Developmental control of spindle positioning in embryos.

胚胎中纺锤体定位的发育控制。

• 批准号: 7730108
• 负责人: LESILEE S. ROSE
• 金额: $ 29.33万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7730108
• 关键词: Ablation; Address; Affect; Anterior; Antibodies; Area; Award; Behavior; Binding; Binding Proteins; Biochemical; Biological Assay; Budgets; Caenorhabditis elegans; Cell Maintenance; Cell Polarity; Cells; Centrosome; Chimeric Proteins; Cues; DNA; Data; Data Analyses; Daughter; Development; Developmental Process; Disease; Drosophila genus; Dynein ATPase; Embryo; Event; Family; Figs - dietary; GTP-Binding Protein Regulators; GTP-Binding Proteins; Generations; Goals; Health Benefit; Homologous Gene; Human; Human Resources; Image; In Vitro; Lasers; Lateral; Lead; Life; Lobular Neoplasia; Maintenance; Malignant Neoplasms; Maps; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitotic spindle; Modeling; Molecular; Motor; Movement; Nuclear; Organism; Pathway interactions; Pattern; Phenotype; Plus End of the Microtubule; Positioning Attribute; Postdoctoral Fellow; Preparation; Protein Family; Protein Isoforms; Proteins; RNA Interference; Reagent; Recruitment Activity; Regulation; Reporter; Research; Residencies; Rotation; Signal Pathway; Signal Transduction; Specialist; Stem cells; Students; System; Testing; Time; TimeLine; Training; Transgenic Organisms; Vertebrates; Work; base; cancer stem cell; cell cortex; cell type; embryo cell; genetic analysis; graduate student; human JTB protein; in vivo; insight; member; mutant; novel; nuclear power; polarized cell; premature; prevent; research study; response; segregation; stem cell biology; transmission process

The long-term goal of this work is to elucidate the mechanisms that control the position of the mitotic spindle during development. Spindle positioning is essential for a number of developmental processes, including asymmetric divisions in which a polarized cell divides to produce daughters with different fates. The proposed project addresses the molecular mechanisms of spindle positioning during asymmetric divisions in the Caenorhabditis elegans embryo. In the C. elegans one-cell embryo, LET-99, a DEP domain containing protein of the DEPDC1 family, is localized in an asymmetric cortical band pattern by the PAR proteins. LET-99 in turn restricts the cortical localization of the positive regulators of G protein signaling, GPR and LIN-5, to certain regions of the cell cortex. G protein signaling is required for cortical pulling forces that act on astral microtubules to position the spindle, and GPR and LIN-5 associate with regulators of the microtubule motor dynein. Homologs of the PAR proteins, GPR and LIN-5 are important for polarity and spindle positioning in several different organisms. However, how GPR and LIN-5 regulate forces that position spindles is not known for any system. Further the molecular mechanism by which asymmetries of GPR and LIN-5 are generated in C. elegans remain to be elucidated. The experiments proposed in Aim 1 will help refine models for the mechanistic basis of force generation by determining how microtubule dynamics and the localization of microtubule binding proteins and motors correlates with the cortical force domains defined by LET-99 and GPR/LIN-5 localization. Live-imaging of GFP-tagged reporters will be used to examine cortical-microtubule dynamics and the localization of dynein and its regulators both at the cortex and on microtubule plus-ends. The hypothesis that the clasp family of microtubule plus-end binding proteins regulates microtubule dynamics to facilitate spindle orientation and then to tether the spindle will also be investigated, using a combination of live-imaging and genetic analysis. The goal of Aim 2 is to determine how binding of LET-99 to G) subunits affects the G protein pathway such that GPR localization is inhibited at the cortex. Quantitative analysis of immunolocalization patterns and double mutant analysis will be used to determine which components of the pathway are regulated by LET-99. Biochemical approaches will be used to determine if LET-99 affects G) activity or its association with other pathway components. Because of the conservation of pathway components, the results of these studies will be relevant to asymmetric division in many systems.

这项工作的长期目标是阐明控制位置的机制 发育过程中有丝分裂纺锤体的变化。主轴定位对于数字化至关重要 发育过程，包括不对称分裂，其中极化细胞 分裂产生了不同命运的女儿。拟议的项目解决了 纺锤体不对称分裂过程中纺锤体定位的分子机制 秀丽隐杆线虫胚胎。在秀丽隐杆线虫单细胞胚胎 LET-99 中， 含有 DEPDC1 家族蛋白的 DEP 结构域，位于不对称区域 PAR 蛋白的皮质带模式。 LET-99 进而限制皮质定位 G 蛋白信号转导的正调节因子 GPR 和 LIN-5 对某些区域的影响 细胞皮质。 G 蛋白信号传导是作用于星体的皮质拉力所必需的 微管定位纺锤体，GPR 和 LIN-5 与调节器相关联 微管运动动力蛋白。 PAR 蛋白 GPR 和 LIN-5 的同源物很重要 用于几种不同生物体中的极性和纺锤体定位。然而，探地雷达如何 LIN-5 调节定位主轴的力对于任何系统都是未知的。进一步的 C 中产生 GPR 和 LIN-5 不对称性的分子机制。 线虫仍有待阐明。目标 1 中提出的实验将有助于完善 通过确定微管如何产生力的机械基础模型 微管结合蛋白和马达的动力学和定位与 由 LET-99 和 GPR/LIN-5 定位定义的皮质力域。实时成像 GFP 标记的报告基因将用于检查皮质微管动力学和 动力蛋白及其调节剂在皮质和微管正端的定位。 微管正末端结合蛋白的扣家族调节的假设 微管动力学以促进纺锤体定向，然后束缚纺锤体 还可以结合实时成像和遗传分析进行研究。目标 目标 2 是确定 LET-99 与 G) 亚基的结合如何影响 G 蛋白 通路使得 GPR 定位在皮层受到抑制。定量分析 免疫定位模式和双突变分析将用于确定哪些 该途径的组成部分受 LET-99 调节。生化方法将 用于确定 LET-99 是否影响 G) 活性或其与其他途径的关联 成分。由于途径成分的保守性，这些结果 研究将与许多系统中的不对称分裂相关。