Investigating Adhesion in Cell Motion

Investigating Adhesion in Cell Motion
Lisa Larrimore
February 10, 2006
Question for the A Exam from Professor Carl Franck
What can we say about the manner in which individual cells,
e.g. amoebae, are able to move about on surfaces? In particular,
what kind of adhesive contacts are possible that enable cells to
secure a foothold in order to move about? Can you suggest an
experiment using a natural or artificial surface, and an electrical
measurement technique that will help us to learn more about the
adhesive contacts that are necessary for cell motion?
Adhesion is critical for cellular motion, but the mechanism by which amoebae adhere to a surface is still poorly understood. Here, we introduce the basics of cell
movement in Section 1, and we describe the molecular machinery used for cell crawling
in Section 2. In Section 3 we focus on what is known about adhesion in the amoeba
Dictyostelium discoideum. Finally, in Section 4, we describe electrical measurement
techniques that have been used to probe biological systems, focusing on carbon nanotube transistors. We discuss the difficulties of using these techniques to learn more
about adhesion in amoebae like Dictyostelium.
1
Introduction to Cell Movement
Individual cells have two basic mechanisms for movement: swimming and crawling.
Cells swim by beating appendages called flagella and cilia, and they crawl by extending protrusions like pseudopods to attach to a surface and drag themselves forward
(Alberts et al., 2002, p. 966-972). Many prokaryotic bacteria1 propel themselves using flagella, while others exhibit a type of crawling known as twitching motility, in
which they extend tiny grappling hooks called type IV pili to drag themselves forward
(Mattick, 2002). Movement is so fundamental to cellular behavior that unicellular eukaryotes, or protists, were classified by their method of locomotion for much of the
1
Biologists have divided living organisms into three domains: eukaryotes (also spelled eucaryotes),
which have membrane-bound nuclei, and prokaryotic bacteria (or eubacteria) and archaea (or archaebacteria), which do not.
1
twentieth century. The animal-like protists, called Protozoa, were divided into swimmers (flagellates and ciliates), crawlers (amoeboids), and the non-motile sporozoa.2
These historical groupings have been replaced by phylogenetic trees based on evolutionary history, but crawling protists generally remain grouped together as amoebozoa
(Baldauf, 2003; Adl et al., 2005).
Crawling is not only important for dinner-seeking bacteria and amoebae, but is
also essential for many individual cells within multicellular eukaryotes. All cell motion
in animals, with the exception of swimming sperm, occurs by crawling. In embryos,
cells crawl as individuals or sheets to form different parts of the growing organism.
For example, neural crest cells in vertebrates crawl from the neural tube to become
skin pigment cells, facial structures, or other features, and the entire nervous system
is constructed by growth cones crawling along developing axons. Adult animals are
also full of crawling cells: white blood cells like microphages and neutrophils crawl to
infection sites to eat invaders, osteoclasts and osteoblasts crawl into bone and renew
it, and fibroblasts crawl through and repair connective tissue. Many cancers spread by
cells crawling from the primary tumor to nearby tissues (Alberts et al., 2002, p. 972).
The importance of cell crawling in human physiology means that understanding this
process is essential for developing treatments for many diseases (Bailly and Condeelis,
2002).
We will focus on eukaryotic cell crawling, the basic steps of which are illustrated in
Figure 1. First, the cell becomes polarized by some external signal that tells it which
way to crawl, resulting in actin polymerization at the leading edge. The cell then
protrudes an actin-rich structure in the intended direction of motion, which in the
case of amoebae is known as a pseudopod. While the protrusion is maintained through
continued actin polymerization in its tip, it makes contact with the extracellular matrix,
or the surface the cell is crawling along. The protrusion adheres to the surface, which
signals motor proteins to contract the actin filaments, putting the cell under tension.
If the cell then releases any adhesion at its trailing edge, the net force will draw it
forward. By repeating this cycle, the cell can progress across a surface (Bailly and
Condeelis, 2002; Alberts et al., 2002, p. 972).
Crawling cells are generally much slower than swimming ones: eukaryotic cells crawl
at speeds ranging from 0.1 to 30 µm/min (the fastest being epidermal keratocytes) and
twitching bacteria can pull themselves along at 60 µm/min; sperm cells, for comparison,
can swim at over 30 µm/sec, and some bacteria can swim at 200 µm/sec (Mattick, 2002;
Alberts et al., 2002, p. 973). Many factors affect the rate of cell crawling, including
the environment and the actions of regulatory proteins. One of the most important
factors is the adhesion between the cell and the surface: with too little adhesion the cell
cannot gain traction, but with too much it becomes glued in place (Friedl et al., 2001).
The regulation of adhesion based on cellular signals is therefore a very important part
of effective crawling.
2
The other protists, algae and yeasts, are not generally motile.
2
Figure 1: The motion of a crawling eukaryotic cell. Step 1: An external signal causes
localized actin polymerization. Step 2: The cell surface rearranges to form an actin-rich
protrusion. This protrusion is called a filopod, lamellipod, or pseudopod depending
on whether it extends in one, two, or three dimensions, respectively. Step 3: The
protrusion contacts and adheres to the surface. Step 4: Tension develops between
adhesion sites. Step 5: If there is de-adhesion at the cell’s trailing edge, the cell
moves towards the signal (A). If there is no de-adhesion, the contraction of the cell can
change the surface geometry as in wound healing (B), or the cell can change shape as
in embryogenesis (C). (Figure from Bailly and Condeelis, 2002.)
2
Cellular Machinery Used for Crawling
The cytoskeleton, molecular motors, and adhesion structures are the main cellular
structures required for eukaryotic crawling, although a multitude of proteins are also
required to regulate the process. Each of these three components is critical for the steps
illustrated in Figure 1: the cytoskeleton gives the cell structure, the molecular motors
move the cytoskeleton around, and the adhesion structures give the cell traction so
that it can pull itself along a surface.
The cytoskeleton has been extraordinarily well-conserved during evolution, particularly in eukaryotic cells. It is composed of actin filaments (diameter 5-9 nm, persistence
length 10 µm) that shape the cell’s surface, microtubules (diameter 25 nm, persistence
length 2 mm) that position organelles and direct transport, and intermediate filaments
(diameter 10 nm, persistence length 1 µm) that provide mechanical strength (Bouxsein
et al., 2004; Alberts et al., 2002, p. 909). Mictrotubules have been found to play an
3
Figure 2: Myosin motor proteins. (A) Cycle of myosin II walking along an actin
filament. ATP binding releases the head from the filament, ATP hydrolysis cocks the
head, and release of the phosphate and ADP moves the filament relative to the motor
protein. The head is bound to the filament only 5 percent of the time, allowing many
myosins to move a filament together. (B) Location of myosin I and myosin II in a
Dictyostelium amoeba crawling toward the upper right. Myosin I (green) is heavily
concentrated at the leading edge of pseudopods, and myosin II (red) is most dense in
the trailing region. (Figure from Alberts et al., 2002, p. 956, 977.)
important regulatory role in cell crawling, inhibiting formation of adhesion structures
and suppressing cell contraction, but actin filaments are the cytoskeleton component
primarily responsible for motility (Ballestrem et al., 2004). Each actin filament is polarized to allow its directional growth and movement: a conformational change in the
subunits that are added to an actin polymer slows the growth at the “minus end,”
while the “plus end” can grow at a rapid rate (Alberts et al., 2002, p. 912). It was
recently discovered that the actin network at the leading edge, which creates random
protrusions in the direction of motion, and the actin network in the bulk of the cell,
which provides tension between adhesion points are actually distinct, weakly coupled
networks, independently promoting cell migration (Ponti et al., 2004).
The protrusion and contraction of the actin network is performed by motor proteins from the myosin family, which use energy from ATP hydrolysis to walk toward
the plus end of an actin filament. Myosin I, which has one head that walks along actin
filaments and a tail that binds to actin or the cell membrane, is important for actin
polymerization and protrusion and is generally located at the leading edge of a migrating cell. The tail end can pull the plasma membrane along while the head walks along
newly-formed actin in a pseudopod. Myosin II, which has two heads that walk along
actin filiments and a long tail that can bind to other myosin II molecules, causes contraction by pulling actin fibers together and is mostly concentrated in the trailing bulk
of the cell (Fukui et al., 1989; Alberts et al., 2002, p. 949-977). The motion of myosin
II along an actin filament and the location of myosin I and myosin II in a migrating
cell are illustrated in Figure 2. Other myosin motor proteins, such as myosin VII, are
also important in cell crawling, and researchers are currently investigating their roles
(Titus, 2004; Tuxworth et al., 2005).
4
Figure 3: Focal adhesions. (A) Cartoon of the proteins composing a focal adhesion.
The transmembrane integrin protein is heterodimeric, being composed of noncovalently
interacting α and β subunits. The integrin connects the extracellular matrix to intracellular anchor proteins (with different combinations of α and β subunits binding to
different matrix proteins), which in turn connect to actin filaments within the cell. (B)
Focal adhesions in a fibroblast. Left: Living cell imaged with interference reflection
microscopy (IRM), in which the light from an inverted microscope reflects from both
the glass substrate and the cell itself. Changes in the distance between the surface
and the cell change the interference between these reflections. Here, the focal contacts
pull the cell membrane closer to the glass surface (10-15 nm instead of 50 nm), causing
them to appear as dark spots. Right: The same cell is visualized after fixation and
staining of the actin filament bundles, revealing their termination at the focal contacts.
(Figure from Alberts et al., 2002, p. 946, 1074.)
The third important class of cellular machinery used for crawling is adhesion structures, which vary with the type of cell. For individual cells crawling inside mammals,
the most important of these structures is called a focal adhesion, which is formed
by a transmembrane protein known as an integrin that connects the surface to the
actin filaments in the cell.3 The spaces between mammalian cells are largely filled by
the extracellular matrix, a network of proteins and polysaccharides that are secreted
by surrounding cells. As illustrated in Figure 3A, the extracellular integrin domains
bind to proteins in the extracellular matrix, which means that different integrins are
adapted to different surfaces. This binding is regulated by internal or external signals
that cause a conformational change in the integrin extracellular domain. Inside the
cell the integrins bind to anchor proteins, which form a dense “adhesion plaque” that
includes structural and signalling proteins. This plaque connects the integrins to actin
filament bundles in the cell (Alberts et al., 2002, p. 1073-1074). The distribution of
focal adhesions in a cell can be seen using interference reflection microscopy (IRM),
and Figure 3B shows the correlation between focal adhesions and the termination of
actin filaments in a fibroblast cell.
While the adhesive properties of cells crawling inside mammals are fairly well3
Integrin proteins can also be used to anchor intermediate filaments in a cell to an extracellular
surface, forming a junction known as a hemidesmosome, but these are not involved in cell migration
(Alberts et al., 2002, p. 1070-1074).
5
characterized, comparatively little is known about the adhesive contacts in amoebae.
The adhesion mechanisms are necessarily different: while mammalian cells can crawl
on an extracellular matrix of proteins that they have secreted, amoebae may encounter
diverse and foreign substrates and require more general mechanisms or adhesion receptors. Mammalian crawling cells usually adhere more strongly to their substrate
than amoebae do, but amoebae can turn adhesion on and off (to change directions,
for example) much more quickly (Friedl et al., 2001). In the following section, we review what has been learned about the adhesion of a particular amoeba, Dictyostelium
discoideum.
3
Adhesion in Dictyostelium
The soil-dwelling Dictyostelium discoideum is one of the most commonly-studied amoebae; in 2005, for example, it became the first protist to have its genome fully sequenced
(Eichinger et al., 2005). Dictyostelium spends most of its life crawling around leaves
and soil to feed on bacteria, but starving Dictyostelium will crawl together into a
mound of 104 -105 cells. This aggregate can form a slug to search for food elsewhere,
or become a fruiting body with a sacrificial stalk supporting a mass of spores (Friedl
et al., 2001). When the cells aggregate, they secrete proteins to form an extracellular
matrix, and a number of receptors for binding to this matrix have been identified. The
mechanism by which an individual crawling Dictyostelium cell adheres to a substrate,
however, remains largely elusive (Titus, 2004).
Part of the difficulty in elucidating the adhesion mechanism is that Dictyostelium
cells can adhere to a vast range of substrates. In nature, they live among leaves and soil,
but they also crawl easily on coverslips and the bottoms of plastic dishes (Uchida and
Yumura, 2004). In early work studying phagocytosis, Vogel et al. (1980) concluded that
Dictyostelium must have at least two kinds of surface adhesion receptors: a nonspecific
hydrophobic receptor, allowing the cells to adhere to latex beads (but not to hydrophilic
particles), and a specific carbohydrate receptor, allowing the cells to adhere to (and
ingest) bacteria with terminal glucose. It was 20 years, however, before much more
was learned about these surface receptors.
No integrin-like adhesion proteins have been found in Dictyostelium, but other
adhesion-related membrane proteins have been recently identified.4 Gp130 (Chia et al.,
2000) and the Phg1 protein family (Cornillon et al., 2000; Benghezal et al., 2003) are
transmembrane proteins that are important for adhesion, and SadA (Fey et al., 2002) is
the only adhesion receptor protein identified so far. SadA contains three extracellular
repeats resembling epidermal growth factor (EGF), which Fey et al. believe may bind
to external molecules, but the exact mechanism is unclear.
Other recent experiments have involved at the dynamics of actin filaments near
the cell membrane, which appear to play an important role in adhesion (Uchida and
Yumura, 2004; Bretschneider et al., 2004). Using IRM and confocal fluorescence microscopy, Uchida and Yumura found that actin foci remain fixed as the cell migrates,
4
A few intracellular proteins that regulate adhesion have also been identified, but they will not be
discussed here.
6
Figure 4: A model of Dictyostelium migration. (Aa) Possible structure of cellsubstratum adhesion sites. Actin filaments link the cytoskeleton to the substratum
through transmembrane adhesion proteins (perhaps including SadA). (b) Dynamic behavior of adhesion structures. The actin focus may bring membrane proteins to the
site, and these proteins remain adhered to the substratum after the actin focus disappears. (B) Coordinated steps of a migrating cell. Blue arrows represent motive force,
and black arrows represent the traction force transmitted through the adhesion sites.
There are separate extension (a-c) and retraction (e-h) phases. (Figure from Uchida
and Yumura, 2004.)
that the cell membrane is closest to the substratum at these points, and that some foci
are left on the surface behind the cell. They also watched fluorescent beads attached to
a flexible silicone substratum and saw that the traction force of the cell is transmitted
to the surface at the actin foci. Figure 4 shows their cartoon model of Dictyostelium
migration. The role of the actin filaments in these cellular “feet” is not understood, but
they suggest several possibilities, including that the actin may link the cytoskeleton to
the substratum and carry components necessary for adhesion to these sites. They also
suggest that the actin foci may act as suction cups to mediate non-specific adhesion;
for example, a small actin protrusion pushing on the surface would push back the surrounding cell membrane, generating a suction force.5 None of their hypotheses have
yet been tested.
Although Uchida and Yumura (2004) do not give a quantitative measurement of the
traction force involved in Dictyostelium migration, a number of researchers have measured the traction force in other crawling cells by using the same technique of placing
5
UC Berkeley bioengineering professor David Fletcher believes that the single-celled parasite Giardia intestinalis can adhere nonspecifically to diverse surfaces using a large suction mechanism (one
suction cup for the cell, as opposed to one at each actin foci), but he has yet to publish results relating
to these experiments (Pescovitz, 2004).
7
the cells on silicone substratums of known stiffness and measuring the displacement of
fluorescent beads attached to the silicone as the cell moves. For example, the traction
generated by locomoting keratocytes has been measured to have a root-mean-squared
magnitude of 0.5 nN/µm2 , or a total force per cell of 10 nN (Oliver et al., 1999).
By examining pictures of the data from Uchida and Yumura, we can estimate that
Dictyostelium cells also generate local forces of order 1 nN.6
Fukui et al. (2000) used a different and creative technique to measure the maximum ability of an entire Dictyostelium cell to propel itself: they placed amoebae on a
centrifuge microscope and increased the rotation speed until no amoebae were able to
crawl outward. They found that a wild-type Dictyostelium could generate more than
2.6 nN of traction force, and that the forces generated by strains lacking certain myosin
proteins were decreased by a factor of ten. This technique is similar in principle to
centrifugal assays, which are used to measure not the force cells need to crawl forward,
but the overall adhesion force needed to stick to the substrate (Lotz et al., 1989).7
In summary, it is clear that Dictyostelium can adhere to diverse surfaces through
some nonspecific binding, and that they can generate traction forces on the order
of 1 nN. They contain at least one membrane adhesion protein, SadA, although the
mechanism by which it binds to the substratum is unclear. Other adhesion-related
proteins have been identified, and biochemical and genetic techniques are being used
to search for more. Finally, actin foci appear to play a critical part in adhesion, but
their exact role also remains unclear. There is still much to be learned about adhesive
contacts in Dictyostelium and other amoebae, and in the following section we discuss
the possibility of studying these contacts using an electrical measurement.
4
Investigating Adhesion Electrically
Several research groups have developed electrical measurements for monitoring adhesive cells. Wegener et al. (2000) have grown cells on arrays of 250-µm diameter gold
electrodes and measured the decrease in the AC (40 kHz) impedance as the cells spread
and covered larger areas of each electrode, a technique known as Electric Cell-Substrate
Impedance Sensing (ECIS). Le Guillou-Buffello et al. (2005) allowed cells to spread on
a thin oscillating quartz crystal and measured the change in resonance frequency as
more cells attached to the surface. But while these measurements allow researchers
to measure spreading cells without optically counting them, they are measurements of
bulk cell properties and provide no new information about the adhesion mechanism of
an individual cell.
There are various other ways in which we could probe a crawling Dictyostelium with
electrical techniques. We could see how the adhesion of a crawling cell is affected by
6
The silicone substratum used by Uchida and Yumura has a stiffness of 4 nN/µm, and the fluorescent beads appear to move by up to 0.3 µm in a given section of the cell.
7
Adhesive and traction forces for various cells have also been measured in many other ways, all
of which involve applying some external force. For example, adhesion forces have been probed with
atomic force microscopes (AFMs), optical tweezers, micropipette suction, and calibrated laminar shear
flows (Simon and Durrieu, 2006). Cells have also been made to crawl across micromachined cantilevers
to measure the traction forces generated by different sections of the cell (Galbraith and Sheetz, 1997).
8
changing the voltage of its substrate, or we could measure the strength of its adhesion
to an AFM tip as a function of the tip voltage.8 Cells can be trapped and manipulated
using dielectrophoresis (DEP), the force on a dipole in a nonuniform electric field
(Gascoyne and Vykoukal, 2002), so we could see how DEP forces affect cells crawling
over an electrode array. It is not clear, however, that we would learn anything new
about adhesive contacts using any of these methods. All of these methods could be
used to quantify the strength of the adhesive force, but this can already be measured
by other techniques, as described in Section 3.
Learning more about the specific adhesion mechanisms of crawling cells is difficult
because adhesion involves the behavior of individual proteins, and proteins are typically
only a few nanometers wide. The size of SadA (the one membrane adhesion protein
that has been identified in Dictyostelium) has not been determined, but it is predicted
to have a molecular weight of 105 kD (Fey et al., 2002). For comparison, the α subunit
of the integrin protein has a molecular weight of about 140 kD and a width of about
5 nm (Alberts et al., 2002). In order to study the behavior of a < 5 nm object, it is
necessary to have a probe that is at least that small; a single-walled carbon nanotube
(SWNT) is therefore a logical choice. To investigate the possibility of using a SWNT
in an electrical measurement of a crawling cell, we will first briefly review previous
biosensing studies using nanotubes.
Semiconducting carbon nanotubes can be used as field-effect transistors (FETs) in
an electrolyte environment, in which a gate wire is used to establish the electrochemical
potential of the solution relative to the nanotube (Krüger et al., 2001; Rosenblatt
et al., 2002). This ability to operate in electrolytes of various salt concentrations and
pH values is critical if nanotubes are to be used as effective biological sensors, and
a number of researchers have demonstrated the use of nanotube transistors to detect
proteins. Electrolyte-gated nanotube transistors have been used to sense nonspecific
protein adsorption (Boussaad et al., 2003; Chen et al., 2003; Bradley et al., 2004) as
well as specific binding of proteins to nanotubes that have been functionalized with the
protein receptors (Chen et al., 2003, 2004). Besteman et al. (2003) have electronically
detected the attachment of glucose oxidase to a nanotube, and have then used the
functionalized nanotube transistor as an electronic pH sensor. Proteins can also be
electronically detected by drying them on a nanotube and then measuring the transistor
with a silicon back gate (Star et al., 2003, 2004).
In all of these experiments, the protein causes either an overall decrease in the
nanotube conductance or a shift in the threshold voltage, and sometimes it is difficult
to distinguish between the two effects. Figure 5A,B shows some examples of these
effects for a nanotube functionalized with glucose oxidase (Besteman et al., 2003). A
decrease in conductance is usually attributed to the potential from the protein causing
scattering of charge carriers in the nanotube, and a threshold voltage shift to electron
transfer from the protein to the nanotube. It is important to be careful in interpreting
these results, however, as we have recently shown that a threshold voltage shift can also
be caused by an electrochemical interaction between the analyte and the electrolyte8
A similar experiment with an AFM tip and a carbon nanotube was used to show that their
attraction is due to electrostatic as well as van der Waals forces (Minot, 2004, p. 56, 106).
9
Figure 5: Nanotube transistors for biological sensing. (A) Immobilization of glucose
oxidase (light blue) decreases the overall conductance of a SWNT from the original
curve (black). The red, green, and dark blue curves show the response of the SWNT
to incubation in dimethylformamide with or without the linking molecule used for
functionalization. (B) The changing charge of glucose oxidase as a function of pH
causes a threshold voltage shift in a glucose oxidase functionalized SWNT; the inset
shows that there is no shift for a bare nanotube (Besteman et al., 2003). (C) Depositing
the cell membrane of Halobacterium salinarum with the cytoplasmic side down on a
transistor made from a network of nanotubes causes a positive threshold voltage shift
(Bradley et al., 2005).
gate wire (Larrimore et al., 2006).
Although unresolved questions remain, researchers are making progress in understanding the interaction between SWNTs and individual biomolecules like proteins.
They are only beginning, however, to integrate nanotube transistors with more complex biological systems like cell membranes. Bradley et al. (2005) have deposited the
membrane from Halobacterium salinarum on top of transistors made from networks of
carbon nanotubes. This membrane contains the protein bacteriorhodopsin, which has
a permanent dipole moment; the researchers were thus able to assemble the membrane
in different orientations on top of the nanotubes by applying different voltages to the
silicon substrate during deposition. They found that the membrane shifted the transistor threshold voltage in different directions depending on which side of the membrane
contacted the nanotubes: the cytoplasmic side caused a positive shift (as shown in
Figure 5C), and the extracellular side caused a slight negative shift. They conclude
that the electrostatic field associated with the bacteriorhodopsin dipole induces charge
in the nanotubes, shifting the Fermi level and thus the threshold voltage, and they use
the differences in threshold voltage shifts to conclude that the electric dipole of the
bacteriorhodopsin is located 2/3 of the way from the extracellular to the cytoplasmic
side of the membrane.
This work is a promising step in understanding the interactions of carbon nanotubes
and cell membranes, and other investigations into this system are underway at Cornell
by Xinjian Zhou in the McEuen Group and Jose Moran-Mirabal in the Craighead
Group. It seems, however, that our understanding of sensing with carbon nanotubes
is not yet at the point where they could be used to provide new information about the
10
adhesion mechanisms in crawling cells. Rather, crawling cells could be used to further
study the response of SWNT FETs to biological systems. We could, for example,
perform a similar experiment to Uchida and Yumura (2004) and observe the dynamics
of actin foci with an inverted confocal microscope while Dictyostelium cells are crawling
over a SWNT FET. It would be necessary to grow the nanotubes on a transparent glass
substrate (as has previously been done in the McEuen Lab) and to cover the electrodes
contacting the nanotube to eliminate leakage currents between them and the cell buffer.
We could then see how the nanotube conductance changes as a cell crawls over it, and
whether it is possible to observe differences in the conductance when the nanotube is
in contact with different sections of the membrane.
5
Conclusion
Although the basic steps of cell crawling (protrusion, adhesion, and contraction) have
been recognized for 35 years, the method by which amoebae such as Dictyostelium
adhere to a surface is still not understood. Amoebae can adhere to diverse surfaces,
which means that unlike crawling mammalian cells, they must have a nonspecific adhesion mechanism. Researchers are beginning to identify adhesion-related proteins
in Dictyostelium, but the method by which they attach to surfaces remains elusive.
Investigating the nanoscale focal adhesions in an amoebae with an electrical measurement would require a nanoscale electrical probe such as a carbon nanotube, but we
do not yet understand nanotube biosensors well enough to use them to learn more
about amoeboid adhesion. Instead, crawling Dictyostelium could be used to learn
more about carbon nanotube transistors so that they might someday be used to study
more complex processes.
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