Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Netrin-1 elicits exocytosis and axonal guidance depending on Sytx1 and TI-VAMP. A–C, Confocal images of live hippocampal growth cones traced with BODIPY (recorded in the red channel). Neurons were labeled with BODIPY ceramide for 30 min at room temperature and then chased for 2.5–3 h at 37°C. Growth cones were treated with control medium, Netrin-1, or Netrin-1 plus BoNT/C1 for 0–30 min. While fluorescent vesicle clusters persist in controls (A), red fluorescence rapidly disappears upon treatment with Netrin-1 (B), indicating the occurrence of secretion events. Treatment with BoNT/C1 prevents the disappearance of red puncta in Netrin-1-treated neurons (C). On the bottom right, merged images showing the BODIPY label recorded separately in the red (high concentration) and green (low concentration) channels. The red spots show Golgi-derived vesicles or vesicle clusters, whereas the green fluorescence outlines the plasmalemma of the growth cone, labeled with small amounts of BODIPY. D, E, Percentage of average intensity of red fluorescent puncta in growth cones for control and Netrin-1-treated hippocampal neurons (normalized to 100% at the onset of treatment) (D). The average intensity of spots recorded in the red channel comes back to control levels after treatment with BoNT/C1 (E). F, Confocal images of hippocampal growth cones immunolabeled for DCC and TI-VAMP. A merged image showing colocalization of both proteins is shown at the bottom. G, Coimmunoprecipitation experiments in HEK293 cells transfected with pCMV6TI-VAMP and Sytx1AEGFP, pCMVDCC, or Friz2-HA DNAs. DCC immunoprecipitation results in coassociation with Sytx1A, visualized with anti-GFP antibodies, and with TI-VAMP (anti-TI-VAMP antibodies). The reverse immunoprecipitation with anti-TI-VAMP antibodies confirms DCC and Sytx1 coimmunoprecipitation. GFP immunoprecipitation of Sytx1 reveals coassociation with DCC and TI-VAMP proteins. Immunoblots show no coimmunoprecipitation of Friz-2 with DCC and the SNAREs Sytx1 and TI-VAMP. H, Coimmunoprecipitation experiments in E15 and adult brain lysates. Sytx1 and TI-VAMP immunoprecipitation yields coimmunoprecipitation of DCC in both samples. DCC and TI-VAMP immunoprecipitation yields coassociation of Sytx1. DCC and Sytx1 immunoprecipitation yields coassociation of TI-VAMP. Immunoprecipitations with anti-myc antibody were used as controls. I, J, Confocal images showing open-book preparations from the chicken spinal cord. The injection of the control pIRES-EGFP construct did not interfere with commissural axon pathfinding (I). Downregulation of TI-VAMP using in ovo RNAi (J) interfered with commissural axon navigation to and across the floor plate, with many fibers being arrested before (arrows) or within the floor plate (FP) (arrowheads). K, Quantification of the axon guidance phenotype in open-book preparations of embryonic chicken spinal cords. RNAi-mediated downregulation of TI-VAMP in chicken spinal cords (n = 139 injection sites in 17 embryos) yielded similar commissural pathfinding errors to those obtained with silencing of Sytx1 (n = 135 injection sites in 18 embryos). We observed strong phenotypes at 37.9% of dsTI-VAMP and 34.6% of dsStx-1, and weak phenotypes at 17.2 and 25.7% of injection sites, respectively. By contrast, electroporation of a pIRES plasmid expressing EGFP under the β-actin promoter (297 injection sites in 28 embryos; 60.1% normal and 21% weak phenotypes) did not differ significantly from untreated controls (6.9% strong phenotypes, 13% weak; n = 489 injection sites in 47 embryos). L–O, Schematic diagram summarizing a model for the regulation of Netrin-1-dependent exocytosis in growth cones. In a steady-state situation, the growth cones present a low release of exocytosis vesicles (L). Netrin-1 activation of DCC receptors result in ligand-dependent clustering of DCC/Sytx1 complexes in activated membrane domains (M). Here, the formation of a SNARE complex between Sytx1 and TI-VAMP proteins occurs, thereby promoting exocytosis of vesicles at DCC-activated domains (N), resulting in membrane expansion (O). Significant differences are labeled by asterisks (**p ≤ 0.001). Scale bars: A, 2 μm; F, 3 μm; I, 50 μm. Error bars indicate SEM.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Labeling, Fluorescence, Concentration Assay, Derivative Assay, Immunolabeling, Transfection, Immunoprecipitation, Western Blot, Injection, Construct, In Ovo, Electroporation, Plasmid Preparation, Expressing, Activation Assay

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Sytx1 coassociates with the DCC receptor in brain tissue and in transfected cells. A, DCC and Sytx1 immunoprecipitation of E15 and adult forebrain homogenates. DCC immunoprecipitation yields coimmunoprecipitation of Sytx1 in both samples (top panels). Sytx1 immunoprecipitation yields coassociation of DCC (bottom panels). Anti-βIII-tubulin antibodies were used as loading controls. B, DCC and Sytx1 immunoprecipitation assays do not result in coimmunoprecipitation of Egr1. Levels of Egr1 protein lysates are shown (bottom). C, DCC associates with A and B isoforms of Sytx1. Forebrain homogenates were immunoprecipitated and immunocomplexes were subjected to urea/SDS-PAGE. DCC coimmunoprecipitates two Sytx1 bands corresponding to Sytx1A and 1B (top panel). D, E, Coimmunoprecipitation experiments in HEK293 cells. DCC immunoprecipitation results in coassociation with Sytx1A, visualized either with anti-GFP (D) or anti-Sytx1 (E) antibodies. The reverse immunoprecipitation with anti-GFP or anti-Sytx1 antibodies also reveals DCC (D, E). Note that the efficiency of the immunoprecipitation with anti-GFP antibodies is consistently higher than when using anti-DCC antibodies, which yields low recovery of proteins in this condition. F, DCC affinity pull-down experiments with purified His-Sytx1A. Incubation with anti-DCC antibody reveals DCC in the beads coupled to His-Sytx1A, but not in control beads. G, Sytx1A was transcribed and translated in vitro in the presence of [35S]Met, and incubated with glutathione-Sepharose beads coupled to GST-DCCCYT or GST-MUNC18a. After SDS-PAGE, gels were exposed to a storage phosphor screen. [35S]Met-Sytx1A binds to DCCCYT (as well as to MUNC18a), but not to empty beads. Binding of [35S]Met-Sytx1A to DCCCYT is decreased in the presence of nonradioactive Sytx1A (Sytx1A*). H, Sensorgram showing binding between the GST-DCCCYT domain and His-Sytx1A. Increasing concentrations of His-Sytx1A were injected into a chip where the GST-DCCCYT was cross-linked; the interaction was recorded as SPR changes [in response units (RU)]. Increasing concentrations of His-Sytx1A yielded higher responses. I, Plot of the steady-state response (in RU) between GST-DCCCYT and a range of concentrations of His-Sytx1A (red) or between control GST and His-Sytx1A (black). No binding is detected when GST protein was cross-linked, whereas a saturable response is observed when GST-DCCCYT was immobilized. J, Pull-down experiments in which brain extracts (P0) were passed through Ni2+-affinity columns to which recombinant His-Sytx1A or His-Sytx1AH3TM proteins were coupled. Western blot analyses show coprecipitation of DCC with similar efficiencies in both cases. No coprecipitation of DCC is detected when extracts are incubated with control GST-GFP or GST-MUNC18a columns, whereas strong coprecipitation was observed with GST-DCCcyt. The high DCC signal in GST-DCCcyt samples is probably due to multimerization of the DCC receptor through the P3 domain. K, Sensorgram showing binding between the T1DCC peptide and purified His-Sytx1A. The peptide was immobilized as described in Material and Methods, and then increasing concentrations of His-Sytx1A were injected. Responses (in RU) increase at increasing concentrations of His-Sytx1A as a function of the time (in seconds). L, Plot of the steady-state response between T1 peptide and His-Sytx1A (red) or between a control peptide and His-Sytx1A (black). While no binding is detected when the control peptide is cross-linked, specific binding is observed when T1 peptide is immobilized.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Transfection, Immunoprecipitation, SDS Page, Purification, Incubation, In Vitro, Binding Assay, Injection, Recombinant, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: The DCC receptor does not coassociate with SNAP25, VAMP2, or Syntaxin 4. A, Coimmunoprecipitation experiments in brain lysates (E15 and adult). Whereas DCC and Sytx1 coimmunoprecipitate, the DCC receptor does not coassociate with SNAP25, VAMP2, or Sytx 4 in brain lysates. The Sytx antibody used for the immunoblot was an anti-Sytx1A antibody in all cases, except in the column immunoprecipitated with anti-Sytx 4 antibodies. Inputs are shown to the right. B, Coimmunoprecipitation experiments in HEK293 cells transfected with pCMVDCC and SNAP25-FLAG, VAMP2-FLAG or Sytx1A-EGFP DNAs. DCC immunoprecipitation results in coassociation with Sytx1A visualized with anti-GFP antibodies, but not with SNAP25 or VAMP2 (FLAG antibodies). The reverse immunoprecipitation with anti-FLAG antibodies did not reveal DCC coimmunoprecipitation.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Western Blot, Immunoprecipitation, Transfection

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Characterization of protein regions required for Sytx1A/DCC interaction. A, Diagram summarizing Sytx1A domains and the truncated Sytx1AEGFP chimeras generated. B, Expression of the several Sytx1AEGFP DNAs in HEK293 cells, showing that they generate proteins of the appropriate Mr between 65 and 27 kDa, as revealed by immunoblotting with anti-GFP antibodies. C, Coimmunoprecipitation experiments in HEK293 cells cotransfected with the distinct Sytx1AEGFP constructs together with pCMV or pCMVDCC. DCC immunoprecipitations (200 μg) were revealed by immunoblotting with anti-DCC or anti-GFP antibodies (left panel). Cells cotransfected with pCMVDCC and Sytx1AFLEGFP or Sytx1AH3TMEGFP show positive coimmunoprecipitation of Sytx1A fusions. The reverse immunoprecipitation assays with anti-DCC antibodies also show coimmunoprecipitation with DCC, exclusively when cells are cotransfected with Sytx1AFLEGFP or Sytx1AH3TMEGFP DNAs (arrows in top right panel). The bands corresponding to the distinct Sytx1AEGFP chimeras are labeled with asterisks in the bottom right panel label. D, Diagram summarizing the DCC domains and the truncated EGFPDCC chimeras generated. E, Western blot, revealed with an anti-GFP antibody, demonstrating appropriate Mr (between 90 and 40 kDa) of the distinct EGFPDCC chimeras expressed in HEK293 cells. F, Coimmunoprecipitation experiments in HEK293 cells cotransfected with the distinct EGFPDCC constructs together with pRcCMV or pRcCMVSytx1A DNAs. Sytx1 immunoprecipitations (200 μg) were revealed by immunoblotting with anti-GFP or anti-Sytx1 antibodies (left panel). All the cells cotransfected with pRcCMVSytx1A and the EGFPDCC constructs show coimmunoprecipitation of EGFP-tagged DCC chimeras (arrows), except when cells are cotransfected with EGFPP1-P3DCC DNA. The reverse immunoprecipitation assays with anti-GFP antibodies also reveal coimmunoprecipitation with Sytx1A (arrows in bottom right panel) in all cotransfected cells, except in those transfected with EGFPP1-P3DCC DNA. The bands corresponding to the distinct Sytx1AEGFP chimeras are labeled with asterisks in the top right panel.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Generated, Expressing, Western Blot, Construct, Immunoprecipitation, Labeling, Transfection

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Netrin-1, but not BDNF, triggers DCC mobilization and DCC/Sytx1 colocalization. A, Confocal images of hippocampal axonal shafts treated with Netrin-1-conditioned media for 0, 15, and 30 min, immunolabeled for DCC (red) and Sytx1A (green). Note that DCC and Sytx1A do not colocalize in axonal shafts in either condition. B, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0–30 min, immunolabeled for DCC and Sytx1. Note increased DCC/Sytx1 colocalization in growth cones incubated with Netrin-1. C, Quantification of DCC/Sytx1 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/Sytx1 colocalization over total Sytx1 signals) in cultures treated with Netrin-1-conditioned (black bars) or control-conditioned (white bars) media. D, Quantification of DCC/Sytx1 coimmunoprecipitation in hippocampal cultures treated with Netrin-1. Sytx1 immunoprecipitation reveals an increase in coassociated DCC in neurons treated with Netrin-1 (black bars). Immunoprecipitation with anti-DCC antibodies shows a marked increase in Sytx1 signals (white bars). E, Quantification of DCC/Sytx1 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/Sytx1 colocalization over total Sytx1 signal) in cultures treated with BDNF (gray bars) or control (white bars). Note that BDNF does not increase colocalization of DCC and Sytx1. F, Western blots from hippocampal cultures treated with BDNF for 15–30 min and immunoprecipitated with anti-DCC or anti-Sytx1A antibodies. Immunoblots reveal that BDNF does not increase the coassociation of DCC with Sytx1A. G, Western blots from hippocampal cultures treated with Netrin-1-conditioned (N) or control-conditioned (C) media for 0–30 min (left), and immunoprecipitated with anti-DCC (top panel) or anti-Sytx1 (bottom panel) antibodies. Immunoblots reveal increased association of DCC and Sytx1 in neuronal cultures treated with Netrin-1. Immunoprecipitations of brain lysates from newborn wild-type, heterozygous and homozygous netrin-1 mutant mice reveal decreased DCC/Sytx1 association in the null mutants (right). Note decreased coimmunoprecipitation of Sytx1 (top) and DCC (middle) in null-mutant brains. H, Quantification of DCC and Sytx1 coimmunoprecipitations in homogenates from newborn wild-type, heterozygous and homozygous netrin-1 mutant mice. Immunoprecipitations with either anti-DCC (white bars) or anti-Sytx1 (black bars) antibodies reveal a marked reduction in DCC/Sytx1 coassociation in netrin-1-null mutants. Significant differences are labeled by asterisks (*p ≤ 0.05; **p ≤ 0.001). Scale bar: A, B, 3 μm. Error bars indicate SEM.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Immunolabeling, Incubation, Immunoprecipitation, Western Blot, Mutagenesis, Labeling

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Netrin-1 does not trigger coassociation of DCC with the SNAREs SNAP25 and VAMP2. A, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0 and 15 min, immunolabeled for DCC and the SNAREs SNAP25 and VAMP2. Note low colocalization signals of DCC and SNAP25 or VAMP2, both in control conditions and after incubation with Netrin-1. B, C, Quantification of DCC/SNAP25 and DCC/VAMP2 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/SNARE colocalization over total SNARE signals) in cultures treated with Netrin-1-conditioned (black bars) or control-conditioned (white bars) media. Note decreased DCC/SNARE colocalization signals after Netrin-1 treatment. D, Western blots from hippocampal cultures treated with Netrin-1- (N) or control- (C) conditioned media for 0–30 min, and immunoprecipitated with anti-DCC or anti-Sytx1A antibodies. Immunoblots reveal no coimmunoprecipitation of DCC with the SNAREs SNAP25 and VAMP2 after incubation with DCC. Note coimmunoprecipitation of Sytx1A with SNAP25 and VAMP2. E–G, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0 and 15 min, and in the presence of BoNT/C1. Cultures were immunolabeled for DCC and stained with phalloidin. Note that the mobilization of DCC to the axonal membrane after incubation with Netrin-1 (F) is not altered by BoNT/C1 incubation (G). H, Quantification of DCC signals in the periphery of and inside growth cones treated with Netrin-1-conditioned media for 0, 15, and 30 min, showing mobilization of DCC to the axonal membrane after incubation with Netrin-1. Whereas no DCC mobilization is detected after incubation with BDNF, treatment with BoNT/C1 does not alter DCC mobilization. I–L, Confocal images of control growth cones (I) and cones incubated with Netrin-1-conditioned media for 15 min (J), and with Netrin-1/BoNT/C1 (K). Cultures were immunolabeled for DCC (red) and Sytx1 (green). Note that the increase in DCC/Sytx1 colocalization in J is blocked after incubation with BoNT/C1 (K). L, Histograms illustrating DCC/Sytx1 colocalization in several experimental conditions. Significant differences are labeled by asterisks (*p ≤ 0.05, **p ≤ 0.001). Scale bar: A, 3 μm. Error bars indicate SEM.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Immunolabeling, Incubation, Western Blot, Immunoprecipitation, Staining, Labeling