Developmental Control of Spindle Positioning in Embryos

胚胎中纺锤体定位的发育控制

基本信息

批准号：
8896808
负责人：
LESILEE S. ROSE
金额：
$ 31.42万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8896808
关键词：
14-3-3 Family Address Affect Animals Anterior Area Back Binding Biochemical Biological Assay Biological Process Caenorhabditis elegans Cell Maintenance Cell Polarity Cell division Cell membrane Cells Centrosome Complex Cues Cytokinesis Data Daughter Development Developmental Process Disease Drosophila genus Dynein ATPase Embryo Family G-Protein Signaling Pathway GTP-Binding Protein Regulators GTP-Binding Proteins Goals Guanosine Triphosphate Phosphohydrolases Health Benefit Human Image Analysis In Vitro Lateral Lead Left Life Lipids Lobular Neoplasia Malignant Neoplasms Malignant neoplasm of urinary bladder Metaphase Microtubules Mitotic spindle Modeling Molecular Monomeric GTP-Binding Proteins Motor Movement Myosin ATPase Nuclear Organism Orthologous Gene Pathway interactions Pattern Phosphotransferases Play Positioning Attribute Protein Binding Protein Family Proteins Relative (related person)Role Signal Transduction Site Stem cells System Testing Transgenic Organisms Vertebrates Work base cancer cell cancer stem cell cell cortex cell fate specification cell type daughter cell embryo cell genetic analysis human JTB protein in vivo insight member mutant novel overexpression polarized cell prevent research study rho rho GTP-Binding Proteins stem stem cell biology

项目摘要

DESCRIPTION (provided by applicant): Asymmetric divisions, in which a polarized cell divides to produce daughters with different fates, contribute to cell fate specification during development as well as stem cell maintenance. The proposed project addresses the molecular mechanisms of spindle positioning and cytokinesis during asymmetric divisions in the Caenorhabditis elegans embryo. As in other systems, asymmetric division in the C. elegans one-cell embryo relies on a conserved pathway in which the PAR polarity proteins regulate the distribution of components of a non-canonical G protein signaling pathway. We identified LET-99, a member of the DEPDC1 family, as a new player in this pathway. LET-99 is localized in an asymmetric cortical band pattern by the PAR-3 and PAR-1 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. The asymmetric localization of these intermediates is an essential feature of spindle positioning, because GPR and LIN-5 associate with regulators of the microtubule motor dynein to generate the asymmetric cortical pulling forces that move the spindle. Once the spindle is positioned, it signals back to the cortex to determine the plane of cleavage. How the PAR proteins promote asymmetry of spindle positioning factors, and how the G protein pathway is integrated with other signaling mechanisms, remains to be elucidated. The experiments proposed in Aim 1 will define the molecular mechanisms by which the PAR proteins regulate LET-99 asymmetry. The hypothesis that PAR-1 directly phosphorylates LET-99 to inhibit its localization at the posterior cortex will be tested using in vitro kinase assays followed by in vivo transgenic studies. PAR-3 inhibits LET-99 localization at the anterior via a separate mechanism, which will be investigated using a combination of live-imaging and genetic analysis. LET-99 interacting proteins will also be tested for a role in LET-99 cortical anchoring. The goal of Aim 2 is to determine how the LET-99/G� pathway is integrated with Rho-family GTPase signaling to properly position the cytokinesis furrow relative to the spindle. Quantitative analysis of localization patterns in mutants combined with biochemical interaction assays will be used to determine how these pathways interact. The hypothesis that LET-99 directly binds Rho GTPases via its partial RhoGAP domain will also be tested. Finally, Aim 3 will test the hypothesis that the human orthologs of LET-99, DEPDC1 and DEPDC1B, have a similar function to LET-99. Specifically we will test the hypothesis that these proteins associate with G� or Rho and are involved in spindle movements or cytokinesis. Because of the conservation of pathway components, the results of these studies will be relevant to asymmetric division in many systems and will define the function of a novel class of proteins, the DEPDC1 family.

描述（由申请人提供）：不对称分裂，其中极化细胞分裂产生具有不同命运的子细胞，有助于发育过程中的细胞命运规范以及干细胞维持。拟议的项目解决了纺锤体定位和胞质分裂过程中的分子机制。与其他系统一样，秀丽隐杆线虫单细胞胚胎中的不对称分裂依赖于 PAR 极性蛋白调节的保守途径。我们发现 LET-99（DEPDC1 家族的成员）是该途径中的一个新参与者，由 PAR-3 定位在不对称皮质带模式中。和 PAR-1 蛋白反过来将 G 蛋白信号传导的正调节因子 GPR 和 LIN-5 的皮质定位限制在细胞皮质的某些区域。中间体是纺锤体定位的一个重要特征，因为 GPR 和 LIN-5 与微管运动动力蛋白的调节器相关联，产生不对称的皮质拉力来移动纺锤体，一旦纺锤体定位，它就会向皮质发出信号以确定纺锤体的位置。 PAR蛋白如何促进纺锤体定位因子的不对称性，以及G蛋白通路如何与其他信号传导机制整合，仍有待阐明目标1中提出的实验。定义 PAR 蛋白调节 LET-99 不对称性的分子机制 PAR-1 直接磷酸化 LET-99 以抑制其在后皮质的定位的假设将使用体外激酶测定进行测试，然后进行体内转基因研究。 -3 通过一种单独的机制抑制 LET-99 在前部的定位，我们将结合实时成像和遗传分析来研究 LET-99 相互作用蛋白的作用。 LET-99 皮质锚定。目标 2 的目标是确定 LET-99/G� 通路如何与 Rho 家族 GTPase 信号整合，以相对于纺锤体正确定位胞质分裂沟。生化相互作用测定将用于确定这些途径如何相互作用。最后，LET-99 通过其部分 RhoGAP 结构域直接结合 Rho GTPases 的假设也将得到测试。目标 3 将检验 LET-99、DEPDC1 和 DEPDC1B 的人类直系同源物与 LET-99 具有相似功能的假设，具体而言，我们将检验这些蛋白质与 G� 或 Rho 相关并参与纺锤体运动或的假设。由于途径成分的保守性，这些研究的结果将与许多系统中的不对称分裂相关，并将定义一类新型蛋白质（DEPDC1 家族）的功能。