Developmental Control of Spindle Positioning in Embryos

不对称分裂有助于胚胎发育和干细胞过程中的细胞命运特化 上维护主轴与极化轴的对齐对于该过程至关重要，并且主轴位置也是 在对称分裂的细胞中至关重要，以维持适当的细胞排列。纺锤体失调 定位和极性与癌症有关。这个建议解决了多重极性线索 协调产生适当的纺锤体对齐，使用早期秀丽隐杆线虫胚胎。不对称 单细胞胚胎的分裂依赖于一个保守的途径，其中PAR极性蛋白调节 G α/GPR/LIN-5复合物的皮质不对称性;该复合物招募微管运动动力蛋白， 产生牵引力来定向纺锤体。LET-99是DEPDC1家族蛋白，定位于不对称的 皮质带模式上的前/后（AP）轴的PAR蛋白，和LET-99反过来限制了 定位GPR/LIN-5以产生不对称拉力。类似的PAR和LET-99结构域是 在第一师的P1女儿中重新建立。P1分为P2和EMS，两者均分为 不对称，但只有P2表现出AP PAR结构域。EMS部门由Wnt和Mes/Src监管 信号通路，其使细胞稳定并使纺锤体在AP轴上对齐。LET-99、LIN-5和PAR 蛋白质存在于EMS中，但这里PAR沿着内/外轴而不是AP轴极化。我们 最近使用快速失活温度敏感等位基因的研究表明，LET-99在以下方面具有主要作用： 纺锤体定位在早期胚胎的许多细胞中，并在EMS中的Mes/Src通路中起作用。我们也 确定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作用于多个极性线索的下游以抑制LIN-1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上游或平行于LET-99的假设。 我们还将确定LET-99、LIN-5、PIG-1和/或DNC（动力蛋白，一种动力蛋白调节剂）是否不对称 定位于EMS，并测试LET-99阻止皮质LIN-5募集的假设。这一目标将使用 光激活荧光蛋白来解决多细胞背景下的皮质不对称性。在目标2中，我们将 使用遗传学和生物化学的组合来确定CED-10/RAC在Mes/Src途径中的作用 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，以测试P1极性重建的几种模型。的 第二次分裂的调节研究很少，LET-99、LIN-5和PIG-1是第一次分裂的中间体。 纺锤体在Mes/Src途径中的定位进行分析。这项研究将大大促进我们的 了解这些途径。由于途径组分是保守的，因此结果将与 在许多系统中不对称的划分和主轴定位。

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 Gα/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.