Project 1. Real-time analysis of the effect of gene manipulation on neuronalmigration and cell morphologyJoseph G. Gleeson, Principal InvestigatorA. Background and SignificanceWe showed that the doublecortin (OCX) gene is mutated in humans with X-lined lissencephaly andsubcortical band heterotopia [1, 2], that it functions as a microtubule associated protein [3], and thatpatient mutations disrupt this function [4]. We also showed that disruption of the murine homologueof OCX results in aberrant brain development due to a defect in migration of neurons from theventricular area into various brain regions. Some of these effects are specific to OCX [5] and someoccur 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 centrosomalassociatedprotein 290 (CEP290) are mutated in humans with Joubert syndrome (JS), characterizedby absence of the cerebellar vermis [7, 8]. Patients with JS display congenital ataxia, mentalretardation, oculomotor apraxia, and frequent retinal blindness and renal failure. Because of theshared phenotypes with these disorders of retinal ciliated photoreceptors, and renal ciliated epithelialcells, and because of localization of this family of genes to the cilia, we and others have proposedthat 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 UCSDNeurosciences Microscopy Imaging Core to advance our research goals. The DeltaVisionand DG5 Spinning Disc Systems have been used in acutely dissociated neurons tomonitor fluorescently-tagged cytoskeletal markers during migration [10-12]. The abilityto obtain images with an environmentallycontrol chamber, with minimal phototoxicity,and sampling from up to 20 cells simultaneously with the automated x-y-zstages had a huge impact on our ability to rapidly evaluate our hypotheses. The FV300and 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 to300 uM has been critical in elucidating how neurons migrate in their natural environment rather thanat the exposed surface of the section (Fig. 1). We have begun to use the MMI Cell-Cut system tosever microtubules (MTs), to acutely injure nerve or growing neurites or to ablate subcellularstructures in neurons such as the centrosome. Although we are still gaining experience with thissystem, we expect it to be perfectly suited to address the next generation of questions inneuroscience cell biology.
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