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期间细胞命运规格维护。与偏振轴的主轴对齐对于此过程至关重要，主轴位置也是在对称分裂的细胞中至关重要，以维持适当的细胞排列。毫无疑问主轴定位和极性与癌症有关。该提议解决了多个极性提示使用秀丽隐杆线虫的早期胚胎，协调以产生适当的纺锤排列。不对称单细胞胚胎的分裂取决于一种保守的途径，在该途径中，极性蛋白调节 GA/GPR/LIN-5复合物的皮质不对称；这个复合物将微管运动动力蛋白招募到产生向定向纺锤体的拉力。 Let-99是DEPDC1家族蛋白，位于不对称 PAR蛋白的前/后轴（AP）轴上的皮质带模式，而Let-99又限制了 GPR/LIN-5的定位以产生不对称的拉力。类似的标准杆和Let-99域是在第一分区的P1女儿中重新建立。 P1分为P2和EMS，两者都分裂不对称，但只有P2表现出Ap par结构域。 EMS部门由WNT和MES/SRC调节信号通路，该路径使电池偏振并在AP轴上对齐主轴。 Let-99，Lin-5和PAR 蛋白质存在于EMS中，但是这里的PARS沿着内部/外部而不是AP轴极化。我们的最近使用快速灭活温度敏感等位基因的研究表明，Let-99在在早期胚胎的许多细胞中纺锤定位，并在EMS中的MES/SRC途径中作用。我们也是确定了EMS中LIN-5和PAR-1相关激酶PIG-1的作用。我们的观察支持我们的中心 Let-99在多个极性提示下进行的假设抑制了lin-的定位或活性 5因此调节不同细胞类型的主轴位置。在AIM 1中，我们将阐明内部的机制使用遗传分析的MES/SRC途径测试PIG-1在上游或平行于Let-99的假设。我们还将确定Let-99，Lin-5，Pig-1和/或DNC（Dynactin，Dynein调节剂）是否不对称局部在EMS中，并检验Let-99阻止皮质LIN-5的假设。这个目标将使用光激活的荧光蛋白可以在多细胞环境中解决皮质不对称。在AIM 2中，我们将结合遗传学和生物化学来定义CED-10/RAC在MES/SRC途径中的作用并区分Let-99和CED-10相互作用的几个假设。在AIM 3中，我们将现场使用成像研究和温度敏感突变体，以测试多种模型是否重建P1极性。这几乎没有研究第二师的调节，而Let-99，Lin-5和Pig-1是第一个中间体在MES/SRC途径中的主轴定位要分析。这项研究将大大推动我们的了解这些途径。由于途径组件是保存的，因此结果将与许多系统中的不对称分裂和主轴定位。