Developmental Control of Spindle Positioning in Embryos

不对称分裂在胚胎发育和干细胞过程中对细胞命运的规定 维修。在这一过程中，主轴与极化轴的对准是必不可少的，主轴位置也是如此 在对称分裂细胞以维持适当的细胞排列方面至关重要。纺锤体失调症 位置和极性与癌症有关。这项建议解决了多重极性如何暗示 使用早期秀丽线虫胚胎，协调以产生适当的纺锤体对齐。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复合体的皮质不对称性；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，定位于不对称的 PAR蛋白在前/后(AP)轴上的皮质条带模式，而let-99反过来限制 GPR/LIN-5的局部化以产生不对称拉力。类似的PAR和LET-99域名是 在第一师的小一女儿中重新建立。P1分为P2和EMS，两者都分为 不对称，但只有P2显示AP-PAR结构域。EMS部门由WNT和MES/Src监管 信号通路，使细胞极化，并使纺锤体与AP轴对齐。让-99，林-5和标准杆 EMS中存在蛋白质，但这里的PARs是沿着内/外而不是AP轴极化的。我们的 最近使用快速失活的温度敏感等位基因的研究表明，let-99在 纺锤体在早期胚胎的许多细胞中定位，并在EMS的MES/Src途径中发挥作用。我们也 确定了LIN-5和PAR-1相关的蛋白激酶PIG-1在EMS中的作用。我们的观察结果支持我们的中心 假设LET-99作用于多极性信号的下游，以抑制LIN的定位或活性- 5，从而调节不同细胞类型的纺锤体位置。在目标1中，我们将阐明 利用基因分析验证PIG-1与LET-99上游或平行作用的假说。 我们还将确定let-99、Lin-5、PIG-1和/或DNC(dynactin，动力蛋白调节因子)是否不对称 定位于EMS，并检验LET-99阻止皮质LIN-5招募的假设。这一目标将使用 光激活荧光蛋白在多细胞环境中解决皮质不对称问题。在目标2中，我们将 结合遗传学和生物化学来确定CED-10/Rac在MES/Src途径中的作用 并区分了LET-99和CED-10相互作用的几种假说。在目标3中，我们将使用LIVE 成像研究和对温度敏感的突变体来测试几个重建P1极性的模型。这个 对第二分裂的调控研究较少，其中let-99、Lin-5和PIG-1为第一中间体。 要分析的主轴在MES/Src途径中的位置。这项研究将大大推进我们的 对这些途径的了解。因为途径成分是保守的，所以结果将与 许多系统中的不对称分割和主轴定位。

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 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.