CAREER: Cytokinesis without an actomyosin ring and its coordination with organelle division

胞质分裂是一个母细胞分裂成两个或多个子细胞的基本细胞过程。今天的所有细胞都通过胞质分裂的连续事件与数十亿年前出现的第一个细胞相连。尽管有这种连续性，但在今天的动物、植物和原核生物中看到的细胞质分裂机制似乎没有潜在的原理，因为大多数细胞质分裂研究集中在一小部分生物和细胞类型上。因此，我们目前对细胞质分裂机制的进化缺乏洞察力。在这个项目中，我们将通过使用绿藻衣藻来解决我们对细胞质分裂的理解中目前的知识差距和固有的偏见。衣藻，像动物一样通过插入质膜进行分裂，但它缺乏典型的动物细胞分裂机制。它的分裂涉及植物分裂机械和许多真核生物中保存的其他成分的混合，包括人类。此外，衣藻只有一个叶绿体，该叶绿体必须与细胞分裂过程相协调地分裂和分离成子细胞。叶绿体来源于蓝藻，蓝藻被早期的真核生物吞噬，成为稳定的内共生体，因此拥有自己的细菌起源的分裂机制。因此，通过研究衣藻的胞质分裂及其与叶绿体分裂的协调，本项目将阐明(I)在大多数真核生物中保守的胞质分裂的原始和基本机制，以及(Ii)真核细胞和细菌分裂系统之间的通信对于建立内共生并随后维持这一光合作用所必需的细胞器至关重要。这项工作的更广泛影响包括研究的内在本质，因为所有细胞都经历了胞质分裂的过程。此外，该项目将为高中生和他们的老师提供研究机会，并教育本科生和研究生关于当前模式生物使用的偏见和新兴模式可能带来的潜在未来机会。该项目的总体研究目标是阐明(I)衣藻中肌动蛋白非依赖的、微管依赖的分裂沟内侵的机制和(Ii)肌动蛋白细胞骨架在胞质分裂和叶绿体分裂的时空协调中的作用。为此，这项研究的一部分将利用光学和电子显微镜研究微管是如何在分裂部位组织起来的，并从遗传学和蛋白质组学角度探讨增强蛋白介导的沟槽相关微管形成的机制。另一方面，该项目将研究叶绿体分裂所需的核编码蛋白如何以F-肌动蛋白依赖的方式转运到叶绿体中，并通过遗传筛选和基于邻近的蛋白质组学识别参与叶绿体分裂的新基因和蛋白质。识别肌动球蛋白非依赖的分裂-皱沟进入机制对于阐明在真核细胞树中发现的似乎多样化和专门化的细胞质分裂模式的普遍、基本规则以及理解这些现代细胞是如何从没有肌动球蛋白环的祖先细胞进化而来的至关重要的。这项研究也与研究早期真核生物的进化事件有关，因为分裂的时空协调是祖先真核细胞和蓝藻细胞之间内共生的关键步骤。衣藻提供了一个极好的系统来填补我们目前理解中的这些空白。这个项目代表了现有文学和课堂教育的重要范式转变。这个奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Cytokinesis is a fundamental cellular process in which a mother cell is divided into two or more daughter cells. All cells today are connected to the first cell that emerged several billion years ago, through the continuous events of cytokinesis. Despite this continuity, the mechanisms of cytokinesis as seen in today’s animals, plants, and prokaryotes appear unrelated without underlying principles, because most cytokinesis research has focused on a small number of organisms and cell types. As a result, we currently lack insights into the evolution of cytokinesis mechanisms. In this project, we will address the current knowledge gap and inherent bias in our understanding of cytokinesis by using the green alga Chlamydomonas reinhardtii. Chlamydomonas, divides by ingressing the plasma membrane like animals, yet it lacks the canonical animal cell-division machinery. Its division involves a hybrid of plant-division machinery and other components conserved in many eukaryotes, including humans. Furthermore, Chlamydomonas possesses a single chloroplast, which must be divided and segregated into daughter cells in coordination with the cell division process. Chloroplasts derive from a cyanobacterium that was engulfed by an early eukaryote and became a stable endosymbiont, and therefore possess their own division machinery of bacterial origin. Thus, by investigating cytokinesis and its coordination with chloroplast division in Chlamydomonas, this project will elucidate (i) the ancestral and fundamental mechanism of cytokinesis that is conserved in the majority of eukaryotes and (ii) the communication between the eukaryotic and bacterial division systems crucial for the establishment of endosymbiosis and subsequently the maintenance this essential organelle for photosynthesis. The Broader Impacts of the work include the intrinsic nature of the research as all cells undergo the process of cytokinesis. In addition, the project will provide research opportunities to high school students and their teachers, and educate undergraduate and graduate students about the current bias in the use of model organisms and the potential future opportunities emerging models can bring.The overall research goals of this project are to elucidate (i) the mechanism of actomyosin-independent, microtubule-dependent cleavage-furrow ingression in Chlamydomonas and (ii) the role of actin cytoskeleton in the spatiotemporal coordination of cytokinesis and chloroplast division. To this end, one part of this research will address how microtubules are organized at the division site using light and electron microscopy, and investigate the mechanisms of augmin-mediated formation of furrow-associated microtubules by genetics and proteomics. In another part, the project will investigate how nuclear-encoded proteins required for chloroplast division are transported to and translocated into the chloroplast in an F-actin-dependent manner, and identify novel genes and proteins involved in chloroplast division through genetic screens and proximity-based proteomics. Identifying the actomyosin-independent mechanisms of cleavage-furrow ingression is critical to elucidate the generalizable, fundamental rules governing the seemingly diverse and specialized modes of cytokinesis found across the eukaryotic tree, as well as to understand how these modern cells evolved from the ancestral ones that did not have an actomyosin ring. This research is also relevant to investigating the evolutionary events in early eukaryotes, because spatiotemporal coordination of division is a critical step in endosymbiosis between an ancestral eukaryotic cell and a cyanobacterial cell. Chlamydomonas provides an excellent system to fill these gaps in our current understanding. This project represents a crucial paradigm shift in the existing literature and classroom education.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.