Ph.D. Projects
Project1: Role of the small GTPaseRac1 in nerve development.
Radial sorting of axons by Schwann cells is a developmental step required for myelination in the peripheral nervous system. Lack of laminin-2 cause an arrest in the process of radial axonal sorting in the neuropathy associated with Congenital Muscular Dystrophy 1A (MDC1A). Our laboratory is devoted to understanding the signals normally initiated by laminins to promote radial sorting, and deficient in MDC1A. We and others have previously shown that two laminins and several laminin receptors are required for radial sorting and myelination to proceed. Among these, integrins of the beta1 class are required for Schwann cells to interact with axons during this process. We also showed that Rac1 is an important signaling molecules downstram of these integrins, as active Rac1 is decreased in beta1 integrin–null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells in vivo delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in β1 integrin–null nerves improves their phenotype (Nodari et al. Journal of Cell Biology , 2007). We now wish to determine the effectors of Rac1 in Schwann cell that allow Schwann cells to myelinate axons. We have generated a list of potential Rac1 effectors based on in-silico studies. We will next isolate Rac1 interactors in Schwann cells using Rac1-GTP affinity chromatography, using specific developmental stages and Rac1-null Schwann cells as control. New interactors, or known interactors present in our list of candidates will be validated by expression studies in developing nerves and functional studies (knock-down studies in myelinating co-colture models and possibly in–vivo perturbation studies).
Project2: Organization and polarization of the Schwann cells internode.
The optimal length of each myelin segment and the correct segregation of proper ion channels at nodes of Ranvier are both required for efficient conduction of nerve impulses. Schwann cells can form long segments of myelin (internodes) and contribute to the polarization of proper axonal domains in and around nodes of Ranvier by organizing their cytoplasm in specific domains. The formation of these domains is regulated in concert with the axon. We and others have shown that in a myelinated fiber microtubules are normally localized in a channel compartment, and postulated that such an organization is required for efficient vesicular transfer. We recently showed that laminins and the dystroglycan complex are involved in the formation of these domains. This may explain why patients lacking laminins have short internodes and impaired nerve conduction velocity (Court et al, J. of Neuroscience 2009). We now want to explore the link between compartments, microtubular organization (including polarity and post-translational modification) and vesicular trafficking in normal and mutant myelinated fibers. Existing mutants lacking components of the dystroglycan complex in Schwann cells will be used for these experiments. We will use morphological studies in vivo, and live-imaging and functional assays in vitro using Schwann-neuronal explants derived from these mutants. One possibility is that laminins receptors cooperate with polarity and junctional proteins to organize and maintain cytoplasmic compartments, therefore we will characterize the role of junctional components in vivo (using available animal models lacking tight junction molecules) and in vitro (expressing loss-and gain of function mutants of junctional proteins in neuronal-Schwann cell co-cultures).
