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Successful axon targeting during development is critically dependent on directionality of axon extension, and requires coordination between the extrinsic cues that provide spatial information to the axon and the intrinsic responses that regulate structural specification of the axon during neuronal polarization. How these responses are coordinated is unclear, but are known to involve aligning the centrosome with the base of the emerging axon. We have used a novel in vitro micropatterning assay that spatially segregates the extrinsic cues used by polarizing cerebellar granule cells to orient axon extension, and used it to investigate the signaling mechanisms responsible for coordinating centrosome positioning with intrinsic responses. The results show that when laminin and/or vitronectin are used as spatially restricted cues in association with substrate-associated sonic hedgehog they are sufficient to induce cell cycle arrest; that laminin and vitronectin then induce integrin-mediated signaling that upregulates PI3 kinase and PKC function to produce PIP3 that is associated with the centrosome; that this PIP3 can interact with PKC-phosphorylated Growth-associated protein GAP-43 and that PKC phosphorylated GAP-43 in turn is required for positioning Par6, Cdc42 and IQGAP1, all intrinsic response components, in proximity to the centrosome, such that, in the absence of GAP-43, they are mislocalized and microtubules are not oriented appropriately. We conclude from these results that GAP-43 plays an important role in coordinating extrinsic signaling and intrinsic responses in polarizing cerebellar granule neurons.
Development of cerebellar granule neurons has been well characterized and their in vitro accessibility makes them an ideal system in which to study regulation of neuronal polarization (Powell et al., 1997). Previous studies have shown the important role played by the centrosome: When granule cells exit the cell cycle, the centrosome is positioned at the pole from which the axon will emerge together with signaling molecules important in establishing the structural polarity of the axon such as Cell division cycle 42 (Cdc42) and the partitioning defective (Par) complex (Zmuda and Rivas, 1998; Higginbotham et al., 2006). This positioning and subsequent emergence of the leading process dictates the direction of axon extension, which is key for correct target identification (Solecki et al., 2004). Previous studies using hippocampal cells in culture have shown an association between the position of the centrosome as polarization is initiated and the orientation of the spindle during the last mitotic division leading to the hypothesis that neuronal polarity is driven intrinsically via microtubule dynamics (de Anda et al., 2005; Calderon de Anda et al., 2008). While this may be true in a simple in vitro situation in which there are no extrinsic cues to dictate axon extension, it cannot be true in vivo where the direction of axon extension is critical for correct target identification. However, the underlying mechanisms that coordinate responsivity to spatially localized extrinsic cues that dictate where the axon will be extended with the intrinsic molecules that regulate the cytoskeletal response are not well understood. Evidence suggests that actin regulation is also involved: key components of the polarity signaling complex that are responsive to extrinsic cues i.e. Cdc42 and Par3/Par6 are linked to actin rather than tubulin polymerization, and when they indirectly respond to spatially localized extrinsic cues, such as during directional outgrowth of the growth cone, they cause localized actin assembly that is then followed by microtubule stabilization (Luo et al., 1997; Zmuda and Rivas, 2000; Alberts et al., 2006; Chen et al., 2006b). The results suggest that other as yet unidentified proteins that are directly responsive to spatially localized extrinsic cues are required to coordinate actin and tubulin regulation in the polarization complex with the membrane. Like Cdc42 and Par6, the membrane-associated Growth Associated Protein – 43 (GAP-43) is also found in the growth cone where it is crucial for remodeling the actin cytoskeleton in response to extracellular signals (Dent and Meiri, 1998). We previously reported the novel finding that, like Cdc42 and Par6, (GAP-43) is tightly associated with the centrosome of cerebellar granule cells, and that in its absence abnormally positioned centrosomes are associated with disruptions to both actin and tubulin polymerization (Mishra et al., 2008). We have developed a micropatterning assay able to spatially deliver extrinsic cues to granule cells to ask whether it regulates positioning of the centrosome and how this is coordinated with intrinsic responses. We further investigated the underlying mechanism, particularly the role of GAP-43 as a coordinator between extrinsically-mediated signaling and intrinsically organized responses via microtubule organization.
GAP-43 deficient mice were generated from targeted CJ7 ES cells in isogenic 129S3/imJ mice (genetic designation +Mgf-SIJ, JAX stock number 002448) and backcrossed for 8–12 generations with C57 BL/66N. Homozygotes (−/−) mice were identified by PCR genotyping as described previously (Maier et al., 1999). Mice were maintained in specific pathogen-free conditions according to NIH and Institutional Animal Ethical Committee-IAEC (NBRC) guidelines. The morning when the vaginal plug was detected was defined as embryonic day E-0.5 and the day of birth as postnatal day P0.
Shh, Vitronectin and Laminin were from R&D (Minneapolis, MN). The laminin receptor blocking antibody α6β1 was from Abcam (Cambridge, MA) and the vitronectin receptor blocking antibody, anti-integrin α5β3 was from Chemicon-Millipore (Billerica, MA). The anti-integrin α5β3 anti-Shh blocking antibodies were from Sigma Aldrich (St Louis, MO). Effectene transfection reagent was from Qiagen (Valencia, MD). All media and supplements were from Gibco Invitrogen (Carisbad, CA). Other chemicals were from Sigma Aldrich (St Louis, MO). For immunostaining, anti-Shh, anti-Tau, anti-AKT, anti-pAKT, anti-PI3K, anti-tubulin, anti-IQGAP and anti-vitronectin antibodies were from Santa Cruz (Santa Cruz, CA), anti-Cdc42, anti-patched, anti-smoothened, anti-MAP2 and anti-Par6 antibodies from Abcam (Cambridge, MA), anti-laminin, anti-BrdU and anti γ-tubulin were from Sigma Aldrich (St Louis, MO) and anti-PIP2, anti-PIP3 were from Echelon (Salt Lake City, UT). Antibodies for GAP-43 and pGAP-43 were from the Meiri lab. All secondary antibodies and Vectashield mounting media with DAPI was from Vector Laboratories (Burlingame, CA).
Lithographically fabricated Silicon masters were generously provided by Prof. David Juncker, McGill University, Montreal, Canada. The stamps were made from Sylgard 184 using SU-88 as a photoresist. (Dow Corning Inc., Midland, TX). Proteins were printed on PDL coated cover slips at 10 μg/ml alone or in combination (Mishra et al., 2008).
Dissociated enriched granule cell cultures were established from P0-WT, P0-KO and P8-WT mouse cerebellum as previously described (Gao et al., 1991). In brief, individual cerebella from P0 and P8 mice were dissected in calcium free CMF-Tyrode and then dissociated by gentle trituration with a fire polished glass pipet. Cultures were enriched for granule cell progenitors by removing glial contamination (Kenney et al., 2003) and plated onto poly-D-lysine (500 μg/ml) coated cover slips in serum (10%) containing media for 24 hours to allow the cells to recover. After 24 hours the media was replaced with serum free media containing DMEM/F12 with N2 and B27 supplement (Invitrogen) with antibiotics. Cells were fixed with 4% paraformaldehyde in microtubule stablization buffer (Zmuda and Rivas, 1998) after another 24 hrs of culture. When anti-vitronectin receptor blocking antibody α5β3, anti-laminin receptor blocking antibody α6β1, Shh blocking antibody (10μl/ml), cyclopamine (5nM), Bisindolylmaliemide (500nM) wortmannin (200nM) (Sigma-Aldrich), PIK 90 (1μM) (Axon Medchem), Go6983 (1μM) (Calbiochem) were added, they were present throughout. For proliferation studies, cells were incubated with 10μM BrDU for 12 hours in serum-containing media.
Cerebella from P8 C57/BL6 mice were removed, kept cold in HBSS solution (Hank's Banck Salt Solution) and then sectioned into 200μm thick slices using a Mclwain Tissue Chopper-800 (Vibrotome-0800) (Gogolla et al., 2006a). The slices were cultured in Nunc 6 well plates with Millicell inserts (0.4 μM, Millipore-PICM03050) in 50% Basal Medium Eagle (BME), 25% HBSS, 25% Horse Serum, 1mM L-Glutamine, 5mg/ml Glucose and 1mM PenStrep (Gogolla et al., 2006b). 200 μL of media was replaced with the fresh media every second day. The slices were fixed after 3 days and processed for immunostaining. When anti-vitronectin, laminin or Shh antibodies (10μl/ml of media) or cyclopamine (5nM), were added, they were present throughout.
Antibodies used were anti-Shh (1:200), -laminin (1:100), -vitronectin (1:100), -γ tubulin (1:400), -par6 (1:100), -cdc42 (1:50), -GAP-43 (1:500), -pGAP-43 (1:500), -patched (1:250), -smoothened (1:250), -α6β1 (1:50), -α5β3 (1:80), -PIP2 (1:100), -PIP3 (1:100), -PI3K (1:100), -MAP2 (1:500), -Tau (1:100) and - IQGAP (1:100). To detect BrdU immunoreactivity, cells were treated with 4N HCl for 4 minutes to denature the DNA followed by sodium borate neutralization (0.1M, pH8.5).
Immunostaining of cerebellar slices was performed according to published protocol (Gogolla et al., 2006b). Slices were fixed in 4% paraformaldehyde (PFA) following which they were incubated with 20% methanol/PBS solution for 5 min. 1% Triton X-100 solution for 12 hrs at 4 °C was used to permeablize the slices. Slices were incubated with primary antibody overnight at 4 °C. Incubation with secondary antibody was done at room temperature for 3 hours.
Enriched granule cell cultures were plated on microcontact printed cover slips, fixed and immunostained to detect printed protein and the centrosome. As depicted in Figure 2A, cells touching the microcontact pattern were designated CONTACT cells, whereas cells not touching the pattern were designated NON-CONTACT cells. Only single cells were included in the analysis. To assess the position of the centrosome relative to the microcontact pattern, a line parallel to each arm of the pattern and passing through the middle of the cells was drawn, and the position of the centrosome with respect to the parallel midline analyzed. Centrosomes positioned in the proximal half of the cell touching the pattern were classified as oriented towards the pattern while those positioned in the distal half of the cell were classified as not oriented towards the pattern. For each field the position of all contact and non-contact cells were analyzed. The total number of cells counted for all combinations of microcontact printed proteins is summarized in Table-1.
Confocal images of slices were taken with a Zeiss LSM 510 Meta microscope and off line analysis was performed using Image J software. From each image three random areas of 625 μm2 were identified and the orientation of centrosome in each was analyzed for each of the cells and classified in two types as follows: Type I cells, in which the centrosome was positioned at the base of the leading process; Type II cells, in which the centrosome was not positioned at the base of the leading process.
Cells were plated in 60 mm dishes at a density of 2 × 106 cells/cm2 and collected into lysis buffer [50 Mm Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 2% SDS, and protease inhibitor mixture] after 48 hours. Western Blotting was performed according to Sharma et al (Sharma et al., 2003). Protein levels were quantified using Chemi Genius2 software (SynGene) to obtain integrated density values (IDV).
GAP-43 was purified from P0 rat brains using reverse phase HPLC (Changelian et al., 1990) and used to probe PIP strips (Echelon Biosciences, Salt lake City UT) according to manufacturer's instructions. Bound total GAP-43 was detected with the 7B10 mab, and bound PKC-phosphorylated GAP-43 was detected with the 2G12 mab. Immunoreactivity was revealed by chemiluminescence, and the film exposed within the linear range of the signal. The experiment was repeated three times using different preparations of purified GAP-43 and results quantitated by densitometry, as above.
The mU6 promoter & GAP-43 ShRNA constructs were cloned into the EGFP-SV40pCMV vector and verified by PCR & sequencing (Mishra et al., 2008). The plasmid vectors pCMV-EGFP (mock) and pCMV-ShRNA plasmid DNA constructs were used for transfection of granule cells after 24 of culture using 0.5 μg of plasmid DNA, 4μl of enhancer (R) and 10μl of Effectene reagent according to the manufacturer's directions. Media was changed after 6 hours of incubation, and cells were fixed with 4% paraformaldehyde after 48 hours of transfection.
The data are expressed as mean ± s.e.m. Data were analyzed with one-way ANOVA (GraphPad Prism version 5 for Windows, GraphPad Software, San Diego, CA). When ANOVA showed significant differences among multiple experimental groups, multiple comparisons between experimental groups were performed using Dunnett's or Bonferroni's post hoc test.
Laminin and vitronectin were differentially localized in the developing cerebellum in vivo at both P0 and P8. Figure 1 shows that at P0 high levels of laminin were found in the pia and the area that will become the Purkinje cell layer (PcL). Conversely levels of vitronectin were lower in the PcL, and strong expression was confined to the deeper layers of the cerebellum. By P8 laminin immunoreactivity extended into the developing molecular layer and the deeper layers of the cerebellum, while levels in the outer external granule layer (oEGL) remained low. Vitronectin was also expressed in the PcL and its arborization in the molecular layer, but was more strongly upregulated in the EGL and in deeper layers of the cerebellum including the inner granule layer (IGL) compared with laminin. In contrast the integrin receptors for laminin and vitronectin (α6β1 and α5β3) were more diffusely localized at both P0 and P8 (data not shown). The results show that laminin and vitronectin are differentially localized in the developing cerebellum in vivo, consistent with a role in influencing polarization of granule cell precursors as they initiate migration from the EGL through the molecular layer.
To assess whether the differential distribution of laminin and/or vitronectin reflects roles as a spatially restricted extrinsic cue to regulate positioning of centrosomes, we developed an in vitro micropatterning assay in which cultures enriched in granule cell precursors were plated onto coverslips on which patterns of either vitronectin or laminin ± sonic hedgehog (Shh) had been laid down by microcontact printing, and centrosome positioning with respect to the printed protein pattern was evaluated. To ensure that equivalent amount of Shh and the ECM molecules where present in micropatterns containing more than one printed the micropatterns were immunostained for the presence of both constituents (supplementary figure S1). Figure 2A depicts a schematic of how centrosome orientation was analyzed. (Details are provided in Methods and details of the number of cells included in the analysis are summarized in Supplementary Table T1). The central red ‘L’ shape represents one printed protein pattern. Cell labeled (a), (b) and (c) are in contact with the pattern, but whereas the centrosome in cell (a) is positioned towards the pattern, centrosomes in cells (b) and (c) are not. Cells labeled (d) and (e) are not in contact with the pattern, but whereas the centrosome in cell (d) is positioned towards the pattern, the centrosome in cell (e) is not. Figure 2B shows a representative image of centrosome position, detected with anti-γ tubulin immunoreactivity, when granule cell precursors were plated on a vitronectin + Shh patterned coverslip. The arrowhead indicates a contact cell in which the centrosome is positioned towards the pattern. Figure 2C shows that the centrosomes in granule cell precursors that contacted a pattern due to Shh alone were not positioned toward the pattern, whereas centrosomes in cells contacting a pattern due to either laminin or vitronectin, were positioned towards the protein patterns, whether or not Shh was present. The results indicate that centrosome position is affected by the presence of laminin and vitronectin, not by Shh.
Developing cerebellar granule cell precursors proliferate under the regulation of Shh, and previous results have shown that while vitronectin can inhibit Shh-mediated mitogenicity, thereby inducing differentiation, laminin substrates potentiate mitogenicity of soluble Shh . Since Shh is thought to interact with the ECM directly (Pons and Marti, 2000; Pons et al., 2001), we used our micropatterning assay to examine how incorporating Shh into laminin and vitronectin micropatterns affected its mitogenic properties. Figures 2D and 2E show that Shh incorporated into a micropattern could still induce proliferation of P0 granule cell, as detected with 5-bromo-2-deoxyuridine (BrDU) labeling. However, when Shh was combined with either laminin or vitronectin the number of contact cells labeled with BrdU was significantly reduced compared with cells contacting Shh alone, or cells contacting poly-D lysine (PDL) control substrate (the NC lanes in Figure 2E). The results, quantitated in Figure 2E, show that under these conditions, laminin as well as vitronectin inhibits the mitogenic activity of substrate-associated Shh. Hence both laminin and vitronectin enhanced the differentiation of granule cell precursors in our micropatterning assay.
We used P8 cerebellar slice cultures as a model that most closely mimics in vivo development, to investigate whether laminin and vitronectin act as extrinsic cues to position the centrosome at the base of the leading process as migration is initiated. We incubated the slices for 48 hours with antibodies having blocking activity against laminin, vitronectin or Shh, or with the pharmacological Shh inhibitor cyclopamine, and then used anti-γ tubulin immunoreactivity to detect the location of the centrosome with respect to the leading process as before (Figure 3). In control slices, 70% of the centrosomes were aligned with the base of the leading process, whereas significantly fewer centrosomes remained aligned after the slices had been treated with either laminin or vitronectin antibodies. In contrast, the percentage of centrosomes aligned with the leading process significantly increased after the slices had been treated with either Shh-blocking antibodies or cyclopamine. The results are therefore consistent with the data from the micropatterning assay and confirm that it provides an appropriate strategy with which to examine laminin and vitronectin function in centrosome positioning during polarization.
Laminin and vitronectin signal directly through the α6β1 and α5β3 integrin receptors respectively, whereas Shh utilizes the patched receptor (Marti and Bovolenta, 2002). We therefore used cognate integrin blocking antibodies to assess whether they inhibited centrosome positioning in the micropatterning assay. In the presence of antibody that recognizes the laminin integrin receptor α6β1, the proportion of contact cells with centrosomes positioned towards the laminin pattern was significantly reduced (0.48 ± 0.03 compared to 0.82 ± 0.02; p< 0.001 by unpaired t-test). In the presence of antibody that recognizes the vitronectin integrin receptor α5β3, the proportion of contact cells with centrosomes positioned towards the vitronectin pattern was likewise significantly reduced (0.48 ± 0.04 as compared to 0.67 ± 0.01; p< 0.01 by unpaired t-test). In addition Figure 4 shows that when the α6β1 integrin was blocked in the presence of vitronectin, there was no effect on centrosome orientation, indicating that the interaction of the receptor with its ligand was specific. Both laminin and vitronectin can modulate Shh signaling, however neither Shh inhibitory antibodies nor cyclopamine were able to inhibit the positioning response to vitronectin, indicating that centrosome positioning requires integrin, not Shh, signaling.
Activation of integrin receptors following ligand binding results in upregulation of PI3K. To determine whether laminin and vitronectin induce PI3K activation we plated granule cells on either laminin, vitronectin or PDL for 48 hours and then monitored PI3K activation on Western blots. Figure 5 shows that cells plated on laminin (Figure 5A) or vitronectin (Figure 5B) had increased levels of PI3K compared with cells plated on PDL alone. Moreover, phosphorylated AKT (pAKT) a downstream target of PI3K was also upregulated on both laminin (Figure 5A, panel c) and vitronectin (Figure 5B panel c). Upregulation of both PI3K and pAKT was inhibited in the presence of blocking antibodies against the laminin and vitronectin integrin receptors α6β1 or α5β3. We next used the PI3K inhibitor wortmannin to determine whether centrosome positioning by laminin and vitronectin require upregulation of PI3K. Figure 5C shows that wortmannin inhibited centrosome positioning in response to both laminin and vitronectin. To confirm the involvement of the PI3K a second PI3K inhibitor (PIK 90) was also used to inhibit centrosome orientation (supplementary figure S2). In further support of the role of PI3K in centrosome orientation, figure 5D shows that PI3K is concentrated at the region of cell contact with the extrinsic cue on either a vitronectin or laminin pattern, whereas it is not concentrated at the region in contact with a Shh micropattern alone. Moreover, in agreement with the foregoing results that Shh does not inhibit the activity of vitronectin, PI3K remained colocalized at the contact point on a micropattern containing both Shh + vitronectin. Activation of PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) and so we also investigated whether PIP3 is located at the region of cell contact with the extrinsic cue. Figure 5E shows that PIP3 was concentrated at the region of contact on the Shh + vitronectin as was shown for PI3K. Taken together these results indicate that centrosome orientation requires activation of the PI3K signaling pathway and show that PI3K activation is associated with PIP3 localization where the centrosome is positioned.
In contrast with PIP2 that can modulate actin dynamics directly, PIP3 modulates actin dynamics via its interaction with actin regulatory proteins. One actin modulatory protein that is associated with centrosomes is the growth-associated protein GAP-43. PKC phosphorylated GAP-43 (pGAP-43) is highly enriched in the EGL along with laminin and vitronectin, and we have previously shown that when it is genetically deleted, centrosome positioning in mitosing granule cell precursors is abnormal both in vitro and in vivo (Mishra et al., 2008). Figure 6A shows that in polarizing precursors too, pGAP-43 is colocalized with centrosomes at the base of primary process. We therefore used the micropatterning assay to determine how extrinsic cues affect the distribution of pGAP-43. Figure 6B shows that pGAP-43 was enriched at the region of contact with either a laminin or vitronectin ± Shh micropattern, but not with a Shh micropattern alone. Previous studies have shown that PKC-mediated GAP-43 is phosphorylated can occur in culture under basal conditions (Dent and Meiri, 1992). The increase in specific activity of GAP-43 phosphorylation in response to laminin or vitronectin, could be prevented by blocking cognate integrin receptors with specific receptor antibodies (Figure 6C, D) or following treatment with bisindolylmaleimide or the PKC inhibitor Gö 6983 to inhibit PKC (Figure 6E and supplementary figure S3). Taken together with Figure 6B the PKC dependent increase in GAP-43 phosphorylation in response to laminin or vitronectin seems to occur at the region of contact. Inhibiting PKC by either bisindolylmaleimide or Gö 6983 also prevented centrosome positioning in response to either laminin or vitronectin (Figure 6 C – F and supplementary figure S2). The results indicate PKC activation is required for laminin and vitronectin to be able to position the centrosome at the base of the primary process, and that this PKC activation in turn causes phosphorylation of GAP-43 in the region where contact occurs.
Figure 7 shows that pGAP-43 in the region of the centrosome colocalizes with PIP3. Moreover, direct binding assays utilizing purified GAP-43 and phospholipids demonstrated that this colocalization is due to a direct interaction with PIP3. Comparing GAP-43 binding using the pGAP-43-specific antibody (2G12) demonstrated that the GAP-43 bound to PIP3 is largely the PKC-phosphorylated form. GAP-43 has previously been shown to interact with PIP2 (Laux et al., 2000), however the specific isoforms of PIP2 had not been investigated. The phospholipid binding assay also revealed that pGAP-43 bound selectively to PIP2(3,4) and PIP2(3,5) whereas GAP-43 bound to PIP2(4,5) was largely unphosphorylated.
To further examine the role of GAP-43 in centrosome positioning toward an extrinsic cue, we performed the micropattern assay in granule cells in which GAP-43 had been genetically deleted (GAP-43 (−/−)). Figure 8A shows that GAP-43 is required for centrosome positioning by both laminin and vitronectin, whereas Shh was unable to position centrosomes whether or not GAP-43 was present. Comparing PIP3 localization in GAP-43 (+/+) and (−/−) granule cell precursors in response to vitronectin + Shh, showed that PIP3 was localized toward the pattern whether GAP-43 was present or not (Figure 8B), hence PIP3 location is independent of GAP-43 and therefore upstream of GAP-43 function. However PIP3 is dependent on PKC function: when PKC is downregulated in response to the PKC inhibitor bisindolylmaleimide, PIP3 fails to be upregulated. The results therefore indicate that while GAP-43 and PKC activity are required to position the centrosome in response to extrinsic cues, GAP-43 is not required for PIP3 upregulation in response to extrinsic cues. This suggests that GAP-43 function is downstream of PIP3, and raises the question of whether PKC phosphorylation of GAP-43 occurs in parallel to integrin signaling rather than in a linear pathway.
Taken together, our results suggest that PKC phosphorylated GAP-43 is an important link between the upstream upregulation of the PI3K pathway in response to laminin and vitronectin and the downstream response that results in centrosome positioning. We therefore investigated how downregulation of GAP-43 affects the localization of Cdc42, Par6 and the IQ motif containing GTPase activating protein 1 (IQGAP1), all of which have been shown to be important for downstream responses to cell polarization and migration signals (Golub et al., 2004; Golub and Caroni, 2005; Chen et al., 2006a; Yoshimura et al., 2006). Figures 9A and 9B (panel a, arrowheads) show that in cells expressing a control Enhanced Green Fluorescent Protein (EGFP) construct, both Cdc42 and Par6 were localized to the base of the leading process. In contrast when GAP-43 was acutely downregulated with a short hairpin RNA (shRNA) construct (Mishra et al., 2008) neither Cdc42 nor Par6 localized to the base of the process (Figure 9A and 9B, panel b, arrowheads). Similarly Cdc42 and Par6 also failed to localize appropriately in GAP-43 (−/−) neurons (supplementary figure S4). Hence failure to express GAP-43 prevents localization of downstream regulators of polarization.
IQGAP1 is important for transducing actin regulatory signals during polarization and in the growth cone. Cdc42 and IQGAP1 were always colocalized at the base of the leading process as well as in the growth cone in GAP-43 (+/+) granule cell neurons (Figure 9C). In this case absence of GAP-43 resulted in two distinct phenotypes: in some GAP-43 (−/−) cells Cdc42 and IQGAP1 did not colocalize either at the base of the primary neurite or at the growth cone (Figure 9C panel c, Cdc42 arrowhead, and IQGAP1 arrow; Figure 9C panel d, Cdc42 arrowhead). In other cells Cdc42 and IQGAP1 did colocalize but not at the region giving rise to the primary neurite (Figure 9C panel d and e, arrows). The results show that GAP-43 is required for the localization of both Cdc42 and Par6, suggesting that it is upstream of both Cdc42 and Par6 function. They also demonstrate that the correct spatial localization of Cdc42 and IQGAP1 requires GAP-43 to be PKC phosphorylated. Taken together they imply that actin regulation is an important aspect of centrosome positioning.
We have previously shown that in the absence of GAP-43 both kinetocore and astral spindles were abnormal in mitosing granule cells, indicating that in addition to its documented effects on actin polymerization GAP-43 also affects microtubules (Mishra et al., 2008). We therefore used the micropatterning assay to determine whether the disrupted response to vitronectin or laminin seen in GAP-43 (−/−) granule cells is also accompanied by abnormal distribution of microtubules. Figure 9D shows that under normal conditions microtubules are oriented toward the extrinsic cue responsible for positioning the centrosome in the micropatterning assay. When GAP-43 is absent, microtubules are still evident, however they are not oriented toward the region where the cell contacts the extrinsic cue (Figure 9D, arrowhead). The results indicate that, in addition to its previously described actin modulatory functions, GAP-43 at the centrosome influences microtubules orientation.
The mechanism(s) by which extrinsic cues are able to determine where the centrosome is positioned, and thereby where the axon will extend during neuronal polarization, are key to target identification during neuronal development, but not well understood. The goal of the present study was to identify relevant extrinsic cues able to position the centrosome in polarizing granule cell precursors, and then to investigate how the underlying signal transduction is coupled to intrinsic regulation of polarity. Previous studies have shown that laminin and vitronectin act in concert with the granule cell mitogen sonic hedgehog (Shh) to regulate cell proliferation and differentiation in granule cells (Liesi, 1992; Pons et al., 2001; Blaess et al., 2004), but how they function in the subsequent centrosome positioning that occurs as a prelude to polarization is unclear. We have shown that both laminin and vitronectin can behave as such extrinsic cues, and that they utilize an integrin-mediated transduction mechanism that is dependant both on PI3K and the growth-associated protein GAP-43. When GAP-43 is absent the cytoskeletal response that results in microtubule organization is disrupted. The results therefore provide an important first step in understanding how extrinsic and intrinsic regulation of neuronal polarity are coupled. How granule cell proliferation is downregulated in order for polarization to be initiated is unclear. Our results showed that at early stages in the differentiation of granule cell neurons in mice (P0 – P8) both laminin and vitronectin reduce the proliferative capacity of substrate-associated Shh in vitro, and also control positioning of the centrosome to the base of the leading process in slice cultures that most closely mimic in vivo development. Our results also show that positioning of the centrosome to initiate polarization is independent of Shh. They therefore confirm previous studies that both proliferation and differentiation are affected by laminin and vitronectin. Those studies showed that while laminin deposition in the external granule layer is important for the directional migration of granule cells toward the internal granule layer (Liesi, 1992; Liesi et al., 1995) presenting Shh in solution to cells in conjunction with substrate-associated laminin potentiates its mitogenic effects (Pons et al., 2001). In contrast, vitronectin interacts with Shh directly to stimulate granule cell differentiation (Pons and Marti, 2000), We found that when Shh is presented in a spatially regulated, substrate-associated format, analogous with its association with laminin and vitronectin in the cerebellar ECM in vivo, both laminin and vitronectin inhibit the proliferative effect of Shh, and both laminin and vitronectin are able to position the centrosome at the base of the primary leading process. Our data therefore suggests that under conditions where both ECM components and Shh are spatially constrained, granule cell maturation leading to polarization occurs as a two - step process with cell cycle exit being controlled by laminin and vitronectin via downregulation of Shh − induced mitogenesis, and subsequent centrosome postioning being controlled by laminin and vitronectin via upregulation of integrin signaling. The current results with regard to the effect of laminin on granule cell proliferation differ from that of Pons et al., (2001). Although Shh was printed on the substrate in the current study versus in solution as in the case of the Pons study, this is not the only difference between the two studies that needs to be taken into account. One major difference is that the Pons study used P6 rat cerebellar cultures whereas the current study uses mouse cerebellar cultures. Perhaps related to this, under basal culture conditions, in the absence of Shh, the Pons study saw no BrdU incorporation and therefore no cell proliferation. In contrast, under our culture conditions we get between 10-15% of the granule cells proliferating without the addition of Shh. This suggests that the response of granule cells may depend on the initial conditions of the cells in vitro and therefore caution should be exercised when comparing results across different culture conditions and across species.
Upregulation of PI3K by integrin receptors (including α5β3) is known to be important in the polarization of hippocampal neurons in vitro (Zheng et al., 2000; Shi et al., 2003; Menager et al., 2004). However, the mechanisms by which receptor-mediated activation of PI3K coordinates with downstream cytoskeletal responses is less clear. We too have shown that laminin and vitronectin signaling via cognate integrin receptors causes an upregulation of PI3K-mediated signaling that is required for granule cell polarization. Our finding that the centrosome positioning that underlies polarization is co-dependant on both upregulation of PI3K and PKC-mediated phosphorylation of GAP-43 identifies for the first time a spatially restricted protein that is both directly responsive to integrin signaling and able to modulate the cytoskeleton in response to extrinsic cues. The present study also shows that phosphorylation of GAP-43 results in a change in the binding affinity for distinct phospholipids and suggesting a potential mechanism whereby a spatially restricted extrinsic signal can given rise to a spatially restricted membrane response (Laux et al., 2000; Golub and Caroni, 2005). In particular we have shown that PIP3, upregulated in response to PI3 kinase activation, appears to be a key player in transduction of the signal from membrane to cytoskeleton. In neutrophils, PIP3-mediated activation of Cdc42 and Rac1, which in turn regulates actin dynamics, drives acquisition of cell polarity and subsequent chemotaxis (Wang et al., 2002; Weiner, 2002). Rac - dependent actin polymerization has also been hypothesized to underlie polarization in hippocampal neurons (Shi et al., 2003). We have also shown here that PIP3 upregulation in the centrosome area occurs in response to laminin and vitronectin, and that inhibition of PI3 kinase prevents centrosome positioning. Moreover we have demonstrated for the first time that PIP3 binds directly to GAP-43, and selectively to pGAP-43. Given that both PIP3 and GAP-43 are required for centrosome positioning, the results suggest that PIP3 binding to GAP-43 may be required for the downstream response to integrin signaling activated by laminin and vitronectin. The results also show clearly that in the absence of GAP-43, extrinsic signaling via upregulation of PI3K is decoupled from the intrinsic cytoskeletal response. Hence PIP3 upregulation is independent of GAP-43, although centrosome positioning is GAP-43 dependent. GAP-43 is localized in distinct membrane microdomains involved in generation of signaling complexes. The involvement of such microdomains in the response to laminin and vitronectin deserves investigation.
The question remains as to how the PKC that phosphorylates GAP-43 is activated. PI3K can activate phospholipase C via diacylglycerol lipase, a pathway previously shown to lead to PKC phosphorylation during granule cell differentiation (Meiri et al., 1998). However GAP-43 is phosphorylated even in the absence of laminin and vitronectin and can also be phosphorylated by PKC in response to treatment with soluble Shh (Meiri et al., 1998; Shen et al., 2008). Whether Shh plays an indirect role in centrosome positioning via GAP-43 phosphorylation that is not sufficient to induce polarization in the absence of the ECM is unclear.
Previous studies showing that the position of the centrosome can be determined by the orientation of the spindle during the last mitotic division have given rise to a model in which orientation of the future axon is intrinsically driven by microtubule dynamics. (Golub et al., 2004; Golub and Caroni, 2005). The model is supported by the finding that several microtubule stabilization factors such as lissencephaly (LIS1), nudE nuclear distribution gene E homolog 1 (NDE1), Adenomatosis Polyposis Coli (APC) as well as members of the kinesin superfamily of proteins also affect cell polarity (Sasaki et al., 2000; Nishimura et al., 2004; Shi et al., 2004; Horiguchi et al., 2006). Other studies demonstrate that Cdc42 and its effector IQGAP1 that are important for polarization anchor microtubules to cortical actin at the membrane (Golub and Caroni, 2005). Cdc42 is particularly key since its activation regulates localization of the Par complex that is essential for the acquisition of cell polarity (Chen et al., 2006a; Yoshimura et al., 2006). Neither of these models, as they stand, account for how the intrinsic regulation of polarization is coupled to the extrinsic cues that are needed to directionally determine polarization as clearly occurs in vivo. Our results here suggest that one clue may lie in the spatial organization of polarizing machinery in lipid microdomains in the membrane that contain not only Cdc42 and IQGAP1 but also GAP-43 (Golub et al., 2004; Golub and Caroni, 2005). The interaction of GAP-43 with Cdc42 and IQGAP1 in lipid microdomains implicates the phospholipid binding properties of GAP-43 as a key element at the intersection between the outside-in signaling via the extrinsic cues that directionally regulates centrosome positioning, and the intrinsic cytoskeletal dynamics that underlie centrosome positioning. In this model (Figure 10) cell polarity in granule cells is acquired through an outside-in signaling pathway that is initiated by extracellular matrix proteins laminin and vitronectin and transduced via integrin-mediated upregulation of PI3K that results in upregulation of PIP3. In parallel with this outside-in signaling PKC-mediated phosphorylation of GAP-43 enables direct interactions with PIP3 at the membrane. The next step will be to determine whether PKC phosphorylation of GAP-43 is itself extrinsically or intrinsically regulated.
We thank Dr. Pierre Gressens for critical reading of the manuscript. We also thank Dr. David Juncker for kindly providing us with the lithographically fabricated Silicon masters. SKG and KM were supported by CSIR, Government of India fellowship awards. Funding for the study was provided by the National Brain Research Centre (SM) and NIH NS33118 (KFM).