Project 1. Real-time analysis of the effect of gene manipulation on neuronal migration and cell morphology Joseph G. Gleeson, Principal Investigator A. Background and Significance We showed that the doublecortin (OCX) gene is mutated in humans with X-lined lissencephaly and subcortical band heterotopia [1, 2], that it functions as a microtubule associated protein [3], and that patient mutations disrupt this function [4]. We also showed that disruption of the murine homologue of OCX results in aberrant brain development due to a defect in migration of neurons from the ventricular area into various brain regions. Some of these effects are specific to OCX [5] and some occur in a redundant fashion with the homologous gene doublecortin-like kinase 1 (DCLK1) [6]. We also showed that the gene Ableson-helper integration-1 (AHI1) and centrosomalassociated protein 290 (CEP290) are mutated in humans with Joubert syndrome (JS), characterized by absence of the cerebellar vermis [7, 8]. Patients with JS display congenital ataxia, mental retardation, oculomotor apraxia, and frequent retinal blindness and renal failure. Because of the shared phenotypes with these disorders of retinal ciliated photoreceptors, and renal ciliated epithelial cells, and because of localization of this family of genes to the cilia, we and others have proposed that JS gene products may function in regulation of cilia structure or function [9]. We have utilized each of the imaging systems that are available in the UCSD Neurosciences Microscopy Imaging Core to advance our research goals. The DeltaVision and DG5 Spinning Disc Systems have been used in acutely dissociated neurons to monitor fluorescently-tagged cytoskeletal markers during migration [10-12]. The ability to obtain images with an environmentallycontrol chamber, with minimal phototoxicity, and sampling from up to 20 cells simultaneously with the automated x-y-z stages had a huge impact on our ability to rapidly evaluate our hypotheses. The FV300 and FV1000 Multiphoton systems have been used predominantly to image living brain slices in which a small subset of neurons have been labeled to mark, to overexpress or to silence a gene of interest. The ability to image cells at depths of up to 300 uM has been critical in elucidating how neurons migrate in their natural environment rather than at the exposed surface of the section (Fig. 1). We have begun to use the MMI Cell-Cut system to sever microtubules (MTs), to acutely injure nerve or growing neurites or to ablate subcellular structures in neurons such as the centrosome. Although we are still gaining experience with this system, we expect it to be perfectly suited to address the next generation of questions in neuroscience cell biology.
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