project 1

项目1

• 批准号: 8120384
• 负责人: JOSEPH G GLEESON
• 金额: $ 62.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8120384
• 关键词: Address; Area; Ataxia; Blindness; Brain; Brain region; Cells; Cellular Morphology; Cellular biology; Centrosome; Cerebellar vermis structure; Cilia; Defect; Development; Environment; Epithelial Cells; Figs - dietary; Gene Family; Genes; Goals; Homologous Gene; Human; Image; Immigration; Joubert syndrome; Kidney; Kidney Failure; Label; Life; Mental Retardation; Microscopy; Microtubule-Associated Proteins; Microtubules; Monitor; Mus; Mutate; Mutation; Nerve; Neurites; Neurons; Neurosciences; Patients; Phenotype; Phosphotransferases; Photoreceptors; Phototoxicity; Principal Investigator; Proteins; Regulation; Research; Retinal; Retinal Diseases; Sampling; Slice; Staging; Structure; Subcellular structure; Surface; System; Testing; Time; Ventricular; base; cellular imaging; cilium biogenesis; experience; injured; interest; lissencephaly; migration; next generation; oculomotor; overexpression

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.

项目1。基因操纵对神经元影响的实时分析 迁移和细胞形态 首席研究员Joseph G. Gleeson A.背景和意义 我们表明，双雌激素（OCX）基因在具有X层lissencephaly的人类中被突变 皮质带异位[1，2]，它起作用的蛋白质[3]，并且 患者突变破坏了此功能[4]。我们还表明了鼠同源物的破坏 OCX导致由于神经元迁移的缺陷而导致脑发育异常 心室区域到各个大脑区域。其中一些影响特定于OCX [5]，有些则是 以多余的方式以同源基因双果素样激酶1（DCLK1）出现[6]。 我们还表明，基因Ableson-helper Integration-1（AHI1）和中心酶相关 蛋白质290（CEP290）在乔伯特综合征（JS）的人类中被突变为特征 由于缺乏小脑vermis [7，8]。 JS患者显示先天性共济失调，精神 延迟，眼动型失调以及频繁的视网膜失明和肾衰竭。因为 与视网膜纤毛感光体的这些疾病共享表型和肾上皮 细胞，并且由于该基因家族将其定位到纤毛，我们和其他人提出了 JS基因产物可能在调节纤毛结构或功能方面发挥作用[9]。 我们利用了UCSD中可用的每个成像系统 神经科学显微镜成像核心以促进我们的研究目标。三角处 DG5旋转盘系统已用于急性解离神经元中 在迁移过程中监测荧光标记的细胞骨架标记[10-12]。能力 要获得具有环境控制室的图像，具有最小的光毒性， 并与自动X-Y-Z同时从20个单元格进行采样 阶段对我们快速评估假设的能力产生了巨大影响。 FV300 FV1000多光子系统主要用于图像活脑切片 一小部分神经元被标记为标记，过表达或沉默 感兴趣的基因。在最多的深处进行图像单元的能力 300 UM对于阐明神经元在其自然环境中迁移而不是 在该部分的裸露表面（图1）。我们已经开始使用MMI细胞切割系统 切断微管（MTS），急性损伤神经或生长神经突或消融亚细胞 神经元中的结构，例如中心体。尽管我们仍在获得这一经验 系统，我们希望它非常适合解决下一代问题 神经科学细胞生物学。