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Article |
Correspondence to Maureen Condic: maureen.condic{at}hsc.utah.edu
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Abstract |
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Introduction |
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Integrins are a major metazoan family of cell adhesion receptors and play key roles in development, immune response, and cancer metastasis (for review see De Arcangelis and Georges-Labouesse, 2000; van der Flier and Sonnenberg, 2001). These heterodimeric transmembrane receptors, composed of an and a ß subunit, bind the ECM and convey signals intracellularly. During vertebrate development, integrins are required at numerous stages for proper cell migration, proliferation, survival, and differentiation of many embryonic cell populations, including the neural crest.
To migrate long distances through diverse tissues in vivo, NCCs must be able to adapt to changing extracellular environments. We have previously shown that embryonic sensory neurons and their immediate embryonic precursors, NCCs, are able to migrate across at least a 10-fold range of ECM protein concentrations in vitro (Condic and Letourneau, 1997; Condic, 2001; Strachan and Condic, 2003). NCCs attain optimal adhesion for sustained motility over a wide range of ECM concentrations by altering surface integrin expression in order to match their adhesion receptor levels to the concentration of ligand. In contrast, many other motile cell types appear unable to modulate surface integrin levels and therefore only migrate on a limited range of ECM concentrations (Goodman et al., 1989; Buettner and Pittman, 1991; Duband et al., 1991; Arroyo et al., 1992; DiMilla et al., 1993; Palecek et al., 1997). These results suggest that rapid NCC motility over a wide range of substratum concentrations is dependent on continuous monitoring of and response to the extracellular environment.
The response of NCCs to the ECM varies along the rostrocaudal axis of the embryo. The neural crest can be divided into four subpopulations (cranial, vagal, truncal, and sacral), each of which occupies its own segment of the neural tube and gives rise to distinct derivatives (Bronner-Fraser, 1993b). We have shown that different crest populations have distinct motility and integrin regulation in culture. For example, cranial and trunk neural crest have similar migratory properties on low concentrations of laminin. Yet, on high concentrations of laminin, cranial NCCs migrate nearly twice as fast as trunk NCCs. Correspondingly, cranial NCCs regulate surface levels of integrin 6 (a laminin receptor) to a greater extent than do trunk NCCs. When integrin
6 is overexpressed in cranial NCCs, their velocity slows to that of trunk NCCs, suggesting that low surface integrin levels are required for rapid motility (Strachan and Condic, 2003). Thus, we focused here on the mechanism cranial NCCs use to modulate their surface integrin levels, thereby promoting rapid cell migration.
One mechanism by which cells can modulate their surface integrin levels is via the clathrin-mediated receptor recycling pathway (Bretscher, 1992; Fabbri et al., 1999; Pierini et al., 2000; Long et al., 2001). Clathrin-mediated endocytosis modulates signal transduction both by controlling the levels of surface signaling receptors and by mediating the rapid clearance and down-regulation of activated signaling receptors. For motile cells, receptor recycling also provides an efficient way to transport receptors from the tailing edge, where the cell is releasing from the substratum, to the leading edge, where new adhesions are being formed (Roberts et al., 2001; Rappoport and Simon, 2003; Powelka et al., 2004). Receptors may either be returned to the surface nearby the site of internalization via fast recycling vesicles, or can be trafficked through the cell via the slower receptor recycling compartment (Sonnichsen et al., 2000). Each endocytic compartment is characterized by the expression of specific rab GTPases (Mellman, 1996; Sheff et al., 1999; Qualmann and Mellor, 2003). Because cranial NCCs down-regulate surface levels of integrin 6 in response to the same conditions under which they migrate relatively quickly (i.e., high laminin concentrations), we questioned whether laminin receptors were being transported through the receptor recycling pathway in rapidly moving NCCs.
In the present work we demonstrate that cranial NCCs regulate their surface integrin expression in a substratum-dependent manner. Using a modified, short-term, biochemical assay we followed the internalization kinetics of 5 (a fibronectin receptor) and
6 (a laminin receptor) integrins in cranial NCCs cultured on fibronectin and laminin. We find that in cells cultured on laminin, but not fibronectin, surface integrin
6 is internalized and intracellular pools of internalized receptor accumulate over a 20-min time period. Internalized laminin receptors colocalize in rab4+ and rab11+ recycling vesicles, indicating that they are transported via the receptor recycling pathway. By labeling surface receptors immunohistochemically, we also demonstrate that internalized receptors are recycled back to the cell surface. Finally, using an acute time-lapse assay we show that inhibiting receptor recycling with bafilomycin A (BafA) significantly slows cranial neural crest migration on high laminin but not on low laminin, suggesting that this pathway is critical for regulating adhesion and supporting rapid motility on this substrata. We conclude that receptor recycling is an important mechanism underlying rapid cranial neural crest migration.
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Results |
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Integrin internalization kinetics are substratum dependent
To analyze the kinetics of surface integrin regulation in cranial NCCs, we followed the internalization of labeled receptors during a short time period. We found a difference between the accumulation of internalized integrin 6 and integrin
5 (Fig. 2). On both low and high laminin concentrations, internalized integrin
6 reached steady-state levels by 20 min; i.e., there was no greater accumulation after 30 or 60 min (unpublished data). In contrast to integrin
6, low levels of integrin
5 accumulated rapidly (within 2 min) in cells cultured on both laminin concentrations, and the internalized pool did not increase over time (Fig. 2, B and D). Thus, cranial NCCs on laminin internalize and accumulate laminin, but not fibronectin receptors.
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Internalized integrins colocalize with receptor recycling pathways
To determine in which intracellular pathway internalized integrins were being trafficked, we investigated the colocalization of receptors with markers of the receptor recycling pathway. Internalized integrins could be targeted for destruction or sequestered in receptor recycling compartments; however, the latter possibility is more likely because synthesis of new receptor molecules is generally incompatible with rapid motility (Bretscher and Aguado-Velasco, 1998). Cranial NCCs were cultured on high concentrations of laminin or fibronectin and colabeled for either integrin 5 or integrin
6, and rab4 and rab11, markers of both the fast and slow recycling pathways. To quantify the colocalization events we used Volocity (Improvision), a program that reconstructs confocal z-series of cells three dimensionally to allow XZ and YZ reslicing in order to verify true colocalization events.
On high laminin concentrations, both integrin 5 and integrin
6 colocalized with vesicles positive for either rab4 or rab11, or double-positive for both (rab+ vesicles), but a significantly greater percentage of rab+ vesicles contained integrin
6 (40%) compared with integrin
5 (21%) (Fig. 3, A and B; Table I). Integrin
5 appeared to be largely localized in intracellular compartments that were neither rab 4 nor rab11 positive (rab vesicles), suggesting that the majority of
5 integrin does not participate in receptor recycling. On high fibronectin concentrations, both integrin
5+ and integrin
6+ vesicles colocalized with rab+ vesicles to similar extents (18 and 17%, respectively) (Fig. 3 B; Table I). Thus, in cranial NCCs cultured on high concentrations of laminin, twice as many recycling vesicles contain the relevant receptor, integrin
6, as the nonrelevant receptor, integrin
5. Furthermore, in cranial NCCs cultured on high concentrations of fibronectin, neither receptor appears to be selectively targeted to the receptor recycling pathway (Table I).
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Internalized integrins are recycled back to the cell surface
Next, we investigated the fate of internalized surface integrin 6 in cranial NCCs cultured on high laminin concentrations. In other fast-moving cells and growth cones, internalized receptors are recycled back to the leading edge rather than being degraded (Lawson and Maxfield, 1995; Kamiguchi and Lemmon, 2000). Using an adapted immunohistochemical technique based on that of Kamiguchi and Lemmon (2000) we examined the recycling of integrin
6 in cranial NCCs. Surface
6 was labeled with anti-
6-Fab while cells were allowed to endocytose and traffic receptors for 30 min at 37°C. Cells were cooled to room temperature to prevent further trafficking and Fab remaining at the surface was blocked with unconjugated secondary antibodies. Subsequently, cells were allowed to traffic internalized receptors at 37°C for 20 or 90 min, and any integrin
6-Fab complexes that returned to the surface were detected with labeled secondary antibodies. Intentionally permeabilized cells (Fig. 4 A, first column; Fig. 4 B) could be distinguished from unpermeabilized cells by significantly higher integrin
6 and actin fluorescence. In the 0-min condition, cells fixed at the beginning of the recycling period and reincubated for 90 min had low fluorescence of both integrin
6 and actin (Fig. 4 C).
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Inhibiting receptor trafficking slows cranial neural crest motility
Because cranial NCCs down-regulate surface levels of integrin 6 (Fig. 1), internalize this receptor only on laminin (Fig. 2 and Fig. 3), and recycle it back to the cell surface when cultured on high concentrations of laminin (Fig. 4), we postulated that rapidly moving cranial NCCs cultured on high concentrations of laminin would be more dependent on receptor trafficking than the same cells on low levels of laminin where they migrate more slowly. To determine whether substratum-dependent receptor recycling affects cranial neural crest motility, we inhibited receptor trafficking using a pharmacologic approach.
Bafilomycin A is a specific inhibitor of the vacuolar type H+-ATPase found in all animal cells, plant cells, and microorganisms. BafA does not prevent endocytosis, yet it prevents the acidification of endocytic structures, which impairs the transport of vesicles out of the early endosome (Thomsen et al., 1999). Because internalized receptors destined for both the fast and slow receptor recycling pathways originate in the early endosome, treatment with BafA allows cells to internalize surface receptors but impairs receptor recycling back to the surface (Fig. 5 A). Previous work has shown that BafA treatment retards the rate of transferrin receptor recycling twofold in CHO cells (Presley et al., 1997). We controlled for any nonspecific effects of BafA treatment by comparing the same cell type cultured on two different concentrations of laminin.
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Discussion |
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To date, only a handful of investigations have examined receptor recycling (Kamiguchi and Lemmon, 2000) and its role in cell motility (Fabbri et al., 1999; Pierini et al., 2000). Previous work in CHO cells and neutrophils has shown that the recycling of integrins to the leading edge of the cell is essential for efficient motility (Fabbri et al., 1999; Pierini et al., 2000). However, these studies were performed using transformed cell lines or adult primary cell populations where large numbers of cells can be easily obtained (Lawson and Maxfield, 1995; Fabbri et al., 1999; Kamiguchi and Lemmon, 2000). In contrast, the single neural tube explant used here will produce, at most, 100,000 NCCs, a limitation that has significantly hampered quantitative assessments of integrin trafficking in this system. The approach described here allows us to gain insight into the molecular mechanisms this developmentally important cell population uses for migration. It is likely that these techniques can be used to examine receptor trafficking and motility in other primary embryonic cell populations.
Integrin recycling and receptor specificity
The first observation that integrins differentially participate in the endocytic pathway was published over a decade ago (Bretscher, 1992). In this paper, Bretscher showed that in CHO cells grown in suspension, integrins 5ß1 and
6ß4 circulate, but
3ß1,
4ß1, and
Lß2 do not. Although it was initially postulated that the ability to participate in endocytic trafficking may be a property of the receptor itself, other groups have since shown that the substratum onto which the cells are adhered also greatly influences this process. When neutrophils are cultured on vitronectin, the vitronectin receptor mediating their migration (integrin
Vß3) is recycled in a polarized manner (Lawson and Maxfield, 1995). In contrast, when the same cells are cultured on fibronectin, integrin
Vß3 is no longer distributed in a polarized manner, and instead integrin
5ß1 (a fibronectin receptor) is internalized into endocytic recycling compartments (Pierini et al., 2000). These studies suggest that cells may preferentially recycle the receptors that are mediating their migration, and this is consistent with our current demonstration of substratum-dependent integrin recycling in cranial NCCs.
Our finding that, in contrast to neutrophils, integrin 5 participates predominantly in short time-frame recycling in cranial NCCs cultured on fibronectin suggests that distinct cell types can regulate the same receptor in different ways. How NCCs modulate adhesion to maintain motility on fibronectin is unknown, but could involve functional modulation of integrin receptors; a possibility we are currently investigating.
Substratum-specific receptor internalization has been shown for nonintegrin receptors as well. For example, insulin receptor internalization was inhibited 4060% in CHO cells adhered to galectin-8 (an ECM protein and an integrin ligand) when compared with the same cells adhered to fibronectin, collagen, or laminin (Boura-Halfon et al., 2003). Other studies have shown that in embryonic dorsal root ganglia neurons, L1, a homophilic cell adhesion receptor that promotes axon growth along preexisting axon bundles, is recycled in a spatially regulated manner within growth cones cultured on L1 but not laminin (Kamiguchi and Lemmon, 2000). It is likely that the internalization of many receptors may depend on the extracellular environment of the cell. In this way, intracellular signaling of downstream events such as migration, differentiation, and apoptosis could be regulated in a region specific manner throughout the embryo.
Recycling kinetics and substratum density
Receptor recycling has not been previously investigated in a single cell population cultured on different concentrations of the same substrata. Our findings suggest that not only is substratum composition a key regulator of receptor recycling, but also that the density of ligands on the substratum can affect the importance of receptor internalization for motility. BafA treatment has a greater effect on cranial neural crest motility when the cells are cultured on high concentrations of laminin compared with low. Thus, when cranial NCCs are cultured on low concentrations of laminin, their dependence on receptor recycling for efficient motility is decreased in comparison to the same cells cultured on high laminin concentrations. These results clearly demonstrate that receptor recycling makes a stronger contribution to NCC velocity on high laminin concentrations, where surface receptor expression levels are low.
These factors suggest a model for NCC migration on laminin (Fig. 6). Our model proposes that efficient motility on laminin is largely regulated by the appropriate matching of cell surface adhesion receptor number to ligand availability. On low concentrations of laminin, there is very little ligand available (calculations from our current measurements of radiolabeled laminin binding indicate that on low laminin only 300 molecules of laminin are bound per µm2) so efficient motility requires a large number of receptors on the cell surface to increase the probability of ligand binding events. In contrast, on high concentrations of laminin (10-fold higher density), ligand is more readily available and fewer receptors are needed on the surface to achieve the same probability of ligand binding. In fact, on high substratum concentrations, high surface receptor number would increase adhesion and concomitantly decrease motility. Thus, on high ligand concentrations, motile NCCs rely on recycling of internal receptor pools to promote ligand binding events at the leading edge of the cell, whereas on low ligand concentrations the receptors are already at the cell surface.
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In summary, we have shown that substratum-dependent receptor recycling is an important molecular mechanism used by cranial NCCs to promote rapid motility on laminin. We have also demonstrated that the same cell population can respond differently to the same substratum, depending on its concentration. Our data suggest that cranial NCCs tightly regulate the cellular mechanisms that support motility in response to the specific conditions the cells encounter. The investigation of receptor recycling in a primary embryonic cell population is both novel and fundamental to the understanding of cell migration during development.
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Materials and methods |
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White Leghorn chicken eggs (supplied by Utah State University, Logan, UT) were incubated at 38°C until the embryos reached stage 8 (Hamburger, 1992). All animal studies were approved by the University of Utah Institutional Review Board. Neural tubes from the mesencephalic level were dissected away from the surrounding tissue with tungsten needles. Neural tubes were cultured at 37°C with 5% CO2 for 4872 h in 250 µl neurobasal medium (GIBCO BRL) supplemented with 25 µM glutamic acid (Sigma-Aldrich), 500 µM L-glutamine (Sigma-Aldrich), 1x B-27 and N-2 (GIBCO BRL), 10 ng/ml NT3 (CHEMICON International), 100 ng/ml EGF (Upstate Biotechnology), 10 ng/ml FGF (Upstate Biotechnology), and 50 ng/ml NGF (R&D Systems). Neurobasal medium without the supplements and buffered with N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (Hepes; USBiological) was used for the time-lapse assays.
Reagents and antibodies
Antibodies against the following proteins were used in these experiments: integrin 5 (D71E2; DSHB, and AB1928; CHEMICON International), integrin
6 (P2C62C4;DSHB, and SC10370; Santa Cruz Biotechnology, Inc.), rab4 (610888; BD Biosciences), rab11 (610656; BD Biosciences), actin (AAN01; Cytoskeleton, Inc.), and
-tubulin (T5168; Sigma-Aldrich). For the integrin recycling experiments a Fab fragment of a monoclonal integrin
6 antibody (MAB13444; CHEMICON International) was generated. Bafilomycin A1 (B1793; Sigma-Aldrich) was dissolved in DMSO (D8418; Sigma-Aldrich). Sodium 2-mercaptoethanesulfonate (MesNa) was purchased from Sigma-Aldrich (M1511) and dissolved immediately before use in Hepes-buffered neurobasal medium.
Immunoprecipitations
Cell surface receptors were labeled with NHS-SS-biotin (Pierce Chemical Co.) on ice and the cells were then lysed on ice in a solution containing 0.1% SDS, 1% Triton X-100, and protease inhibitors. Protein concentration in each cell lysate was determined with a BCA protein assay (Pierce Chemical Co.) and equalized across conditions. Integrins were immunoprecipitated by using standard protocols (de Curtis et al., 1991), and immunoprecipitated proteins were size fractionated under nonreducing conditions on acrylamide gels and electrophoretically transferred to nitrocellulose membranes. Biotinylated proteins were detected by using Strep-Avidin conjugated to HRP and a chemoluminescent reagent (Pierce Chemical Co.) followed by exposure to film. For the internalization assays (Fig. 2), where reducing agents interfere with protein concentration assays, 15 µl of lysate was removed before immunoprecipitation as a protein loading normalization control, size fractionated on an acrylamide gel, transferred to nitrocellulose, and revealed with an anti--tubulin antibody (T5168; Sigma-Aldrich). Using nonreduced lysates, BCA assays were performed alongside
-tubulin Western blots to ensure that
-tubulin expression was not different in cells cultured on LM1, LM20, or FN20. Comparisons of protein concentrations (BCA) and
-tubulin band intensities as a measure of protein loading give identical results. Exposures of blots that were in the linear range were quantified using GelDoc and QuantityOne (Bio-Rad Laboratories).
Internalization assay
For the analysis of receptor internalization, we adapted a biochemical assay (Fabbri et al., 1999) for NCCs. Cranial neural crest cultures were grown for 48 h, the neural tubes were removed, and the remaining crest cells were scraped off the coverslip (Bronner-Fraser, 1996). This procedure yields relatively pure NCC populations as determined by HNK-1 staining (Bronner-Fraser, 1986). The cells were spun, resuspended in neurobasal medium with supplements, and plated (equal number of cells per dish) as dissociated cells overnight or until the cultures reached 70% confluency. Cell surface receptors were labeled with biotin at 37°C for 0, 2, 5, 10, or 20 min. For the 0-min controls, cells were gently rinsed once with biotin. After biotinylation, the cells were placed on ice, rinsed gently, and treated with a noncell-permeant reducing agent (MesNa) to remove the biotin from the receptors that remained on the cell surface. After MesNa treatment, cells were lysed on ice and immunoprecipitations and analysis were then performed as detailed above.
Recycling assay
For the analysis of recycling we adapted an immunohistochemical assay based on that of Kamiguchi and Lemmon (2000). Cranial neural crest cultures were grown on high laminin concentrations for 72 h. Live cells were incubated with anti-integrin 6 Fab (0.5 µg/ml) for 30 min at 37°C to allow for optimal internalization of the integrin
6Fab complex. To avoid internalization induced by cross-linking caused with bivalent antibodies, Fab fragments are routinely used to follow receptor internalization and trafficking. Although it is possible these fragments may induce receptor internalization through an unknown mechanism, and that ligand binding could reverse this effect, it is unlikely. The cells were then cooled to RT to stop further endocytic trafficking and were incubated with unlabeled antimouse IgG (1:100; Jackson ImmunoResearch Laboratories) for 30 min at RT to block the Fab remaining at the cell surface. After gentle washing, the cells were then reincubated at 37°C for 20 or 90 min in neurobasal medium to allow for trafficking of endocytosed integrin
6Fab complexes. The cells were fixed with 2% PFA with 30% sucrose for 20 min at RT, and were incubated with an anti-actin antibody to reveal whether the cells had been permeabilized during fixation. Cells treated with 0.1% Triton X-100 (22686; USBiological) were used as permeabilization controls (Fig. 4 A, first column). Cells fixed but not permeabilized before the reincubation period were used as 0-min time points (Fig. 4 C). The recycled integrin
6 on the cell surface was then detected by visualizing the anti-integrin
6 Fab that had not been blocked with the unconjugated secondary. The unpermeabilized cells were incubated with Alexa-conjugated secondary antibodies (1:500; Molecular Probes, Inc.) for 1 h at RT.
To confirm that the detection of recycled receptors was not due to the loss of unconjugated secondary antibody over time, we performed the following control experiment. Cells were labeled live with anti-6 Fab, fixed, incubated with unconjugated secondary antibodies, allowed to sit for up to 2 h, and then incubated with labeled secondary antibodies. After 2 h we still did not detect any
6 staining, indicating that the unconjugated secondary antibody remained complexed with the Fab antibody over the time course of our assay. Images were acquired from all conditions of a single experiment on the same day and were collected with a confocal laser microscope as detailed below. Average intensity per cell area was determined using MetaMorph 5.0 (AG Heinz) software. In brief, cell outlines were traced from phase images and applied to corresponding fluorescent images, and average pixel intensity was determined as a function of cell area.
Immunohistochemistry and colocalization analysis
For the analysis of integrin and rab colocalization, neural crest cultures were grown for 48 h and then were fixed with 2% PFA (Sigma-Aldrich) for 1 h at RT. The cultures were then rinsed and incubated in blocking buffer (PBS with 0.1% Triton X-100, 1% BSA, and 5% normal goat serum) for 1 h at RT. Antibodies were diluted (1:100) in blocking buffer and added to the cells for 1 h at RT. After rinsing, Alexa-conjugated secondary antibodies (Molecular Probes, Inc.) were diluted (1:500) in blocking buffer and were added to the cells for 1 h at RT. Images were acquired with an Olympus IX70 confocal laser microscope (Fluoview 4.3) using an argon or HeNe laser (excitation lines, 488 and 543 nm) and a 60x Plan-APO (NA 1.15) water immersion objective. All images that were compared were acquired in the same session using the identical PMT, laser power, pinhole, gain, and offset settings. For the colocalization analysis, rab+ and integrin+ vesicles were selected independently using an intensity-based classifier to apply segmentation to the image (Volocity 2.5; Improvision). Anything smaller than 10 voxels was eliminated from the analysis. The number of rab+ vesicles that overlapped with integrin+ vesicles was divided by the total number of rab+ vesicles to determine the percentage of rab+ vesicles with integrin colocalization. XZ and YZ reslicing was performed to show the position of these vesicles along the apicalbasal axis of the cell.
BafA treatment
Cranial neural crest were cultured for 4872 h. The neurobasal media was replaced with Hepes-buffered neurobasal media for filming. Independent cells at the outer edge of the explant were videographed at 10x for 30 min to establish the pre-treatment velocity. Phase images were acquired (Metamorph 5.0) every 150 s. BafA (100 nM) or DMSO alone (vehicle) was then added, and after 15 min the post-treatment velocity was determined by filming the cells for an additional 60 min. Cell velocity was determined by tracking the position of the nucleus over time (Metamorph 5.0). Cumulative distribution graphs were generated by sorting each cell's velocity from highest to lowest speed, and determining the percentage of cells (in the same condition) that traveled slower than that cell. The speed of the cell was plotted against the percentage of cells traveling slower in order to show the velocity of each cell in relation to the whole population. Specific points in each graph are not directly comparable to corresponding points in other graphs, as each point merely reflects a cell's speed in relation to the whole population.
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Acknowledgments |
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This work was supported by a Basil O'Connor Award from the March of Dimes to M.L. Condic and by a National Institutes of Health grant (NIH-F31-NS43849-01) to L.R. Strachan.
Submitted: 4 May 2004
Accepted: 5 October 2004
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