Probing the spatiotemporal regulation of cell division

项目总结 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，该收缩环位于和 由纺锤体微管发出的信号控制。胞质分裂需要高度的空间和时间 分子调控，以确保每个子细胞继承一个细胞核。尽管必要的分子 胞质分裂所需的参与者是已知的，许多胞质分裂蛋白动态定位到多个 整个细胞分裂过程中的亚细胞生态位，潜在地允许多种亚细胞功能，并使这些 蛋白质很难用传统的遗传方法进行研究。光遗传学通过以下方式实现时空研究 局部靶向光线以控制特定亚细胞区域的蛋白质功能。出于这个原因，我们开发了 FLORT(快速局部红外线热遗传学)，它使用红外(IR)激光来局部加热，从而局部加热 高时空失活基因编码的快速反应温度敏感(TS)突变蛋白 精确度。调情也是可逆的：红外激光可以在任何时候关闭，以停止局部加热并允许 用来重新激活蛋白质。此外，使用调情，非ts突变体(野生型)可以用作任何 由特定的调情程序引起的激光损伤。在使用线虫胚胎的初步数据中，我们 校准了使用调情技术实现的温度感应，在亚细胞水平上验证了调情技术的使用 在1-细胞胚胎和16-细胞胚胎中都是如此，并证明调情可以抑制其他细胞的生物学特性。 多细胞胚胎中的细胞命运信号和成人性腺的膜分裂等过程。 在为进一步的研究奠定了基础之后，我们现在提议进行实验来定义时空 肌动蛋白收缩能力和纺锤体微管相关信号复合体的调节 细胞质分裂和解决该领域长期存在的问题，例如赤道和赤道的相对贡献。 细胞分裂过程中极肌动球蛋白的收缩能力。调情实验也将在C. 线虫的早期胚胎和体细胞中具有多细胞发育的蠕虫组织。这些实验将 定义细胞质分裂中关键角色的精确时空调节，测试关于以下方面的特定假设 它们的作用机制，并测试控制细胞质分裂的规则的普遍性，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.