| Abstract No.: | B-A2017 |
| Country: | Canada |
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| Title: | A COMPLEX OF DCC AND COOL-1/-PIX, A CDC42 AND RAC SPECIFIC GEF, IS INVOLVED IN COMMISSURAL AXON CHEMOATTRACTION TO NETRIN-1 |
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| Authors/Affiliations: | 1 Sonia P. Rodrigues, 1 K. Adam Baker, 1Karen Lai Wing Sun, 1 Zhijin Chai, 1,2 Artur Kania, 1,2 Timothy E. Kennedy
1Montreal Neurological Institute, McGill University,
Montréal, Quebec, H3A 2B4 Canada. *Institut de recherches cliniques de Montréal and Université de Montréal, Montréal, Quebec, H2W 1R7 Canada. 2Department of Anatomy and Cell Biology and Experimental Medicine, McGill University, Montréal, Quebéc, H3A 2B2 Canada.
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| Content: | Objective: What signalling events take place in a growth cone to promote cytoskeletal rearrangements during chemoattraction? Netrin-1 secreted by the floor plate is essential for spinal commissural axon guidance during neural development. We have previously reported that activation of the RhoGTPases Cdc42 and Rac1, and recruitment of their downstream effectors p21-activated kinase (Pak-1) and N-WASP, is required for the netrin-1 receptor DCC to regulate growth cone morphology. We therefore investigated the mechanisms that regulate Cdc42 and Rac1 activation downstream of DCC.
Materials and Methods: Commissural neurons were dissected from E13 rat spinal cord and cultured for 2 DIV prior to immunoprecipitations, transfections and immunofluorescence assays. E11 and E13 rat embryos were fixed in 4% paraformaldehyde and sectioned. -Pix and neurofilament-M proteins were detected using specific anti-sera. In ovo electroporation of -Pix functional mutants’ expression plasmids was performed at stage 18. At stages 24-25 chick spinal cords were dissected and axonin-1 was detected using specific anti-bodies/sera. GST-CRIB pulldowns were performed as described in Del Pozo MA et al, EMBO 2000.
Results: We report that a protein complex of the Rac and Cdc42 specific GEF, Cool-1/-Pix and Git-2/PKL is associated with DCC in commissural neurons. Our findings indicate that dimerization of Cool-1/-Pix and its association with Pak-1 is required for netrin-1 induced filopodia formation and growth cone expansion. Additionally, we provide evidence that Cool-1/-Pix function is required for commissural axon extension to the floor plate of the embryonic spinal cord. These findings implicate both the adaptor and GEF functions of Cool-1/-Pix in the activation of Cdc42 and Rac1 by DCC.
Conclusion: Our findings provide evidence the activation of Cdc42 and Rac1 in neuronal growth cones by netrin-1 via DCC requires the Cdc42 and Rac specific GEF Cool-1/-Pix.
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