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：
10386679
负责人：
LESILEE S. ROSE
金额：
$ 8.84万
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 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10386679
关键词：
Address Alleles Animals Anterior Award Binding Biochemistry Caenorhabditis elegans Cell Maintenance Cell Nucleus Cell Separation Cell division Cells Centrosome Complex Cues Data Daughter Development Developmental Process Disease Dynein ATPase Embryo Embryonic Development Ensure Excision Exhibits Family Fertilization Genetic Guanosine Triphosphate Phosphohydrolases Human Inherited Knowledge Lead Lobular Neoplasia Malignant Neoplasms Microtubules Mitotic spindle Modeling Morphogenesis Motor Nuclear Organism Parents Pathway interactions Pattern Phosphotransferases Positioning Attribute Process Protein Family Proteins Regulation Research Role Signal Pathway Signal Transduction Signal Transduction Pathway Study models System Temperature Testing Tissues WNT Signaling Pathway Work base cell fate specification cell type daughter cell dynactin embryo cell genetic analysis imaging study insight novel polarized cell prevent recruit response sperm cell stem cells temperature sensitive mutant

项目摘要

Asymmetric divisions contribute to cell fate specification during embryonic development and stem cell maintenance. Spindle alignment with a polarized axis is essential for this process, and spindle position is also crucial in symmetrically dividing cells to maintain proper cellular arrangements. Disregulation of spindle positioning and polarity has been implicated in cancer. This proposal addresses how multiple polarity cues coordinate to produce proper spindle alignment, using the early Caenorhabditis elegans embryo. Asymmetric division of the one-cell embryo relies on a conserved pathway in which the PAR polarity proteins regulate cortical asymmetry of the Ga/GPR/LIN-5 complex; this complex recruits the microtubule motor dynein to generate pulling forces that orient spindles. LET-99, a DEPDC1 family protein, is localized in an asymmetric cortical band pattern on the anterior/posterior (AP) axis by the PAR proteins, and LET-99 in turn restricts the localization of GPR/LIN-5 to generate asymmetric pulling forces. Similar PAR and LET-99 domains are reestablished in the P1 daughter of the first division. P1 divides into P2 and EMS, which both divide asymmetrically, but only P2 exhibits AP PAR domains. The EMS division is regulated by Wnt and Mes/Src signaling pathways, which polarize the cell and align the spindle on the AP axis. LET-99, LIN-5 and the PAR proteins are present in EMS, but here the PARs are polarized along the inner/outer instead of the AP axis. Our recent studies using fast-inactivating temperature sensitive alleles reveal that LET-99 has a primary role in spindle positioning in many cells of the early embryos, and acts in the Mes/Src pathway in EMS. We also identified roles for LIN-5 and the PAR-1 related kinase PIG-1 in EMS. Our observations support our central hypothesis that LET-99 acts downstream of multiple polarity cues to inhibit the localization or activity of LIN- 5 and thus regulate spindle position in different cell types. In Aim 1, we will elucidate mechanisms within the Mes/Src pathway using genetic analysis to test the hypothesis that PIG-1 acts upstream or parallel to LET-99. We will also determine if LET-99, LIN-5, PIG-1 and/or DNC (dynactin, a dynein regulator) are asymmetrically localized in EMS, and test the hypothesis that LET-99 prevents recruitment of cortical LIN-5. This aim will use photo-activated fluorescent proteins to resolve cortical asymmetry in a multicellular context. In Aim 2, we will use a combination of genetics and biochemistry to define the role of CED-10/RAC in the Mes/Src pathway and distinguish between several hypothesis for how LET-99 and CED-10 interact. In Aim 3, we will use live imaging studies and temperature sensitive mutants to test several models for P1 polarity reestablishment. The regulation of the second division is little studied, and LET-99, LIN-5 and PIG-1 are the first intermediates for spindle positioning in the Mes/Src pathway to be analyzed. This research will significantly advance our knowledge of these pathways. Because the pathway components are conserved, the results will be relevant to asymmetric division and spindle positioning in many systems.

不对称分裂有助于胚胎发育和干细胞过程中的细胞命运规范维护。主轴与偏振轴对齐对于此过程至关重要，并且主轴位置也对于对称分裂细胞以维持适当的细胞排列至关重要。主轴失调位置和极性与癌症有关。该提案解决了多重极性线索如何使用早期秀丽隐杆线虫胚胎进行协调以产生适当的纺锤体排列。不对称单细胞胚胎的分裂依赖于 PAR 极性蛋白调节的保守途径 Ga/GPR/LIN-5 复合物的皮质不对称性；该复合体招募微管运动动力蛋白产生定向主轴的拉力。 LET-99 是一种 DEPDC1 家族蛋白，定位于不对称 PAR 蛋白在前/后 (AP) 轴上形成皮质带图案，而 LET-99 反过来又限制了 GPR/LIN-5 的定位以产生不对称的拉力。类似的 PAR 和 LET-99 域是重新建立了P1女儿甲级联赛。 P1分为P2和EMS，两者都分为不对称，但只有 P2 表现出 AP PAR 域。 EMS部门受Wnt和Mes/Src监管信号通路，使细胞极化并使纺锤体在 AP 轴上对齐。 LET-99、LIN-5 和 PAR EMS 中存在蛋白质，但此处 PAR 沿内/外轴而不是 AP 轴极化。我们的最近使用快速失活温度敏感等位基因的研究表明，LET-99 在纺锤体定位于早期胚胎的许多细胞中，并在 EMS 中的 Mes/Src 通路中发挥作用。我们也确定了 LIN-5 和 PAR-1 相关激酶 PIG-1 在 EMS 中的作用。我们的观察支持我们的中央假设 LET-99 在多个极性线索下游发挥作用，抑制 LIN- 的定位或活性 5 从而调节不同细胞类型中纺锤体的位置。在目标 1 中，我们将阐明 Mes/Src 通路使用遗传分析来检验 PIG-1 作用于 LET-99 上游或平行的假设。我们还将确定 LET-99、LIN-5、PIG-1 和/或 DNC（dynactin，动力蛋白调节剂）是否不对称定位于 EMS，并检验 LET-99 阻止皮质 LIN-5 募集的假设。这个目标将使用光激活荧光蛋白可解决多细胞环境中的皮质不对称性。在目标 2 中，我们将结合遗传学和生物化学来定义 CED-10/RAC 在 Mes/Src 通路中的作用并区分 LET-99 和 CED-10 如何相互作用的几种假设。在目标 3 中，我们将使用 live 成像研究和温度敏感突变体来测试 P1 极性重建的几种模型。这第二分裂的调控研究很少，LET-99、LIN-5和PIG-1是第一个中间体要分析的 Mes/Src 路径中的主轴定位。这项研究将极大地推进我们的对这些途径的了解。由于途径成分是保守的，因此结果将与许多系统中的不对称分割和主轴定位。