Probing the spatiotemporal regulation of cell division

项目摘要 在动物细胞中，胞质分裂是由肌动球蛋白收缩环的收缩驱动的，肌动球蛋白收缩环定位并 由纺锤体微管发出的信号控制。胞质分裂需要高度的时空 分子调控以确保每个子细胞继承单个细胞核。虽然基本的分子 胞质分裂所需的参与者是已知的，许多胞质分裂蛋白质动态定位于多个 亚细胞壁龛在整个细胞分裂，潜在地允许多种亚细胞功能，使这些 使用传统的遗传学方法很难研究蛋白质。光遗传学使时空研究成为可能 局部靶向光以控制特定亚细胞区域中的蛋白质功能。因此，我们开发了 FLIRT（快速局部红外热遗传学），它使用红外（IR）激光局部加热，从而局部 具有高时空特异性的基因编码的快速作用温度敏感（TS）突变蛋白 精度FLIRT也是可逆的：红外激光器可以在任何时候关闭，以停止局部加热， 用于蛋白质再活化。此外，使用FLIRT，非ts-突变体（野生型）可以用作任何突变的对照。 由给定FLIRT程序引起的激光诱导损伤。在初步数据中，使用C。elegans胚胎，我们 校正了FLIRT的温度诱导效应，验证了FLIRT在亚细胞水平上的应用 在1-细胞胚胎和16-细胞胚胎中，并证明FLIRT可以抑制其他细胞生物学活性， 多细胞胚胎中的细胞命运信号和成年性腺中的膜分配等过程。 在为进一步的研究奠定了基础之后，我们现在提出实验来定义时空 肌动球蛋白收缩性和纺锤体微管相关信号复合物的调节 胞质分裂，并解决长期存在的问题，在该领域，如赤道与相对贡献。 在细胞分裂期间的极性肌动球蛋白收缩性。FLIRT实验也将在C. 线虫早期胚胎和体细胞中的多细胞发育蠕虫组织。这些实验将 定义胞质分裂中关键参与者的精确时空调节，测试关于 它们的作用机制，并测试管理胞质分裂规则的普遍性，是否相同 适用于早期胚胎的原理也适用于多细胞环境中的体细胞。

PROJECT SUMMARY In animal cells, cytokinesis is driven by constriction of an actomyosin contractile ring, which is positioned and controlled by signaling from spindle microtubules. Cytokinesis requires a high degree of spatial and temporal molecular regulation to ensure each daughter cell inherits a single nucleus. Although the essential molecular players required for cytokinesis are known, many cytokinesis proteins localize dynamically to multiple subcellular niches throughout cell division, potentially allowing multiple subcellular functions and making these proteins difficult study using traditional genetic approaches. Optogenetics enable spatiotemporal studies by locally targeting light to control protein function in specific subcellular regions. For this reason, we developed FLIRT (Fast Local Infrared Thermogenetics), which uses an infrared (IR) laser to locally heat and thus locally inactivate genetically-encoded fast-acting temperature sensitive (ts) mutant proteins with high spatiotemporal precision. FLIRT is also reversible: the IR laser can be turned off at any point to stop local heating and allow for protein reactivation. Furthermore, using FLIRT, non-ts-mutants (wildtype) can be used as controls for any laser-induced damage induced by a given FLIRT procedure. In preliminary data using C. elegans embryos, we calibrated the temperature induction achieved using FLIRT, validated the use of FLIRT on the subcellular level in both the 1-cell embryo and the 16-cell embryo, and demonstrated that FLIRT can inhibit other cell biological processes such as cell fate signaling in multicellular embryos and membrane partitioning in the adult gonad. Having laid the foundation for further studies, we now propose experiments to define the spatiotemporal regulation of actomyosin contractility and spindle microtubule-associated signaling complexes during cytokinesis and address longstanding questions in the field, such as the relative contributions of equatorial vs. polar actomyosin contractility during cell division. FLIRT experiments will also be conducted on cells in C. elegans early embryos and in somatic cells with multicellular developing worm tissue. These experiments will define the precise spatiotemporal regulation of key players in cytokinesis, test specific hypotheses regarding their mechanisms of action, and test the universality of rules governing cytokinesis, whether the same principles that apply to the early embryo also apply to somatic cells within a multicellular context.