Developmental Control of Spindle Positioning in Embryos

胚胎中纺锤体定位的发育控制

• 批准号: 8435313
• 负责人: LESILEE S. ROSE
• 金额: $ 31.65万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435313
• 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

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. PUBLIC HEALTH RELEVANCE: Asymmetric divisions and spindle positioning are critical for generating cell diversity during normal development. Recent studies also highlight the importance of asymmetric division in stem cell maintenance and cancer. All of the proteins under study are conserved. Further, the analysis of LET-99 in C. elegans shows that LET-99, a DEPDC1 family protein, is a novel regulator of G protein signaling. Therefore, the proposed studies of the mechanistic basis of spindle positioning in C. elegans and the biochemical function of LET-99 will greatly benefit health related-areas such as cancer and stem cell biology. In addition, DEPDC1 is upregulated in bladder cancer. The studies of DEPDC1 outlined in the proposal will begin to characterize the cell biological functions of this protein, which will help elucidate its role in cancer cells.

描述(由申请人提供)：不对称分裂，即极化的细胞分裂产生不同命运的子代，在发育过程中对细胞命运的规定以及干细胞的维持都有贡献。该项目旨在研究线虫胚胎不对称分裂过程中纺锤体定位和胞质分裂的分子机制。与其他系统一样，线虫单细胞胚胎的不对称分裂依赖于一条保守的途径，在该途径中，PAR极性蛋白调节非规范G蛋白信号通路的成分分布。我们确定了DEPDC1家族的成员let-99是这一途径中的一个新的参与者。LET-99被PAR-3和PAR-1蛋白定位在不对称的皮质条带中。LET-99反过来又将G蛋白信号的阳性调节因子GPR和LIN-5的皮质定位限制在细胞皮质的某些区域。这些中间产物的不对称定位是纺锤体定位的一个基本特征，因为GPR和LIN-5与微管马达动力蛋白的调节器结合在一起，产生移动纺锤体的不对称皮质牵引力。一旦纺锤体被定位，它就会向皮质发回信号，以确定卵裂的平面。PAR蛋白如何促进纺锤体定位因子的不对称，以及G蛋白途径如何与其他信号机制整合，仍有待阐明。目标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以抑制其在后皮质定位的假设将通过体外激酶分析和体内转基因研究来验证。PAR-3通过一种单独的机制抑制let-99在前部的定位，这一机制将结合活体成像和遗传分析进行研究。还将测试let-99相互作用蛋白在let-99大脑皮层锚定中的作用。目标2的目的是确定let-99/G？通路如何与Rho家族GTP酶信号整合，以适当地定位胞质分裂沟相对于纺锤体的位置。对突变体定位模式的定量分析将与生化相互作用分析相结合，以确定这些途径如何相互作用。Let-99通过其部分RhoGAP结构域直接与Rho GTP酶结合的假设也将得到检验。最后，目标3将检验这样一个假设，即LET-99的人类同源基因DEPDC1和DEPDC1B具有与LET-99类似的功能。具体地说，我们将检验这一假设，即这些蛋白质与G？或Rho相关，并参与纺锤体运动或胞质分裂。由于途径组分的保守性，这些研究的结果将与许多系统中的不对称分裂相关，并将定义一类新的蛋白质--DEPDC1家族的功能。 与公共卫生相关：在正常发育过程中，不对称分裂和纺锤体位置对于产生细胞多样性至关重要。最近的研究也强调了不对称分裂在干细胞维持和癌症中的重要性。所有研究中的蛋白质都是保守的。此外，对线虫LET-99的分析表明，LET-99是一种新的G蛋白信号转导调节因子，属于DEPDC1家族蛋白。因此，对线虫纺锤体定位的机制基础和LET-99的生化功能的研究将对癌症和干细胞生物学等健康相关领域有很大的帮助。此外，DEPDC1在膀胱癌中表达上调。该提案中概述的DEPDC1的研究将开始表征该蛋白质的细胞生物学功能，这将有助于阐明其在癌细胞中的作用。