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Supplementary Data
Neurite consolidation is an active process requiring constant repression
of protrusive activity
Ana Mingorance-Le Meur, and Timothy P. O'Connor
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I. Supplementary Materials and Methods
Construction of plasmids. Calpain-2 cDNA (OriGene) was subcloned into pEGFP-N1
(Clontech). cDNA encoding protease-dead mutant (Arthur et al., 1995; Nuzzi et al.,
2007) was generated by polymerase chain reaction (PCR) using site directed
mutagenesis. Cortactin cDNA was amplified from cortactin-RFP (Cao et al., 2003) vector
and HA and myc tags were added by PCR. HA-cortactin-myc was then cloned into the
pCIG vector (Megason and McMahon, 2002). HA-NTA contains the nucleotides 1-84 of
cortactin N-terminus and was cloned following the same protocol.
Antibodies. The primary antibodies used where: anti-Phospho-Erk, PKA substrate, fulllength fodrin, calpain-1and -2 (all rabbit, Cell Signaling Technologies); mouse anticalpain-2 (Abcam); rabbit anti GAP-43 and mouse anti PSA-NCAM (Chemicon); rabbit
anti PSD-95 and rabbit anti NCAM (Abcam); mouse anti-cortactin 4F11 (Upstate); goat
anti-Arp3 (Santa Cruz); mouse anti-III tubulin (Covance); mouse anti-HA and antiActin (Sigma); mouse anti-myc (ATCC CRL-1725). Rabbit anti-proteolyzed Fodrin
Ab37 was a gift of Dr. Siman (University of Pennsylvania). Alexa 488 and 546–
conjugated phalloidin and Alexa 488and 546–conjugated goat antibodies to mouse or
rabbit immunoglobulin G (IgG) were from Molecular Probes.
Pharmacological reagents
The following pharmacological reagents were bath-applied to cultures: 10-50 µM ALLM,
25 µM Calpeptin and 25 µM PD150606 (Calbiochem), which inhibit Calpain-1 and -2;
100-500 nM BAPTA-AM (Calbiochem), an intracellular Calcium chelator; 10-50 µM
PD-98059 (Calbiochem), which inhibits Erk-1/2; 50 µM 8Br-cAMP (Calbiochem), a
cAMP analogue; and 1 nM PKI (Calbiochem), a PKA inhibitor.
Western blot
Cell lines or primary cultures were lysed in a solution containing 50 mM Hepes (pH 7.8),
150 mM NaCl, 1% Triton X-100, 3 mM MgCl2, 150 mM NaCl, 1 mM EGTA, and 10%
glycerol, supplemented with protease and phosphatase inhibitors cocktails (Roche). After
incubation for 10 min on ice, each homogenate was centrifuged at 10,000 × g for 10 min
at 4°C. The cell and tissue extracts were then subjected to SDS-PAGE, and the separated
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proteins were transferred to a nitrocellulose membrane (Amersham Biosciences) and
probed with antibodies. Immune complexes were detected with ECL (Amersham
Biosciences).
Immunocytochemistry and image acquisition
Neurons were fixed with 4% paraformaldehyde and 4% sucrose in 0.1M phosphate buffer
for 30 min at room temperature. Fixed cells were incubated with primary antibodies in
the presence of 0.1% Triton X-100. Immune complexes were visualized with Alexa 488–
or Alexa 546–conjugated goat antibodies to mouse or rabbit IgG, and F-actin with Alexalabelled Phalloidin (Molecular Probes) as described (Mingorance-Le Meur et al., 2007).
In vivo images using calpain-2, PSD-95 and PSA-NCAM antibodies, as well as Figure
2A-B images, were obtained with an Olympus inverted confocal microscope IX81
controlled by FluoView 1000 (Olympus). For PSA-NCAM-positive neurons
morphological reconstruction, a series of stacks spanning the entire 30m of the tissue
section were acquired (1 image/m) and either projected, to generate Supplementary
Figure 5, or used to trace the morphology of every neuron using FluoView 1000.
Phalloidin images were visualized with a Nikon DIAPHOT 200, Kodak chip KAF 1400
CCD camera and MetaView Imaging System 3.6 (Universal Imaging Corporation), and
all the other images with a Zeiss Axioplan 2 Imaging microscope using a Retiga 1350EX
camera (Quantitative Imaging Corporation) with Northern Eclipse software (Empix
Imaging). Negative controls in which the secondary antibody or the fluorescent
phalloidin were omitted were included in every experiment.
Analysis and quantification of immunofluorescence
For immunofluorescence quantification, a region of interest (ROI) was used to measure
grey intensity within the cell, for example along a neurite, and also on the side to obtain
the background levels using ImageJ (NIH). Background values were then subtracted from
the experimental values.
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For quantification of GAP43-possitive terminals in the stratum lucidum of adult mice, we
selected ROIs of 650x650 pixels along this stratum and counted the number of GAP43
puncta per ROI as illustrated in the insert in F. Two to three ROIs per hippocampus were
selected (8 hippocampus -4 mice- per group) and combined to calculate the final average
number of GAP-43 positive fibers. For quantification of PSD-95 intensity in the CA3
field of adult mice, images were imported into ImageJ (NIH) and an outline of the PSD95-immunoreactive area was manually drawn to obtain the average intensity of
fluorescence. A portion of the stratum oriens (above the pyramidal layer) was also
quantified the same way and subtracted from the PSD-95 intensity measurement to
normalize for variations in overall intensity levels between tissue sections. One
measurement was taken from each hippocampus of each mice (2 measurements per mice,
4 mice per group) rendering a total of 8 data points per group.
Branching quantification
For the short term pharmacological experiments, 1 day in vitro neurons were treated with
DMSO or ALLM for 30 min, fixed, and stained with Phalloidin-Alexa-488 or -546.
Neurites with a regular length of 60-80m, which represented most of the population
(Supplementary Figure 3), were used for quantification. Protrusions were classified as
"branches" when a sub-branch or a growth cone was visible, or when longer than 10 m.
Less developed protrusions were classified as filopodia. For each neurite, the number of
filopodia and branches was noted and used to generate an average of each type of
protrusion for every data set. Six data sets, resulting of 3 independent experiments with
duplicates were used to generate the final average and standard error of the mean. For
statistical tests, all data was pooled (see below).
For 24 h transfection experiments, only thick protrusions ("branches") and fully
developed branches were quantified as described. For 1 h treatment of transfected
neurons with Calpain inhibitors, NT3, BDNF or netrin-1, only thick protrusions
("branches") were considered.
For the PSA-NCAM in vivo branching experiment, the morphology of young (PSANCAM-positive) neurons, containing the soma and dendritic tree, was traced using
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confocal stacks spanning the entire 30m of brain sections. By moving up or down the zstack, we could discriminate between several neurons with overlying neurites and trace
each one separately. All PSA-NCAM-positive neurons present in 2 brain slices per mice
(2 hippocampus per mice) that seemed entire were traced and considered for
quantification. Because neurons that were exposed to calpain inhibitors also displayed
greater dendritic length, we adjusted the branching number to branches per 100m in
Figure 3N.
Statistical methods
Data was analyzed using InStat3, from GraphPad Software (San Diego, CA). We used a
Kruskal-Wallis test with Dunn's multiple comparison post-test (nonparametric ANOVA)
to compare the mean number of filopodia or branches among data sets and for t-Boc
experiments. A nonparametric test was required because most datasets had non-normal
distributions. This is often the case of biological data sets where numbers are close to
zero, such as branches or filopodia, and therefore have a lower limit at zero but no upper
limit. Results are displayed as mean ± s.e.m using measurements from of 2-3 independent
experiments with duplicates. Significance was notated as * for p<0.05, ** for p<0.01, and
*** for p<0.001. The number of measurements has been included in every bar. For
comparison of only two groups, which included the number of GAP-43 puncta, PSD-95
fluorescence and PSA-NCAM branching, the Mann-Whitney test was chosen
(nonparametric equivalent to the t test). Significance and number of measurements were
notated as before.
Time-lapse fluorescence imaging
Live cell imaging was performed with an Olympus inverted microscope IX81 using a 40x
oil objective and 3x digital zoom. Images were acquired every 10 seconds and played
every 25msecs. The camera, shutters and lasers were all controlled by FluoView 1000
(Olympus). To maintain cell viability, the microscope was equipped with a chamber to
regulate temperature and CO2. Single frame images were visualized with the FV10-ASW
software (Olympus) and exported as individual images for Figure 3J. For Figure 5T-U, 3
frames (first, middle one and last of a 5-minute recording) were exported as red, green
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and blue images and then merged in Photoshop to generate the final display. In these
images, those regions that don't change their position overlap with one another and
generate white, while motile regions are seen in color (red, green or blue). ImageJ (NIH)
and FluoView 1000 (Olympus) were used to generate the supplementary videos.
As an internal control of neurite health, selected neurites with growth cones close by and
monitored the mobility of the growth cone, which was included in all our videos (shown
or not shown in the processed images), since growth cones are sensitive to alkalinization
of the media and not disrupted by calpain inhibitors. We also limited the time the cells
were incubated in the microcope chamber to 45-50 minutes. For the addition of the
inhibitors, 10l of solution containing the amount of inhibitor for a final dose of 25M
was applied though a whole in the tissue culture lid.
F-actin patch classification (Figure 5E)
For the classification of F-actin patches, 1 day in vitro neurons were treated with DMSO
or ALLM for 30 min, fixed as described, and labeled with a cortactin antibody followed
by an Alexa-488 tagged secondary, and Phalloidin-Alexa-546. For every experiment, the
first 50 random patches of F-actin along neurites were analyzed. For every patch, the
presence of protrusions (filopodia) and cortactin accumulation were noted. The
experiment was independently performed twice. Patches with protrusions but not visible
cortactin accumulation accounted for a very small percentage and are not represented in
the graph. The percentage of patches in each category in the final graph shows the
average of the 2 experiments ± standard error of the mean.
Calpain activity assay.
10 µM of BOC-LM-CMAC (Molecular Probes) was added to living neurons 10 minutes
prior to visualization of Calpain activity. For each sample, a pair of coverslips (control
and treatment) were used and imaged in parallel under UV light by inverting the
coverslips over a microscope slide. Exposure times were kept constant and at a medium
grey scale saturation to detect both increase and decrease in fluorescence. When imaged
at 10-15 minutes after addition of tBoc, the observed intensity was highly reproducible
between experiments and therefore the data were combined. After a number of pilot
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studies, we determined that measurements from neuronal somas, as opposed to neurites,
would be more appropriate for the following reasons. As tBoc is a soluble molecule, it
distributes uniformly within the cell, and therefore measurements taken from neurites
produced very similar results to those taken from the cell soma. In this situation a
compartment of less variable size (or width), as in the case of the soma, is more desirable
than neurite because it minimizes the differences in fluorescence due to changes in
neurite thickness. In addition, the tBoc fluorescence faded very quickly when imaged,
thus at the higher magnification and precise focus required to image neurites the accuracy
of the data was unreliable, while at low magnification images of full cells could be taken
with minimum exposure time. Last, because the fluorescence observed is the
accumulation of proteolyzed tBoc produced over a 10-15 minutes time throughout the
entire cell, this reporter technique is not very accurate and high definition data cannot be
expected. We therefore interpret the data as "activation" or "inhibition" of calpain, but
not, for example, as a 33% reduction of calpain activity. Thus, we measured the grey
level of several neurons per coverslip using ImageJ as described above for
immunofluorescence (the final number is indicated in each bar graph). Because the
luminosity of the background varied slightly between coverslips, background
measurements were also taken for each coverslip. After subtracting background levels,
we referred every data point of the "control" and "treatment" coverslips to the average
value of the control as percentage of change (with respect to control).
When PKA or Erk inhibitors were included, a set of 12-well plates was treated in parallel
with the same inhibitors and/or NT3 and P-Erk and PKA phosphorylated substrate levels
analyzed by western blot to confirm inhibitor efficiency. Chelation of Calcium with
BAPTA and PD150606 efficiency was assessed in coverslips treated with Semaphorin
5B (To et al., 2007).
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II. Supplementary Figures
Supplementary Figure 1. Calpain is expressed in growing axons in vivo.
Immunohistochemistry of calpain-2 in mouse cortex at days 15 (E15) and 18 (E18) of
embryonic development. Calpain staining is faint at E15 (A) and concentrates in fibrelike structures that populate the intermediate zone (arrows in A and insert). This cortical
layer contains the axons of the cortical neurons situated in the cortical plate. (B) At E18,
calpain staining is stronger and clearly localizes to the axon tracts in the intermediate
zone (arrows). Double color images show calpain in red and the nuclear label Hoescht.
Abbreviations: CP: cortical plate, IZ: intermediate zone, VZ: ventricular zone.
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Supplementary Figure 2. Neurites display two kinds of protrusions by 24 h in vitro.
Representative images of neurites used in Figure 1. Hippocampal neurons were cultured
for 24 hours and stained for F-actin. Only neurites with a regular neurite length were used
for quantification in pharmacological treatment experiments. At this time, two kinds of
protrusions were differentiable and were counted separately. Branched protrusions or
those with growth cones were counted as “branches” (noted as “b”), while the rest were
considered to be “filopodia” ("f").
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Supplementary Figure 3. Validation of Calpeptin as a pharmacological inhibitor of
neuronal calpains. (A-B) t-Boc raw imaging (A) and quantification of fluorescence (B)
of hippocampal neurons treated with DMSO or 25M of Calpeptin for 30 minutes. After
loading with t-Boc for 10 minutes, the fluorescence of Calpeptin-treated neurons is much
lower than that of the control cells (A-B), indicating calpain activity has been reduced.
(C) 1 hour treatment with Calpeptin (25M) promotes neurite sprouting in hippocampal
neurons (1div) at a degree indistinguishable of that induced by the inhibitor ALLM (also
25M).
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Supplementary Figure 4. Inhibition of calpain promotes neurite plasticity of
hippocampal neurons in vivo. (A) Schematic showing the treatment time-line. Eight
mice received 2 injections of salinum or calpain inhibitor, one per day, followed by 2
days without treatment before sacrifice. These mice (4 mice per group) were used for
innmunohistochemistry. Four additional mice (2 per group) were sacrificed 90 minutes
after a single injection and their hippocampus were immediately removed and lysed for
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western blot analysis. (B-C) Western blot analysis of a control (salinum) and a calpain
inhibitor treated mice showing that calpain inhibition does not affect calpain levels (top)
but reduces calpain activity, as evidenced by decreased proteolyzed fodrin (smear below
the big band) and increased cortactin levels (B). An in crease in the plasticity-related
polysialylated form of NCAM (PSA-NCAM), but not in total NCAM, is also observed at
this time (C). (D-E) Calpain-2 is expressed in several neuron populations in the adult
hippocampus, such as the granule cells of the dentate gyrus (D, arrows point to calpain-2
expressing neurons) and the pyramidal neurons of the CA3 field gyrus (E, arrows point to
calpain-2 expressing neurons). Calpain-2 is also detected in the axons that cross the
stratum lucidum (arrowheads in E). (F-G) The same calpain-2 expressing terminals are
labeled with the sprouting marker GAP-43 (SL layer). In mice that received two
injections of calpain inhibitor, the number of these terminals is significantly increased in
the stratum lucidum of the hippocampus (an example of Region Of Interest indicated in
F, quantification in G). (H-I) The synaptic marker PSD-95 is also increased in the
hippocampal dendrites of treated animals (H and quantification in I), indication plastic
reorganiztion. Results displayed as mean ± s.e.m. Asterisks denote statistical significance
between groups (Mann-Whitney test, * p < 0.05, *** p< 0.001). Abbreviations: GCL:
granule cell layer, ML: molecular layer, SL: stratum lucidum, SO: stratum oriens, SP:
stratum pyramidale, SR: stratum radiatum. Scale bars = 25m.
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Supplementary Figure 5. Inhibition of calpain promotes neurite sprouting of
hippocampal neurons in vivo. Examples of PSA-NCAM-positive cells in the
subgranular zone of the dentate gyrus in control and calpain inhibitor treated mice.
Images correspond to different mice. PSA-NCAM-positive cells are young granular
neurons that are in the process of growing their neurites and were therefore exposed to
inhibition of calpain during this period of neurite outgrowth. A greater density of
processes (due to a higher number) is observed in all animals treated with calpain
inhibitors and is quantified in Figure 3K-N. Abbreviations: GCL: granule cell layer, H:
hilus, ML: molecular layer. Scale bar = 25m.
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Supplementary Figure 6. Arp3 expession in neurons and collocalization with
Cortactin. Immunostaining of Arp3, a component of the Arp2/3 complex, in cultured
hippocampal neurons, showing Arp3 only localizes to those regions of F-actin
accumulation, where it also collocalizes with cortactin (B). Scale bars = 10m.
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Supplementary Figure 7. Identity of cortactin bands labeled by the cortactin
antibody. (A) Western analysis showing cortactin antibody specificity in 293 cells
transfected with HA-cortactin-myc (+) and control cells (-). Endogenous cortactin is
already expressed by this cell line (Ctn band). The cortactin antibody (left) produces a
non-specific low molecular weight band (asterisk), that is not recognized by anti-HA
(center) or myc (right) antibodies and that is not affected by treatment with calpain
inhibitors (not shown). (B) Complete western blot of Figure 5C showing cortactin levels
after treatment with different doses of ALLM. While full length cortactin levels are
significantly increased in a dose-response manner, the non-specific band (asterisk) is not
affected and behaves as a loading control. Cortactin proteolytic fragments are not always
observed and therefore were omitted from figures, but could be detected in this blot as
well as after incubating the membrane with cortactin antibodies a second time and reexposing. Cortactin fragments do show a regulation complementary to the full length
form and have higher molecular weight than the non-specific band.(C) Western blot
showing full-length cortactin and one detectable fragment (after re-incubation and reexposure) in neurons treated with ALLM for 5 minutes, 30 minutes or 5 minutes
followed by a wash out (and lysed after 25 minutes to be comparable to the 30 minutes
treatment). Cortactin levels are increased as fast as 5 minutes after inhibition of calpain
(also visible in the reduction of the fragment levels) and remain increased for 30 minutes.
If the inhibitor is washed out after the initial 5 minutes, proteolysis of cortactin is
restored. The experiment was done in parallel to that of Figure 3H (main text).
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Supplementary Figure 8. Morphology of neurons that overexpress cortactin or the
dominant negative of cortactin, NTA. Additional images of hippocampal neurons
transfected with the yellow fluorescent protein YFP (A-B), cortactin-RFP fusion protein
(C-D), or NTA cloned in an IRES vector that also expresses GFP (E-F). Transfected cells
were imaged taking advantage of the fluorescent proteins they expressed, without using
any immunostaining and are shown at two magnification levels (B, D and F are higher
magnification). While NTA-transfected neurons have the same number of branches as
control neurons (Figure 5 main text), they tend to be longer (E-F). Overexpression of
cortactin induces a very characteristic overbranched and spiny morphology (C-D). Scale
bars = 25m.
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Supplementary Figure 9. Netrin-1 treatment increases cortactin levels in neurons.
Western blot showing cortactin levels after treatment with 200ng/ml of mouse netrin-1.
As a positive control, we detected a transient increase in phospho-Erk in response to
Netrin, consistent with previous reports. In the same cultures, full-length cortactin levels
were elevated matching the regulation of cortactin by NT3 (Figure 7). Loading control
(asterisk) corresponds to cortactin antibody non-specific band.
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Supplementary Figure 10. Morphology of transfected neurons exposed to branching
factors. Raw images of hippocampal neurons transfected with the yellow fluorescent
protein YFP, Cpn-2-GFP, pCIG or HA-NTA, one hour after treatment with neurotrophins
or control (phosphate buffer). Images correspond to the experiments shown in Figure 7IJ. Scale bar = 25m.
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Supplementary Figure 11. Analogy between cell polarity and neurite consolidation.
Polarized epithelial cells (left) have two functionally and structurally distinct surface
domains, termed apical (green) and basolateral, that are determined by the interplay
between membrane and cytoskeleton (Fais et al., 2000). Directed cell migration (middle)
also involves the establishment of polar structures, the leading edge (green) and the
trailing edge, with membrane protrusions restricted to the former (Ridley et al., 2003).
(Right) Comparable domains are also apparent in neurons, were actin polymerization
drives membrane protrusion at the growth cone (green). We propose that consolidated
areas can be considered equivalent to the trailing edge, while deconsolidated areas
(growth cones) are analogous to the leading edge. Because the cytoskeleton components
involved in critical steps of both processes are shared, the signaling pathways that govern
cell polarity and neurite consolidation are also likely to be similar.
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III. Supplementary Videos (legends)
Supplementary Movie 1. Calpain inhibition induces neurite sprouting. Time lapse of
a pDsRed transfected neuron during the 5 minutes prior and 40 minutes after addition of
the calpain inhibitor Calpeptin (1 frame/10 seconds). The movie shows the portion of an
axon of a 5div hippocampal neuron and its reaction to the inhibitor (added when the
image jumps). Following addition of Calpeptin, a great number of filopodia start growing
from the previously non protrusive neurite and immediately retract. Later in the movie,
some filopodia start growing at a slower rate, covering more distance and remaining for
longer time. This pattern of early transient protrusions followed later by more stable
protrusions supports the results with fixed cells shown in Figure 3 H-I.
Supplementary Movie 2. Phenotype of control neurons. Time lapse of a pDsRed
transfected neuron imaged for 5 minutes (1 frame/10 seconds, repeated 4 times). The
movie shows the mobility of a region containing several neurites (likely to be part of the
axon) of a 5div hippocampal neuron. A few filopoda emerge during the 5 minutes of
recording and are quickly retracted.
Supplementary Movie 3. Phenotype of cortactin overexpressing neurons. Time lapse
of a cortactin-RFP transfected neuron imaged for 5 minutes (1 frame/10 seconds,
repeated 4 times). The movie shows the mobility of a region containing several neurites
(likely to be part of the axon) of a 5div hippocampal neuron similar to that shown in
Video 2. The neurites are covered by numerous protrusions from the beginning of the
video and these all show great activity during the entire imaging period.
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IV. Supplementary references
Arthur, J.S., Gauthier, S. and Elce, J.S. (1995) Active site residues in m-calpain:
identification by site-directed mutagenesis. FEBS Lett, 368, 397-400.
Cao, H., Orth, J.D., Chen, J., Weller, S.G., Heuser, J.E. and McNiven, M.A. (2003)
Cortactin is a component of clathrin-coated pits and participates in receptormediated endocytosis. Mol Cell Biol, 23, 2162-2170.
Fais, S., Luciani, F., Logozzi, M., Parlato, S. and Lozupone, F. (2000) Linkage between
cell membrane proteins and actin-based cytoskeleton: the cytoskeletal-driven
cellular functions. Histol Histopathol, 15, 539-549.
Megason, S.G. and McMahon, A.P. (2002) A mitogen gradient of dorsal midline Wnts
organizes growth in the CNS. Development, 129, 2087-2098.
Mingorance-Le Meur, A., Zheng, B., Soriano, E. and del Rio, J.A. (2007) Involvement of
the myelin-associated inhibitor Nogo-A in early cortical development and
neuronal maturation. Cereb Cortex, 17, 2375-2386.
Nuzzi, P.A., Senetar, M.A. and Huttenlocher, A. (2007) Asymmetric localization of
calpain 2 during neutrophil chemotaxis. Mol Biol Cell, 18, 795-805.
Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G.,
Parsons, J.T. and Horwitz, A.R. (2003) Cell migration: integrating signals from
front to back. Science, 302, 1704-1709.
To, K.C., Church, J. and O'Connor T, P. (2007) Combined activation of calpain and
calcineurin during ligand-induced growth cone collapse. Mol Cell Neurosci.
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