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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 1 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 2 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 30m 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. 3 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 60-80m, 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 4 confocal stacks spanning the entire 30m 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 100m 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 5 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, 10l of solution containing the amount of inhibitor for a final dose of 25M 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 6 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). 7 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. 8 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"). 9 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 25M 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 (25M) promotes neurite sprouting in hippocampal neurons (1div) at a degree indistinguishable of that induced by the inhibitor ALLM (also 25M). 10 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 11 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 = 25m. 12 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 = 25m. 13 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 = 10m. 14 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). 15 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 = 25m. 16 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. 17 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 = 25m. 18 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. 19 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. 20 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. 21