for tips in the preparation of this interview.
REFERENCE
Berger KM, Roderick J (2014). National and transnational security implications of Big Data in the life sciences. Available at www.aaas.org/report/
national-and-transnational-security-implications-big-data-life
-sciences (accessed 19 March 2015).
Biosecurity in the age of Big Data
| 25
MBoC | ARTICLE
2015 MBoC PAPER OF THE YEAR
Subcellular optogenetic inhibition of G proteins
generates signaling gradients and cell migration
Patrick R. O’Neilla and N. Gautama,b
a
Department of Anesthesiology and bDepartment of Genetics, Washington University School of Medicine, St. Louis,
MO 63110
ABSTRACT Cells sense gradients of extracellular cues and generate polarized responses
such as cell migration and neurite initiation. There is static information on the intracellular
signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert
spatial and temporal control over specific signaling molecules inside a living cell. Here we
introduce optogenetic tools that act downstream of native G protein–coupled receptor
(GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein
subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS)
protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results
in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform
chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling
activity. Migratory responses generated by this approach show that a gradient of active G
protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide
the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the
mechanistic basis of other GPCR-modulated cellular functions.
INTRODUCTION
A cell’s function often depends on its ability to sense gradients of
external cues and generate a polarized response such as directed
migration or neurite initiation. There is a limited understanding of
how dynamic networks of intracellular signaling molecules generate
polarized cell behaviors. Network motifs have been proposed that
can give rise to some of the features of cell migration, such as directional sensing, adaptation, and amplification of an external gradient
(Xiong et al., 2011; Wang et al., 2012). However, existing experimental methods have provided mostly static information on the relevant signaling molecules, making it difficult to examine whether
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E14-04-0870) on June 11, 2014.
Mol Biol Cell 25, 2305–2314.
Address correspondence to: N. Gautam ([email protected]).
Abbreviations used: CRY2, cryptochrome 2; GAP, GTPase-accelerating protein;
GPCR, G protein–coupled receptor; GRK, G protein–coupled receptor kinase;
OA, optical activation; RGS, regulator of G protein signaling.
© 2014 O’Neill and Gautam. This article is distributed by The American Society for
Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
the Cell®” are registered trademarks of The American Society of Cell Biology.
Monitoring Editor
Peter Van Haastert
University of Groningen
Received: Apr 9, 2014
Revised: May 16, 2014
Accepted: Jun 5, 2014
and how specific molecular interactions map onto these dynamic
network motifs. In particular, there has been a lack of methods to
exert spatial and temporal control over the activity of select signaling molecules inside a cell.
Optical manipulation of signaling presents an attractive approach
for achieving such control (Toettcher et al., 2011). We recently used
color opsins to spatially confine G protein–coupled receptor (GPCR)
activity to a selected region of a single cell and gain optical control
over immune cell migration (Karunarathne et al., 2013b) and neurite
initiation and extension (Karunarathne et al., 2013a). The opsin approach optically activates an entire signaling pathway to orchestrate
cell behavior, but new tools that provide optical control of downstream signaling molecules are required to dissect the network of
dynamic interactions triggered inside a cell.
Here we create new optogenetic tools that enable light-triggered inhibition of endogenous G protein subunits in a selected
region of a cell. We use them to generate reversible intracellular
signaling gradients in cells exposed to a uniform extracellular stimulus. We apply this approach to study cell migration in a macrophage
cell line, RAW 264.7.
GPCRs control migration of a wide variety of cell types, but the
dynamic roles of the G protein α and βγ subunits in directing cell
27
one side of a cell. We applied two approaches: light-triggered acceleration of
GTP hydrolysis on the α subunit, and optical
recruitment of a βγ-sequestering domain.
Design of an optically controlled
GTPase-accelerating protein
GTPase-accelerating proteins (GAPs) act allosterically on G protein α subunits to accelerate the transition from active αGTP to
inactive αGDP (Ross and Wilkie, 2000). Spatially localized acceleration of GTP hydrolysis at the α subunit can potentially reduce
signaling by both the α and βγ subunits because deactivated αGDP rapidly rebinds
the βγ complex and prevents its interaction
with effectors (Lin and Smrcka, 2011). We
sought to gain optical control over regulator
of G protein signaling 4 (RGS4), which has
GAP activity on both the αi and αq subunit
types (Berman et al., 1996; Hepler et al.,
1997). In yeast, exogenously expressed
RGS4 has been shown to localize to the
plasma membrane and inhibit the GPCRregulated mating pathway (Srinivasa et al.,
1998). A truncated mutant, RGS4(Δ1-33),
FIGURE 1: Generating intracellular signaling gradients by localized optical inhibition. (A) Optical did not localize to the plasma membrane
recruitment of an RGS protein to a spatially confined region of the plasma membrane generates and did not exhibit GAP activity. Its function, however, was rescued by addition of
localized GAP activity, resulting in deactivation of the α subunit and the βγ complex. (B) Local
inhibition of βγ signaling by optical recruitment of a βγ-sequestering peptide. Both approaches
an alternative, C-terminal membrane–tarprovide spatial control over G protein subunit activity downstream of uniform GPCR activation.
geting domain (Srinivasa et al., 1998). These
results suggested that it might be possible
to gain optical control over the GAP activity of RGS4 by replacing its
migration remain unclear. Signaling by βγ subunits is generally recnative membrane-targeting domain with a light-induced memognized as a requirement for GPCR-stimulated chemotaxis (Bagorda
brane-targeting domain.
and Parent, 2008), and multiple βγ effectors have been implicated in
The CRY2PHR and CIBN domains from Arabidopsis thaliana
cell migration (Li et al., 2003; Yan et al., 2012; Runne and Chen,
proteins cryptochrome 2 (CRY2) and CIB1 exhibit blue light–depen2013). However, it is unknown whether a gradient of active βγ is sufdent binding and can be used for light-triggered recruitment of a
ficient to trigger a directional response. Recent work in neutrophils
CRY2-fused protein to the plasma membrane (Kennedy et al.,
suggests that βγ signaling may be primarily involved in controlling
2010). We fused CRY2PHR-mCherry to RGS4(Δ1-33) to make CRY2the motility rather than the directionality of a migrating cell
mCh-RGS4Δ. We then coexpressed this construct in HeLa or RAW
(Kamakura et al., 2013). Meanwhile, there have been conflicting re264.7 cells with a construct containing CIBN fused to the plasma
ports on the requirement of G protein αi subunit signaling in
membrane–targeting C-terminal domain from KRas (CIBN-CaaX;
chemotaxis (Neptune et al., 1999; Kamakura et al., 2013), and there
Idevall-Hagren et al., 2012). We found that CRY2-mCh-RGS4Δ
remains the possibility that GPCR activation of non–G protein pathtranslocated from the cytosol to the plasma membrane on photoways also contributes to chemotaxis (Neptune et al., 1999; Van
activation with 445-nm light (Figures 2 and 3).
Haastert and Devreotes, 2004).
Here we use our new optogenetic tools to address fundamental
Optical control over the GAP activity of an RGS protein
questions about chemotaxis: can a gradient of heterotrimeric G procan be demonstrated using a G protein βγ subunit
tein subunit activity stimulate all of the processes required for GPCR
translocation assay
mediated chemotaxis, or is there an additional requirement for a
We used a βγ subunit translocation assay to test whether light-actigradient of G protein–independent signaling stimulated by the revated recruitment of CRY2-mCh-RGS4Δ to the plasma membrane
ceptor? Is a gradient of βγ activity sufficient for directional sensing?
could regulate its GAP activity in a living cell. This assay leverages
Does G protein subunit activity at one end of a cell lead to inhibition
the αGDP-dependent plasma membrane targeting of βγ subunits to
of responses such as increased phosphatidylinositol (3,4,5)-trisphosdetect changes in the relative amounts of αGTP and αGDP in a
phate (PIP3) and lamellipodia formation at the opposite end?
living cell.
We previously showed that βγ subunits translocate reversibly
RESULTS
from the plasma membrane to intracellular membranes upon GPCR
Creating intracellular signaling gradients using uniform
activation (Akgoz et al., 2004; Azpiazu et al., 2006; Saini et al., 2007;
ligand stimulation and confined optical inhibition
Karunarathne et al., 2012). In unstimulated cells, G protein α and βγ
The general scheme used in our experiments is shown in Figure 1.
subunits are primarily found as heterotrimers anchored to the plasma
We combined spatially uniform stimulation of GPCRs by a chemoatmembrane by the lipid modifications on the α and γ subunits
tractant with confined optical inhibition of G protein signaling on
28 | P. R. O’Neill and N. Gautam
detected as a loss of YFP fluorescence from
the plasma membrane (Figure 2, B and C).
Localized optical activation (OA) of CRY2 resulted in localized accumulation of CRY2mCh-RGS4Δ at the plasma membrane. This
was accompanied by an increase of YFP-γ9
at the region proximal but not distal to the
optically activated area. The light-triggered
reverse βγ translocation occurred in the
presence of continued receptor activity. No
reverse βγ translocation was observed when
CRY2-mCh lacking the RGS4 domain was
optically recruited to the plasma membrane
(Supplemental Figure S1). Thus the spatially
confined reversal of βγ translocation detected here is consistent with optical recruitment of CRY2-mCh-RGS4Δ to the plasma
membrane being able to locally trigger its
GAP activity on α-GTP and thereby increase
the concentration of α-GDP in that region.
Optically generated Gi protein
signaling gradients direct migration
of RAW 264.7 macrophage cells
The foregoing results showed that local optical activation of CRY2-mCh-RGS4Δ can
trigger deactivation of αi-GTP and Gβγ in a
selected area of a cell. This capability provides a way to create an intracellular G proFIGURE 2: Optical control of GTP hydrolysis with CRY2-mCh-RGS4Δ. (A) CXCR4 activation by
tein subunit activity gradient and examine
SDF-1α triggers G protein activation, dissociation, and βγ translocation to intracellular
polarized cell behaviors. We used it to exmembranes. OA of CRY2-mCh-RGS4Δ recruits it to the plasma membrane, where it can
amine the migratory response in RAW 264.7
accelerate GTP hydrolysis on the α subunit. The increased concentration of αGDP at the plasma macrophage cells. RAW cells are known to
membrane results in reverse βγ translocation due to reformation of heterotrimers. (B) Live-cell
exhibit GPCR stimulated chemotaxis (Wiege
imaging of a HeLa cell transiently transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, and YFP-γ9.
et al., 2012), and we found that their low
Activation of endogenous CXCR4 receptors with 50 ng/ml SDF-1α triggered γ9 translocation
basal motility compared with commonly
from the plasma membrane to intracellular membranes. Photoactivation-stimulated
studied HL-60 neutrophils and Dictyostetranslocation of CRY2-mCh-RGS4Δ to the plasma membrane. There it catalyzed hydrolysis of
lium cells simplifies the use of localized OA
αGTP to αGDP, leading to reverse translocation of γ9 back to the plasma membrane due to the
to control membrane recruitment of CRY2
reformation of heterotrimers. Scale bar, 10 μm. (C) Time course of plasma membrane intensity
for CRY2-mCh-RGS4Δ and YFP-γ9 in the photoactivated region.
constructs.
Directionally responsive spatial gradients
of PIP3 are believed to be one of the mediators of chemotaxis (Cai
(Wedegaertner et al., 1995). GPCR activation triggers nucleotide
and Devreotes, 2011; Weiger and Parent, 2012). We examined
exchange on the α subunit, resulting in dissociation of the αGTP
whether local inhibition of G protein subunit activity could be used
and βγ subunits (Bondar and Lazar, 2014). The prenylated C-terminal
to direct the formation of PIP3 gradients in RAW cells exposed to a
domain of the γ subunits provides βγ subunits some membrane afuniform extracellular stimulus. We examined the PIP3 response in
finity, but it is insufficient for permanent anchoring in a membrane
RAW cells transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, PH(Akt)(O’Neill et al., 2012). As a result, free βγ subunits diffusively transloVenus, and CXCR4. PIP3 dynamics in a live cell can be measured by
cate to intracellular membranes (Saini et al., 2007; O’Neill et al.,
imaging the translocation of a PH(Akt)-Venus sensor from the cyto2012). When receptors are deactivated, rebinding of βγ to αGDP
sol to the plasma membrane (James et al., 1996; Meili et al., 1999).
results in their return to the plasma membrane.
We used the chemokine receptor CXCR4 to activate G proteins
Because reverse translocation of βγ subunits to the plasma memglobally, since activation of this receptor by a gradient of the chebrane occurs through rebinding to αGDP, accelerating GTP hydrolysis
mokine SDF-1α stimulates migration in many cell types (Bleul et al.,
on the α subunit should be capable of triggering reverse βγ transloca1996; Klein et al., 2001; Molyneaux et al., 2003).
tion even if the receptors remain activated. We leveraged this feature
First, we used localized OA to recruit CRY2-mCh-RGS4Δ to the
of βγ translocation to test whether optical recruitment of CRY2-mChplasma membrane at one side of a cell and followed this with global
RGS4Δ to the plasma membrane can control its GAP activity.
CXCR4 activation using 50 ng/ml SDF-1α (Figure 3). Before receptor
We measured βγ translocation in HeLa cells by imaging a yellow
activation, localized plasma membrane recruitment of CRY2-mChfluorescent protein (YFP)–tagged version of γ9, a fast-translocating
RGS4Δ did not produce any detectable PIP3 generation or cell
subunit (Figure 2). Consistent with previous observations (Karunarashape changes. On receptor activation, cells responded by generatthne et al., 2012; O’Neill et al., 2012), activation of endogenous
ing PIP3 gradients and initiating migration in the direction opposite
CXCR4 receptors with 50 ng/ml SDF-1α triggered βγ9 translocation
to the CRY2-mCh-RGS4Δ gradient (Figure 3A and Supplemental
from the plasma membrane to intracellular membranes, which was
Optical inhibition of G protein subunits
| 29
respond in opposite directions (Supplemental Figure S2). The same directional
control was observed when OA was applied
after the uniform extracellular stimulus, with
migration being initiated at the time of OA
(Supplemental Movie S2). Furthermore, the
direction of PIP3 accumulation and lamellipodia formation could be reversed by
switching the location of OA to the opposite
side of the cell (eight of eight cells) (Supplemental Figure S3).
Cells did not exhibit any of these directional responses to localized OA when a
CRY2 construct (CRY2-mCh) without RGS4Δ
was expressed in the cells or when a CRY2mCh construct containing the cDNA for a
glycolytic enzyme, PGK1, was expressed
(CRY2-mCh-PGK1; Figure 3, B and D, and
Supplemental Figure S4). These cells exhibited uniform PIP3 responses (29 of 36 cells)
or polarized spontaneously in directions that
did not depend on the side of OA with reference to the cell (7 of 36 cells). Compared
to neutrophils, spontaneous polarization in
response to a uniform stimulus appears to
be much less common in RAW macrophage
cells. This is consistent with their general
lack of basal polarization and their greatly
reduced basal motility compared with neutrophils. These controls show that the directional responses observed with CRY-mChRGS4Δ are due to localized inhibition of αi
and βγ subunit activity by RGS4Δ rather than
a nonspecific effect due to localized OA or
accumulation of the CRY protein at the
membrane.
We performed identical experiments using activation of endogenous C5 receptors
to ensure that the migratory response induced by localized GAP activity was not peculiar to the CXCR4 receptor or due to overexpression of a GPCR. The anaphylatoxin
C5a is known to stimulate chemotaxis of all
myeloid cell lineages (Gerard and Gerard,
FIGURE 3: Cell migration driven by localized Gi protein inhibition. (A) Image sequence of a live
1994), and it has been shown to induce cheRAW 264.7 cell transiently transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, PH(Akt)-Venus, and
motaxis of RAW 264.7 cells (Wiege et al.,
CXCR4. Local OA was applied to generate a CRY2-mCh-RGS4Δ gradient before uniform
2012). We activated endogenous C5a readdition of SDF-1α. Scale bar, 10 μm. (B) Negative control expressing CRY2-mCh-PGK1 instead
of CRY2-mCh-RGS4Δ. (C, D) The t-stacks corresponding to the data in A and B. Localization of
ceptors with 10 μM FKP-(D-Cha)-Cha-r, a
the RGS construct, but not the PGK construct, results in a PIP3 gradient, directional cell
peptide derived from the C-terminus of the
protrusions, and migration. White boxes correspond to OA regions. Yellow boxes show regions
full-length, 74–amino acid C5a. It has been
selected for generating the corresponding t-stacks.
reported to be a full agonist of the C5a receptor, eliciting responses comparable to
Movie S1). Of 43 cells that provided a PIP3 response, all exhibited
those of full-length C5a in several assays, including chemotaxis
PIP3 gradients and directed lamellipodia. Of these, seven migrated
(Konteatis et al., 1994). Localized OA of CRY2-mCh-RGS4Δ with uniat least 1 cell diameter in 15 min, 10 migrated between 1/2 and
form activation of endogenous C5a receptors generated directional
1 cell diameter, and 26 migrated <1/2 cell diameter. Of those that
responses similar to those seen with uniform activation of transmigrated <1/2 cell diameter, five extended the front by at least 1/2
fected CXCR4 (Supplemental Figure S5).
cell diameter but did not retract the back, and five initiated migraThe ability to locally inhibit G protein signaling and generate a
tion before snapping back to their initial positions, perhaps due to
migratory response in immune cells showed that an internal gradistrong adhesion to the uncoated glass surface.
ent of αi and βγ activity is sufficient to direct cell migration in the
The directional responses were not due to unintended SDF-1α
absence of an external gradient. The results with endogenous C5a
gradients, because two cells in close proximity could be made to
receptors showed that these internal gradients are sufficient to drive
FIGURE 4: Localized Gβγ inhibition directs PIP3 gradients and lamellipodia. (A) Light-triggered recruitment of CRY2mCh-GRK2ct to the plasma membrane allows for spatially confined inhibition of βγ signaling. (B) Live-cell imaging of a
RAW cell expressing CRY2-mCh-GRK2ct, CIBN-CaaX, PH(Akt)-Venus, and CXCR4. (C) The t-stack corresponding to the
data in B. Localization of the GRKct construct generates reversible lamellipodia and PIP3 responses.
cell migration at levels of signaling activity normally achieved within
a cell.
Gβγ signaling gradients generated by CRY2-mCh-GRK2ct
direct PIP3 gradients and lamellipodia formation
To further dissect the roles of G protein subunits in cell migration,
we sought to develop an optogenetic tool to specifically inhibit βγ
signaling. We created a CRY2-mCh-GRK2ct construct that could be
optically recruited to one side of a cell to produce a gradient of βγ
activity (Figure 4A). The C-terminal domain of G protein–coupled
receptor kinase 2 (GRK2ct) is capable of inhibiting responses downstream of βγ without inhibiting those generated by α subunit effectors (Koch et al., 1994). It has been widely used to sequester Gβγ
and inhibit its activity, but this is the first time it was used asymmetrically within a single cell to study a polarized cell behavior.
In RAW cells transiently transfected with CRY2-mCh-GRK2ct,
CIBN-CaaX, PH(Akt)-Venus, and CXCR4, spatially confined OA
resulted in localized recruitment of CRY2-mCh-GRK2ct from the
cytosol to the plasma membrane. Subsequent activation of
CXCR4 receptors with 50 ng/ml SDF-1α resulted in generation
of a PIP3 gradient and lamellipodia toward the side of the cell
that was opposite to the location of the OA (40 of 50 cells; Figure
4B). The direction of the lamellipodia and the PIP3 gradient
could be reversed by switching the location of OA with reference
to the cell (6 of 12 cells; Figure 4, B and C, and Supplemental
Movie S3).
To ensure that these responses occurred due to localized sequestration of βγ and not some peculiar effect of GRK2ct, we performed identical experiments with a homologue, GRK3ct. GRK3ct
binds to βγ subunits in biochemical (Daaka et al., 1997) and live-cell
imaging assays (Hollins et al., 2009). GRK2 and GRK3 sequences are
85% identical, but their βγ-binding regions are only 52% identical
(Daaka et al., 1997). The CRY2-mCh-GRK3ct construct was capable
of producing similar directional (15 of 19 cells) and reversible (9 of
13 cells) responses (Supplemental Figure S6). The ability of both
CRY2-GRKct constructs to elicit these directional responses, but not
CRY2-mCh or CRY2-mCh-PGK1, confirmed that the directional responses occurred due to sequestration of Gβγ. Similar CRY2-mChGRKct directed responses were also observed when endogenous
C5a receptors were activated with 10 μM FKP-(D-Cha)-Cha-r
(Supplemental Figure S7).
Whereas the CRY2-mCh-GRKct constructs were capable of generating PIP3 gradients and directional lamellipodia similar to those
generated by the CRY2-mCh-RGS4Δ construct, none of these cells
exhibited appreciable cell migration. This difference could potentially be due to different magnitudes of βγ inhibition achieved by the
GRKct versus RGS constructs, or it could it be that a gradient of αi
activity is additionally required for migration. We suspect that the
latter explanation is more likely, given that recent studies using a
variety of cell types reported roles in chemotaxis for αi subunit interactions with proteins such as GIV (Ghosh et al., 2008), ELMO1/
Dock180 (Li et al., 2013), and AGS3/mInsc (Kamakura et al., 2013).
| 31
FIGURE 5: Local optical inhibition of Gi activity after adaptation to uniform stimulus. (A) Image sequence showing a
RAW cell transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, PH(Akt)-Venus, and CXCR4. Addition of uniform SDF-1α
(2:05) resulted in PIP3 accumulation and generation of cell protrusions (3:20). After several minutes, the PIP3 level and
cell shape resembled those seen before the uniform stimulus (11:40). Localized OA of CRY2-mCh-RGS4Δ (12:55) was
applied in this adapted state to inhibit Gi activity at one end of the cell. This resulted in the generation of a PIP3
gradient and cell migration directed toward the far side of the cell. Scale bar, 10 μm. (B) An illustration of the expected
time dependence of an activator (A), inhibitor (I), and downstream response (R) in a LEGI model (Xiong et al., 2010) in
which Gi signaling generates both A and I. Uniform activation of Gi signaling produces a transient downstream response
that returns to the basal level due to the delayed increase in I. Subsequent optical inhibition of Gi signaling at the back
causes a reduction in the level of Ifront but not Afront due to the differential movement of I and A throughout the cell.
This leads to an increase in R at the front of the cell, resulting in directional migration.
Overall, these results suggest that a gradient of activated Gβγ subunits stimulated by endogenous receptors is sufficient to elicit directional PIP3 responses and cell protrusions in the absence of an external gradient.
Generating light-triggered gradients in cells that have
adapted to a uniform stimulus: evidence of global inhibition
mediated by G protein subunits
Directional sensing in migratory cells is believed to be intimately
related to their ability to adapt to a spatially uniform stimulus (Parent
and Devreotes, 1999; Van Haastert and Devreotes, 2004; Levchenko
and Iglesias, 2002). In this context, adaptation refers to a cell’s ability
to generate transient responses that return to near-basal levels after
a uniform increase in chemoattractant concentration. This occurs
through a mechanism other than desensitization, and it allows a cell
to sense gradients over a wide range of background chemoattractant concentrations. The mechanisms that control adaptation in migratory cells are not fully understood.
An incoherent feedforward loop (IFFL) has been identified as a
signaling motif capable of generating adaptive responses (Ma et al.,
2009). In the IFFL, the input signal generates an activator with fast
kinetics and an inhibitor with slower kinetics that converge on a
downstream response such as PIP3. At short times after application
of the stimulus, the activator generates an increase in PIP3 levels,
but over time, the rising level of the inhibitor causes the PIP3 to
decay back to its prestimulus level. Recent studies show that an IFFL
can explain adaptation of PIP3 and Ras responses in Dictyostelium
(Takeda et al., 2012; Wang et al., 2012)
A local-excitation global-inhibition (LEGI) mechanism that incorporates the IFFL motif has been proposed that can account for both
adaptation and directional sensing (Parent and Devreotes, 1999;
Levine et al., 2006). In the LEGI model, the activator signals locally,
while the inhibitor diffuses throughout the cell to signal globally. As
a result, downstream responses adapt to a uniform stimulus but exhibit sustained intracellular gradients in response to a gradient stimulus. Several models of chemotaxis incorporate the LEGI motif to
account for directional sensing and adaptation, combining it with
motifs that account for additional features of chemotaxis, such as
basal motility, cell shape changes, or amplification of the external
gradient (Xiong et al., 2011; Wang et al., 2012; Shi et al., 2013).
However, a specific global inhibitor has not yet been identified. It is
not known whether an inhibitor is generated by Gi signaling or by an
independent pathway triggered by the GPCR.
We designed an experiment to determine whether Gi signaling
by itself leads to global inhibition (Figure 5, Supplemental Figure
S5, and Supplemental Movie S4). First we exposed RAW cells to a
uniform chemoattractant, either 50 ng/ml SDF-1α to activate transfected CXCR4 or 10 μM FKP-(D-Cha)-Cha-r to activate endogenous
C5a receptors. This resulted in translocation of PH(Akt) to the plasma
membrane and generation of cell protrusions. After the cells had
adapted, as indicated by PH(Akt) returning to the cytosol and the
cell protrusions subsiding, CRY2-mCh-RGS4Δ was optically recruited
to one side of the cell to induce localized inhibition of αi and βγ activities. This resulted in the formation of a PIP3 gradient and initiation of cell migration in a direction that was opposite to the location
of the OA (seven of eight cells).
The ability to generate responses at the front of a cell simply by
inhibiting G protein activity at the back provides direct evidence
that Gi signaling can act at a distance to inhibit “frontness” signaling
pathways. This result is consistent with a LEGI model in which both
the local activator and the global inhibitor are generated by Gi signaling. Figure 5B shows schematic plots that illustrate the time dependence of the activator, the inhibitor, and the downstream response. Application of a uniform input initially leads to rapid
generation of the activator and the downstream response. The delayed accumulation of the inhibitor causes the response to return to
its prestimulus level. The levels of both activator and inhibitor remain high throughout the cell in the adapted state. When the cell is
in this state, applying localized OA to inhibit Gi signaling causes the
levels of activator and inhibitor to decrease on one side of the cell.
Because the inhibitor acts globally, the cell encounters the reduced
level of inhibitor over its entire space. In contrast, the level of activator is only reduced on one side. As a result, the level of activator
overwhelms that of the inhibitor on one side of the cell, leading to
the generation of downstream signaling gradients that drive cell
migration.
et al., 1985; Hartmann et al., 1997). However, the dynamic roles for
the αi and βγ subunits are not known, and it has not been possible
to test whether a gradient in the activity of αi and βγ subunits is sufficient to generate cell migration. There have been suggestions that
other G protein subunit types may also be required. For example, in
N-formyl-methionyl-leucyl-phenylalanine (fMLP)–stimulated neutrophil chemotaxis, it has been reported that Gi signaling regulates
“frontness,” whereas G12/13 regulates “backness” pathways (Xu
et al., 2003). Cell migration could additionally require gradients of
GPCR-triggered but G protein–independent signaling (Ge et al.,
2003). It could also potentially require interactions between ligandbound GPCRs and accessory proteins that modulate G protein–
mediated signaling, for example by bringing specific effector molecules closer to the activated G protein (Ritter and Hall, 2009).
Here we activated receptors that couple to Gi heterotrimers. By
breaking spatial symmetry downstream of the receptor, directly at
the level of the Gi protein, we were able to identify molecular and
cellular responses generated by a gradient of αi and βγ activity. The
ability of optically localized CRY2-mCh-RGS4Δ to generate directional cell migration shows that a gradient of αi and βγ activity is
sufficient to elicit the entire gamut of migratory responses, including
generation of lamellipodia at the front of a cell, retraction of the
back, directional changes, and ability to respond directionally after
adapting to a uniform stimulus.
DISCUSSION
Optical control of cell signaling by inhibition of endogenous
proteins
Directional sensing by a Gβγ signaling gradient
Most of the current information about signaling molecules involved
in cell migration comes from genetic manipulations that establish
whether a given protein is required for migration and biochemical
studies that identify its relevant interactions. Imaging methods have
provided additional information about the localization of several
signaling molecules to the front or back of a migrating cell. This information is valuable, but new kinds of information are required in
order to understand how a network of dynamic interactions shapes
the cellular response. Obtaining this kind of information has been
limited due to a lack of methods to exert spatial and temporal control over the activity of intracellular signaling molecules.
Here we developed optogenetic tools that provide such control
by locally inhibiting the activity of specific G protein subunits. We
showed that light-triggered membrane recruitment of a truncated
RGS4 can be used to spatially localize G protein subunit activity
within a cell. We also showed that similar optical recruitment of
GRK2ct to a spatially confined region of the plasma membrane can
locally inhibit Gβγ-signaling activity. We combined the capabilities
of these optogenetic tools with spatially uniform activation of GPCRs to generate intracellular gradients of G protein subunit activity.
An advantage of the optical inhibition approach used here is that
it enables spatial and temporal control over the activity of endogenous untagged proteins. Inhibition is achieved by expression of a
CRY2-tagged protein, but the cellular response is elicited by a pathway that is entirely in its native state at the distal end of the cell with
reference to the site of OA. This ensures that the targeted protein retains all of its native signaling properties. It also provides control over
intracellular signaling at levels that reflect those driving native cell behavior because all of the signaling is done by endogenous proteins.
A gradient of G protein αi and βγ activity is sufficient
to drive cell migration
Inhibition by pertussis toxin showed that Gi signaling is required for
cell migration toward many different chemoattractants (Spangrude
It has been shown that βγ inhibition by sequestering proteins
(Arai et al., 1997; Neptune and Bourne, 1997) or small molecules
(Lehmann et al., 2008; Kang et al., 2014) suppresses chemotaxis in
many cell types. It was unknown, however, which features of cell
migration are controlled by βγ signaling. Some reports implicated βγ
signaling in directional sensing, whereas others proposed that it is
primarily involved in controlling cell motility (Kamakura et al., 2013).
Our results with CRY2-mCh-GRK2ct and CRY2-mCh-GRK3ct show
that a gradient of activated βγ is sufficient to generate a PIP3 gradient and lamellipodia formation directed toward the side with a
higher level of βγ activity. This directly demonstrates a role for βγ
signaling in directional sensing.
Overall these results with CRY2-RGS and CRY2-GRKct suggest
that in immune cells sensing a chemoattractant gradient, the occurrence of a gradient of activated G protein subunits is sufficient to
initiate directionally sensitive migration.
Adaptation of cell migratory responses involves
Gi-mediated global inhibition
There is limited understanding of the molecular interactions that
allow eukaryotic migratory cells to adapt to uniform stimulation.
Dynamic control over receptor activation using microfluidics provides evidence that these cells use an IFFL network motif for adaptation (Takeda et al., 2012; Wang et al., 2012). Examining whether and
how specific signaling molecules map onto the IFFL motif can be
aided by methods that provide acute control over their activities
within a living cell.
Previously it had not been possible to test directly whether Gi
activity generates the inhibitory signaling that is required for adaptation in a migratory cell. It was only known that an inhibitory pathway should be present downstream of the receptor. Our results
show that Gi signaling is capable of triggering a delayed inhibitory
pathway that acts throughout the entire space of a cell. The ability
to generate a postadaptation PIP3 gradient by local suppression of
αi and βγ activity shows that Gi stimulates a signaling pathway capable of inhibiting PIP3 globally. The response is reflected at the
| 33
cellular level, because the cells demonstrate directional migration.
Many downstream responses have been observed to adapt, and
there is evidence that pathways acting in parallel to PIP3 signaling
are involved in controlling cell migration. The ability of local Gi inhibition in an adapted cell to elicit a directional migratory response
suggests that all of the relevant pathways are under control of G
protein αi and βγ subunit activity.
G proteins remain active when downstream responses adapt
A fluorescence resonance energy transfer–based G protein sensor
in Dictyostelium indicated that G protein heterotrimers remain dissociated after transient downstream responses such as PIP3 have
adapted (Janetopoulos et al., 2001). This suggested that adaptation
does not require deactivation of G protein subunits. There are examples, however, such as the response to mating pheromone in
yeast, in which adaptation occurs at the level of the G protein (Cole
and Reed, 1991). In the case of mammalian migratory cells, it has
not been clear whether G protein subunit deactivation plays a role
in adaptation. It has not been directly tested whether G protein subunit activity continues after an immune cell adapts to a uniform signal (Iglesias, 2012). Here, in immune cells that have adapted to a
uniform stimulus, asymmetric G protein deactivation triggered a directional migratory response. This showed that in a fully adapted
cell, G protein subunits continue to be in the activated state.
Optical control over G protein subunits to dissect their
dynamic signaling roles
GPCRs have been implicated in other polarized cell behaviors, such
as yeast budding (Bi and Park, 2012), neurite outgrowth (Fricker
et al., 2005; Georganta et al., 2013), and orientation of asymmetric
cell divisions (Yoshiura et al., 2012). The ability of our optogenetic
tools to locally inhibit G protein subunits can be used to help determine their dynamic roles in these polarized responses.
G protein subunits were classically believed to carry out all of
their signaling functions at the plasma membrane, but mounting
evidence suggests that they can have signaling activities at other
locations within a cell (Hewavitharana and Wedegaertner, 2012).
There is a lack of methods to determine the functions of G protein
subunit signaling at intracellular locations. Existing methods to interfere with G protein signaling act over an entire cell. Optical recruitment of the CRY2-RGS to specific intracellular locations could be
achieved through the use of appropriately targeted CIBN constructs.
This could provide a means to acutely perturb G protein subunit
activity at different locations within a cell. This could help dissect the
functions of GPCR stimulated signaling at different locations of a
cell. For example, signaling can be perturbed at a growth cone or a
synapse. It can also be used to examine the temporal role of GPCR
signaling in cell differentiation or development by inhibiting it at
specific time points.
MATERIALS AND METHODS
Reagents
SDF-1α/CXCL12 (S190; Sigma-Aldrich, St. Louis, MO) was dissolved
to 10 μg/ml in 1× phosphate-buffered saline (PBS) containing 0.1%
bovine serum albumin and stored as aliquots at −20°C. The C5a
receptor agonist FKP-(D-Cha)-Cha-r (65121; Anaspec, Freemont,
CA) was dissolved to 2.5 mM in 1× PBS containing 0.1% albumin
and stored as aliquots at −20°C.
DNA constructs
CRY2PHR-mCh was obtained from AddGene (Cambridge, MA) (plasmid #26866). CIBN-CaaX was a kind gift from the lab of P. Di Camilli
(Boyer Center for Molecular Medicine, Yale School of Medicine, New
Haven, CT) (Idevall-Hagren et al., 2012). CXCR4 was a kind gift from
the lab of I. Schraufstatter (Torrey Pines Institute for Molecular Studies, San Diego, CA) (Zhao et al., 2006). YFP-γ9 has been described
before (Saini et al., 2007). A PCR product of PGK1 (38071; Addgene)
was inserted into the KpnI and XbaI sites of CRY2PHR-mCh to create
CRY2-mCh-PGK1. A PCR product of GRK2ct (Irannejad and Wedegaertner, 2010) was inserted into the KpnI and XbaI sites of CRY2PHR-mCh to make CRY2-mCh-GRK2ct. A PCR product of GRK3ct
(Hollins et al., 2009) was inserted into the KpnI and XbaI sites of
CRY2PHR-mCh to make CRY2-mCh-GRK3ct. A PCR product of RGS4
lacking residues 1–33 was inserted into the KpnI and XbaI sites of
CRY2PHR-mCh to make CRY2-mCh-RGS4Δ. PH(Akt)–green fluorescent protein (GFP; 18836; Addgene) was cut with BamHI and XbaI to
release GFP, and a PCR product of Venus was inserted in its place to
make PH(Akt)-Venus.
Tissue culture
HeLa cells were obtained from ATCC and cultured in MEM (CellGro
10-010-CM) supplemented with 10% dialyzed fetal bovine serum
(FBS; Atlanta Biologicals) and 1× antibiotic-antimycotic solution
(CellGro) at 37°C and 5% CO2. RAW 264.7 cells were obtained from
the Washington University Tissue Culture Support Center and cultured in DMEM supplemented with 10% dialyzed FBS and 1× antibiotic-antimycotic solution at 37°C and 5% CO2. RAW cells ranging
from passage 3 to passage 12 were used for experiments.
Transfections
HeLa cells were transfected using Lipofectamine 2000. Cells were
plated at 2 × 105 cells/dish in 29-mm glass-bottom dishes (In Vitro
Scientific) 1 d before transfection. RAW cells were transfected by
electroporation using Cell Line Nucleofection Kit V (Lonza) with a
Nucleofector II device (Amaxa). For each sample, 2 × 106 cells were
pelleted by spinning at 90 × g for 10 min, resuspended in 100 μl of
Nucleofection solution containing between 0.2 and 2.5 μg of each
plasmid DNA, depending on the specific construct (0.2 μg of
PH(Akt)-Venus, 2 μg of CXCR4, and 2.5 μg of others), and electroporated using program D-032. Immediately after electroporation,
500 μl of prewarmed medium was added to the cuvette, and this
was split among 29-mm glass-bottom dishes (8–10 dishes) containing 500 μl of prewarmed medium in the center well. After transfection, dishes were kept in a 37°C, 5% CO2 incubator until imaging.
Live-cell imaging and optical activation
All imaging was performed using a spinning-disk confocal imaging
system consisting of a Leica DMI6000B microscope with adaptive
focus control, a Yokogawa CSU-X1 spinning-disk unit, an Andor
iXon electron-multiplying charge-coupled device camera, an Andor
fluorescence recovery after photobleaching–photoactivation unit,
and a laser combiner with 445-, 448-, 515-, and 594-nm solid-state
lasers, all controlled using Andor iQ2 software. This system allows
live-cell imaging to be combined with localized OA within a selected
region of the sample that can be redefined in between images in a
sequence. For OA of CRY2, the 445-nm laser was used at 5 μW and
scanned across the selected region at a rate of 0.9 ms/μm2. This was
performed once every 5 s. Solid-state lasers with wavelengths of
515 and 594 nm were used for excitation of Venus and mCherry,
respectively. Emission filters were Venus 528/20 and mCherry 628/20
(Semrock). All images were acquired using a 63× oil immersion objective. A single confocal plane was imaged at a rate of 1 frame/5 s.
All imaging was performed inside a temperature-controlled chamber held at 37°C. Imaging of HeLa cells was performed 1 d after
transfection with Lipofectamine 2000. Imaging of RAW 264.7 cells
was performed 2–10 h after electroporation. Before imaging, the
culture medium was replaced with 500 μl of Hank’s balanced salt
solution supplemented with 1 g/l glucose (HBSSg). An equal volume of agonist in warm HBSSg was added at the time specified in
the figures to achieve the final concentration given in the corresponding figure legends. Only cells that exhibited a detectable PIP3
response were included in the analysis. Approximately 10% of the
cells did not exhibit any detectable PIP3 response to SDF-1α. This
was true regardless of which CRY construct was expressed.
ACKNOWLEDGMENTS
We thank W.K.A. Karunarathne and V. Kalyanaraman for useful discussions, Y. Ordabayev for assistance in making a construct, and
R. Gereau for use of an electroporator. We thank P. De Camilli,
P. Wedegaertner, N. Lambert, and I. Schraufstatter for DNA constructs. This work was supported by National Institutes of Health
Grants GM069027 and GM080558 to N.G. and National Research
Service Award Postdoctoral Fellowship GM099351 to P.R.O.
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