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：
8554366
负责人：
LESILEE S. ROSE
金额：
$ 30.47万
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 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8554366
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（由适用提供）：不对称的划分，其中一个极化细胞分裂产生了具有不同命运的女儿，在发育过程中以及干细胞维持过程中有助于细胞命运规格。提出的项目介绍了秀丽隐杆线虫胚胎中不对称分裂期间主轴定位和细胞因子的分子机制。与其他系统一样，秀丽隐杆线虫中的不对称分裂一细胞胚胎依赖于一种保守的途径，在该途径中，极性蛋白调节非经典G蛋白信号通路的组件分布。我们确定了DEPDC1家族的成员Let-99是该路径的新玩家。 Let-99通过PAR-3和PAR-1蛋白在不对称皮质带模式中定位。 Let-99反过来限制了G蛋白信号传导GPR和LIN-5的正调节剂的皮质定位到细胞皮质的某些区域。这些中间体的不对称定位是主轴定位的重要特征，因为GPR和LIN-5与微管运动动力蛋白的调节剂相关联，以产生移动纺锤体的不对称皮质拉力拉力。主轴放置后，它将信号回到皮层以确定裂解平面。 PAR蛋白如何促进主轴定位因子的不对称性，以及G蛋白途径如何与其他信号传导机制集成在一起，尚待阐明。 AIM 1中提出的实验将定义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抑制其在后皮层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。 PAR-3通过单独的机制抑制在前部处的Let-99定位，该机制将通过实现现象和遗传分析的组合进行研究。 Let-99相互作用的蛋白也将在let-99皮质锚定中的作用进行测试。 AIM 2的目的是确定如何与Rho-family GTPase信号集成Let-99/G途径，以正确定位相对于纺锤体的细胞因子沟。对突变体中与生化相互作用分析结合的定位模式的定量分析将用于确定这些途径的相互作用。 Let-99通过其部分RhoGap结构域直接结合Rho GTPase的假设也将进行测试。最后，AIM 3将检验以下假设：Let-99，DEPDC1和DEPDC1B的人类直系同源物具有与Let-99相似的功能。具体而言，我们将检验以下假设：这些蛋白质与G g或Rho相关，并参与纺锤体运动或细胞因子。由于途径成分的保护，这些研究的结果将与许多系统中的不对称分裂有关，并将定义新型蛋白质（DEPDC1家族）的功能。