Student-Directed Investigations of the Actin Cytoskeleton in Chinese

This article reprinted from:
Connerly, P. 2010. Student-Directed Investigations of the Actin Cytoskeleton in Chinese
Hamster Ovary Cells. Page(s) 462-468, in Tested Studies for Laboratory Teaching,
Volume 31 (K.L. Clase, Editor). Proceedings of the 31st Workshop/Conference of the
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462
INDIANA UNIVERSITY
SOUTHEAST
Student-Directed Investigations of the Actin Cytoskeleton in
Chinese Hamster Ovary Cells
Pam Connerly, Indiana University Southeast
Abstract
Materials and methods
As part of an upper-l",'el Cell Bio;>logy rourse, students utilize n""""""ntly la-
beled phalloidin 10 'isualiu act;n in fixed Chinese hamster "'''lfy (CHO) cen•.
Slu~nts are first introduced 10 the gro"1h of animal cells in culture through an
aseptic I~hnique practice cxcreise. Next. tlK-y learn logrow and stain the cells
Cells lines. growth media and growth co"ditioll5. Chincsc bamster (Cricemlus grise....) O\lIry (C.IQ-K,)cells were purebasOO from ATCC ("CCIA,,). Cells ....eregro....n in
FI2-K media supplernentnl ..~tb 10910 Fetal Clone 11 (a fetal bmineserurn substitute optimized for CHO cells). Cells ...... regro....n in sterile Pctri plates or inT2Scell culture
flasks (A) in a 37"C incubator "ith 5" C02{a). Cell media was replaced approximately we..y 3 da)"8 and cultures ....ere split approximat..lyonce per week. All manipulations
...-ere perfo""ed in a tissue culture bood (C). Cells "ill be stored for future use in a liquid nitrogen de...." .. {D).
forobservalion and charactcri:tation of chl'llmosomc number. The 1eclure POl'·
lion of the class discusses the C)1oskelcton in sorn<! depth, SO students are asked
to design their own experiment tomanipulalc SOme aspect ofthcCHOcell
Cell <Iaining alld chromosomespreads. CHO""lls gro....n on eo..-erslips ....ere stained ...ith cl)"5tal \"iolet and Giemsa stains according to protocols described in FreshllC)'< Cu/lut-e 0/ Animal Cells: A Manual a/Bu8ic T....hnique. Digital photos ofstain~.,) cells ,,-ere taken by mounting a digital camera In place of one ocular 1"lI8on a light microscope
To ",nend the CharaClenl'.3tion of the cells. mitotic chromosomes ....ere prel"lred and spread on glass slides. again acrording to protocols described in Freshney. Cells ....ere then
stained ..ith el)"5tal ,iolet and obser>'oo by light microscopy.
gro",h eondilion \0 see if changes in actin C)1oskeletal stn..:Wrt'Scan beob!lCI"Td. All grouJlS usc the same actin \isualization procedure andcach group
gets time On the nuoresttnee micl'06COpe, using digital micto6COpy to reoord
data. The nUol'1':S<.'Cnt ,'isualization of cortical actin is always dramatic, and students sain insights into cell culture methods. e..po.'Timental design. fluorescence
micmscopy. and data analysis. Samplesize is often limited. and variable results
allow for important discllSSions about the importance of quantitation and ",producibility.
Cell manipulations. Students ....ere enoouraged to d"'·e!optbeiro....n manipulation thattbeythought might ba'-e an effect on the O'-erall morpbologyoftbeactin C)1oskelelonln
CHO cells. In the course oft....oy..ars. student groups ha'-estudioo ''3rioUSC)1oskeleton disrupting drugs (e.g. C)1ochalasin D. ookhicine). differences in mnlia conditions. density of cells. and presence of antibioties. Treatments likel)' to pre...ent culture gro..1b ( g. C)106kelelon--disrupting drugs) ",,", applied hours before obse"lItion.....hile those
less likely to <lamage the cells permanantl)" (e.g. ''lll)ing cell densities) were applinl s erJl days before obse"·ation.
Acti" oisua/aanon. The F-aelinvisualization Biocbem Kit (Cat ,BKooS) from Cytoskeleton. Inc. was IISO.'d for fluorescent ,isualization of61amentousaetin in C.IOcells. The
kit includes rhodamine-phalloidin. a chemical "ith a phalloidin moiety to bind to filamentous actin linknl to the flooropbore rhodamine. Steps in the 'isuahuotion process included first gro...ing ""lis on sterile glass co,...r slips. fixing the eells. permeabilizing the oolls. incubating ...ith rhodamine-pballoidin. extensive ....""hing. mounting onto glass
slides and obser>'3tion by floorescence micmscop)" (E).
Introduction
Images of the C)106keleton are anlOng the most compelHng 'isuals in
modern cell biology. The lab exereises prescntnl here were designed to
gi"e upper-le--el biology majors experi"r>ee using animal cell tissue coltu",
and the opportunity to create and analyze their own images of the C)106keleton. BioI 1.-312 Cell Biology Lectures is anupper-le-'el course required for
the BS in Biology at Indiana University Southeast. The assoeiatnllaboratory course (Bioi 1.-3'3) is optional for most biology majors but fulfils an
electi'-eand is required for the BS in Oini.... l Laboratory Sciences. TIle
class sire is typically quile small (S-IO), containing mainly stud..nts \\'110
enjoy laboratory lXIUr8eli. 1be laboratorycurrieulum isorganiu.,) around
severalmulti-\\,,,,,kseries of experim..nts, ...ith each series including a
student--direct~'<I component. Ik-cause ...... han' two tissue culture hoods
and a ....ater-j;!cketnl CO2 incubator, one of the lab series in,'OI,·es the
gro...1h and analysis of animal culture oolls. Chincsc hamsterovary(CHO)
cells were chosen for the investigation because th..y were commercially
available and. as non-human ""lls. pose little risk to student researehers.
The CHOcelilabseries inw.h-ed four main e..ercises: (I) an Introduction to
aseptic technique, (2)
staining ....ith el)"5tal ,·iol..t and Giemsa stains, (3)
mitotic chromosome pn..-parntion and anal)"5is. and (4) ,~sualizatlon of fila"",ntous actin C)1oskeletoll. Th.. studcnt--directnl asllCCl of the ill\"CStigation focused on the actin 'isualization component. Students designnl various treatmcnts tbey heli",'oo would influenoe tbe structure of the actin cytoskel..ton in theCHO oolls and IISO.'d rhodan,ine-phalloidin tn lluorescently
lahel filamentous actin in tbecells.
INDIANA UNIVERSITY
SOUTHEAST
Data ocquisih·o". Each b1udent group bad time toobse,,·e their slides by floorescenoe microscopy. Additionally. a digital camer.l ....as insert~"lI in p1aoeof One of the ocular
l.. nses on the microscope, alln"ing for acquisition of digital images ol ....rio'-"' preparations. Image fil"" .......... nploaded to a classroom manag..",..m site and stndents retri",'OO
tbeir photos for anal)"sis
""n
Figure 3. Fluorescence micrographs ofCHO cells 'isualized ..itb rhodaminephalloidin. These images represent the 'ie.... offlooresooent cells in the
microscope's field of ,-lew. The digital camera could be further zoomed to capture more cellular detail. These images represent multiple student prel"lrations using a IOOX objeel:i,-e.
Filture4. t-1ooreseence micrographs ofCHO cells visualized with rhodamine-phalloidin. These are examples ofimag... oollectcd by students using a digital camera mounted in the ocular lensofthe microscope. Each group had uso..,) '"3riousd~'gteeS
ofO';(l()m on thecam.. ra to focus onlhe cells. 111e lOOX objecth-e ""3S used forcach ~mpl..
Results
Figure I, Micrograpbsof el)"5tal ,iolet (ev) stained CliO oolls. Cells ....ere fu:ed ontoOO\-erslips. stained. and
mount~.,) forobse,,·ation. CV is a monochromatic stain. Darker areas "ithin the c.,ntral nucleus represent
nucleoli. Images represent mnltiple student preparations and magnifications.
Discussion
Students ,,""' able to successfully manipulate th.. CHO cells in C1Jltnre.
No m-ert examples of contamination ...... re obsen'oo ..ither semester the lab
"<lSmn.
• Students gain~.,) experi..nce using aseptic technique and working in the
tissue culture hood.
• Students successfully stained and observoo cells they had grow" in culture
(see Figures, and 2.)
• C'hromolSOme spreads w..re not reproducibly successful. Onesemestcr's
stud..nts produced ni.... slid.... but the n..xt did not.
• Students design~.,) and earri~") out theiro"ll in,...- stigation intoa cell manipulation that might affect the C)t06keletal structure of CHO oolls.
• Students follo"-ed oompk'll directions from a kit for ,~sualization of a lIuotese('nl marker. gainnl experience using the lIuorescence microscope (see
figures 3 and 4), documenting and interpreting their results.
Resources
Alberts. B et al. 200'7. Molecular Bioi09Y oJthe Cell. 5th edition. Garland
Science.
• Freshney.1R. 2OOS. Culture 0/Animal Cells: A Mmlual o/Basic Technique, 5th nlition. Wiley-Uss.
• F-aetin ,isualization Bioch..m Kit (Cat #BKooS), C}1oskeleton. Inc.
(www·CJ10iSkelcton.oom)
• NOTE: I am hapPl' send electronic files of my lab handouts for As<'ptic
Technique Practice, Cell Staining, Chromoson'e Sp....ads. Culture Splitting.
and Actin Visualization 'ia ernail.
'''''eTI
• Students gainnl ..xperiencedoing tissue
culture. a fairly specialized skilltbatllOt all
biology majors experience.
• The importance of group ....ork was .. mphaslzed.
• Students designnltheirown experiments.
llli.')' had to consider controls. replicates.
time managem..nt. and oth..r issues
• Students gainnl experience ...ith lluoreseence micmscopy and produced compelling
digital photos of their cells
• Students had a real introduction to the
multiple steps. long time-fr'Jme. and attention to detail required for a significant cell
biological experiment. Th~')" nOW ha,-e personal experience to ....late to t..xtbook fluorescence mieroocopy images.
COMI'UCATIONS
• Some groups allowed the most ~",pericnced group
m..mberdnall of the ....ork in the tissue culture hood
ratber tban taking tbe opportunitr to get their own
experi..nce.
• Students han diff....ultrcollCehing toorO\\ll experiments. They ....ant direction in what would be in_
teresting to study as ...-ell in details ofho.... to set up
the experiment.
• Ilceau:;eoflabtime constraints. ,,-e ...-ere not able
to troubleshoot and detennine reasons for unexpednl results (J"'lrticularly absence of cells.)
• Photodocumentation is time consuming and challenging.
• Students need assistanee to arri..e at ....asonable
oonclusions. Although many manipulations resnlt~.,)
in no obse"-ed diffe....nce bet"...... n t.....J tments. many
students found that conclusion to be unsatisfactol)·
and would often m-en;tate their results.
IDEAS FORTHEFlITURE
• !lequire group oral presentation of results in a lab mcetingattl1OSph..re
• Aliow more time for modification and repetition of tbe experiment.
• Relluire all students to demonstrate competenC)· in the tissue cuhure
hood.
• Design a praetice pootodocumentation exercise to hclp students
become oomfortable "ith the fluorescen"" microscope and the camera. as
.....11 as to emphasize the n~-ed for and complexity of quantitati'-e anal)"ls of
results.
Acknowledgements
Figure 2. Micrographs ofGiemsa stained CHO eells. Cells ...·ere fixnl ontoOO\·erslips.
stained and mounted On glass slides forobse,.,..ation. Giemsa is a dichromatic stain. marking
the nucleus pink and both th.. eytnplasm and nncleoli purpl... Images ....present multiple stu·
dent preparations and magnifications.
Indiana Unh-ersitr Soutbeast Celi Biology Laboratory (BioI 1.-313) students from Spring 2008 and Spring 2009 for their patiencedllringd~'\-elopmentof this lab S<.'Ties.
Mr. Jon Norman for assista""" ...ith cell cultu,," ordering, equipment, and maintenar>ee.
Dr. Adam Hammond for my long ago and '-ery brief training in animal cell culture and tbe assurance that it is actually fairly hard to kill SOme cells.
463
Student-Directed Investigations of the Actin Cytoskeleton in Chinese Hamster
Ovary Cells
Pamela L. Connerly
Biology Department
Indiana University Southeast
4201 Grant Line Rd.
New Albany, IN 47150
[email protected]
Abstract
As part of an upper-level Cell Biology course, students utilized fluorescently labeled phalloidin
to visualize actin in fixed Chinese hamster ovary (CHO) cells. Students learned aseptic technique, then
grew and stained CHO cells for observation and characterization of chromosome number. Students next
designed their own experiment to manipulate CHO cell growth condition to induce changes in actin
cytoskeletal structure. All groups used the same actin visualization procedure, using fluorescence
microscopy and digital photography to record data. The fluorescent visualization of cortical actin is
always dramatic, and students gained insights into cell culture methods, experimental design,
fluorescence microscopy, and data analysis.
Introduction
The advent of fluorescent markers that bind specifically to components of the cytoskeleton has
revolutionized cell biology and produced some of the most compelling visuals in modern biology. The
actin cytoskeleton in particular is often easily visualized as various bundles and networks of fibers in the
cell cortex, underlying the plasma membrane (Alberts et al., 2007.) The lab exercises presented here
were designed to give upper-level biology majors experience using animal cell tissue culture and the
opportunity to create and analyze their own images of the cytoskeleton. Biol L-312 Cell Biology
Lectures is an upper-level course required for the BS in Biology at Indiana University Southeast. The
associated laboratory courses (Biol L-313) is optional for most biology majors but fulfils an elective and
is required for the BS in Clinical Laboratory Sciences. The class size is typically quite small (5-10),
containing mainly students who enjoy laboratory courses. The laboratory curriculum is organized
around several multi-week series of experiments, with each series including a student-directed
component. Because we have two tissue culture hoods and a water-jacketed CO2 incubator, one of the
lab series involves the growth and analysis of animal culture cells. Chinese hamster ovary (CHO) cells
were chosen for the investigation because they were commercially available and, as non-human cells,
pose little risk to student researchers. The CHO cell lab series involved four main exercises: (1) an
introduction to aseptic technique, (2) cell staining with crystal violet and Giemsa stains, (3) mitotic
chromosome preparation and analysis, and (4) visualization of filamentous actin cytoskeletal elements.
The student-directed aspect of the investigation focused on the actin visualization component. Students
designed various treatments they believed would influence the structure of the actin cytoskeleton in the
CHO cells and used rhodamine-phalloidin to fluorescently label filamentous actin in the cells.
464
Instructor's Notes
Materials and Methods
Cell lines, Growth Media and Growth Conditions
Chinese hamster ovary (Cricetulus griseus) ovary cells (CHO-K1) were purchased from ATCC
(#CCL-61). Cells were grown in F12-K media supplemented with 10% Fetal Clone II (a fetal bovine
serum substitute optimized for CHO cells.) Cells were grown in sterile Petri plates or in T25 cell culture
flasks in a 37°C incubator with 5% CO2. Cell media was replaced approximately every three days and
cultures were split approximately once per week. All manipulations were performed in a tissue culture
hood. Cells were stored for future use in a liquid nitrogen dewar.
Cell Staining and Chromosome Spreads
CHO cells grown on cover slips were stained with crystal violet and Giemsa stains according to
protocols described in Freshney (2005). Digital photos of stained cells were taken by mounting a digital
camera in place of one ocular lens on a light microscope. To extend the characterization of the cells,
mitotic chromosomes were prepared and spread on glass slides, again according to protocols described
in Freshney (2005). Cells were then stained with crystal violet and observed by light microscopy.
Cell Manipulations
Students were encouraged to develop their own manipulation that they thought might have an
effect on the overall morphology of the actin cytoskeleton in CHO cells. In the course of two semesters,
student groups have studied various cytoskeleton disrupting drugs (e.g. cytochalasin D, colchicine),
differences in media conditions, density of cells, and presence of antibiotics. Treatments likely to
prevent culture growth (e.g. cytoskeleton-disrupting drugs) were applied hours before observation, while
those less likely to damage the cells permanently (e.g. varying cell densities) were applied several days
before observation.
Actin Visualization
The F-actin visualization Biochem Kit (Cat #BK005) from Cytoskeleton, Inc.
(www.cytoskeleton.com) was used for fluorescent visualization of filamentous actin in CHO cells. The
kit includes rhodamine-phalloidin, a chemical with a phalloidin moiety, which binds specifically to
filamentous actin, linked to the fluorophore rhodamine. Steps in the visualization process included first
growing cells on sterile glass cover slips, fixing the cells, permeabilizing the cells, incubating with
rhodamine-phalloidin, extensive washing, mounting onto glass slides, and observation by fluorescence
microscopy.
Data Acquisition
Each student group had time to observe their slides by fluorescence microscopy. Additionally, a
digital camera was inserted in place of one of the ocular lenses on the microscope, allowing for
acquisition of digital images of various preparations. Image files were uploaded to a classroom
management site and students retrieved their photos for analysis.
Results
465
Each group of students was able to successfully manipulate the CHO cells in culture. No overt
examples of contamination were observed either semester the lab was run. Students gained experience
using aseptic technique and working in the tissue culture hood. Student successfully stained and
observed cells they had grown in culture with both crystal violet (Figure 1) and Giemsa (Figure 2).
Figure 1. Micrographs of crystal violet (CV) stained CHO cells. Cells were fixed onto
cover slips, stained, and mounted for observation. CV is a monochromatic stain. Darker
areas within the central nucleus represent nucleoli. Images represent multiple student
preparations and magnifications.
Figure 2. Micrographs of Giemsa stained CHO cells. Cells were fixed onto cover slips,
stained, and mounted on glass slides for observation. Giemsa is a dichromatic stain,
marking the nucleus pink and both the cytoplasm and nucleoli purple. Images represent
multiple student preparations and magnifications.
466
Mitotic chromosome spreads were not reproducibly successful. One semester's students
produced nice slides, but the next did not. Students designed and carried out their own investigation into
a cell manipulation that might affect the cytoskeletal structure of CHO cells. Students chose to use
variables including addition of cytoskeleton-disrupting drugs, changes in cell culture density, and
presence of antibiotics in the growth medium. Students followed complex directions from a kit for
visualization of fluorescent rhodamine-phalloidin, a fluorescence marker specific for filamentous actin.
Students also gained experience using the fluorescence microscope to document their results (Figures 3
and 4).
Figure 3. Fluorescence micrographs of CHO cells visualized with rhodamine-phalloidin.
These are examples of images collected by students using a digital camera mounted in the
ocular lens of the microscope. These images represent the view of fluorescence cells in
the microscope's field of view. The digital camera could be further zoomed to capture
more cellular detail. These images represent multiple student preparations.
Figure 4. Fluorescence micrographs of CHO cells visualized with rhodamine-phalloidin.
These are examples of images collected by students using a digital camera mounted in the
ocular lens of the microscope. Each group used various degrees of zoom on the camera
to focus on the cells.
467
Discussion
Benefits of this series of experiments analyzing the actin cytoskeleton of CHO cells were
extensive. Students gained experience doing tissue culture, a fairly specialized skill that not all biology
majors experience. The importance of group work was emphasized. Students designed their own
experiments in which they had to consider controls, replicates, time management, and other issues.
Students gained experience with fluorescence microscopy and produced compelling digital photos of
their cells. Students had a real introduction to the multiple steps, long time-frame, and attention to detail
required for a significant cell biological experiment. They now have personal experience to relate to
textbook fluorescence microscopy images.
In addition to the many benefits described above, this activity does have several complications.
Some groups allowed the most experienced group member to do all of the work in the tissue culture
hood rather than encouraging all individuals to take the opportunity to get their own experience.
Students had difficulty conceiving their own experiments, requesting direction in what manipulation to
study as well as details of how to set up the experiment. Because of lab time constraints, we were not
able to troubleshoot and determine reasons for some unexpected results (such as absence of cells in
some treatments). Photo documentation was time consuming and challenging without dedicated
equipment. Students required significant assistance to arrive at reasonable conclusions. Although many
manipulations resulted in no observed difference between treatments, many students found that
conclusion to be unsatisfactory and would often overstate their results. All of these complications can
be addressed by the instructor. In fact, conversations regarding misconceptions about the results offer a
powerful teaching tool, but sufficient time must be allowed for discussion and re-evaluation of results.
As I continue to refine this series of lab activities, I plan to make several changes to address
some of the mentioned complications while maintaining the positive aspects. To address student
hesitancy to work in the tissue culture hood, I plan to require all students to demonstrate their
competency at manipulating cells in the tissue culture hood before proceeding with their projects. To
help all groups achieve interpretable photos of their cells, I am designing a practice photodocumentation
exercise to help students become comfortable with both the fluorescence microscope and the digital
camera, as well as to emphasize the need for and complexity of quantitative analysis of results. To
facilitate communication about results and reasonable interpretations, I will require group oral
presentation of results in a lab meeting atmosphere. I also plan to allow more time for modification and
repetition of the experiment.
Acknowledgements
I would like to than the Indiana University Southeast Cell Biology Laboratory (Biol L-313)
students from Spring 2008 and Spring 2009 for their patience during development of this lab series, Mr.
Jon Normal for assistance with cell culture ordering, equipment, and maintenance, and Dr. Adam
Hammond for my long ago and very brief training in animal cell culture and the assurance that it is
actually fairly hard to kill some cells.
468
Literature Cited
Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2008. The Cytoskeleton.
(Chapter 16). Pages 965-1052, in Molecular Biology of the Cell, Fifth edition. Garland Science,
Taylor & Francis Group, New York, NY, 1268 pages.
Freshney, RI. 2005. Culture of Animal Cells: A Manual of Basic Technique, Fifth edition. Wiley-Liss,
New York, NY, 672 pages.
About the Author
Pamela L. Connerly is an Assistant Professor of Biology at Indiana University Southeast, where
she teaches Cell Biology and Introduction to Biological Sciences I. She actively involves
undergraduates in two ongoing research projects investigating the relationship between the Golgi
apparatus and the process of endocytosis in the methylotrophic budding yeast Pichia pastoris and the
discovery and comparative genomics of novel bacteriophages infecting the gram negative bacterium
Acinetobacter baylyi.