EAGER: Cytokinesis mechanisms and cytoskeletal dynamics in Chlamydomonas

细胞质分裂是细胞分裂的最后阶段，单个母细胞在物理上分裂成两个子细胞，对于任何形式的生命细胞的正常增殖和分化都是必不可少的。从历史上看，细胞质分裂的研究只集中在两类生物上：动物、真菌和它们的亲戚，它们的细胞通过形成一个向内生长的“卵裂沟”分裂；陆地植物，它们的细胞通过形成一个向外生长的“细胞板”分裂。因此，目前在细胞生物学的核心存在一个未解决的悖论：现代细胞的共同祖先是通过什么机制分裂的？这种机制是如何演变成今天看到的看似非常不同的机制的？本项目将利用莱茵衣藻（Chlamydomonas reinhardtii）的实验优势和有利的进化地位（与陆地植物密切相关，但像动物一样通过形成卵裂沟进行分裂）来帮助阐明祖先细胞中细胞分裂的共同核心机制，尽管它们表面上存在差异，但仍然在现代生物中共享。了解这些核心机制不仅可以更好地理解动物、真菌和植物细胞，还可以更好地理解其他细胞类型，这些细胞类型在很大程度上被细胞生物学家所忽视，但具有巨大的生态和经济重要性。除了基础知识之外，该项目还将提供在基础研究和应用研究（特别是生物燃料开发）中都有价值的新实验工具，并将为博士后、研究生、本科生和高中生（包括来自代表性不足群体的学生）提供学习遗传学、分子生物学和细胞生物学的机会，作为继续从事科学和工程职业的前奏。该项目的重点是阐明衣藻细胞在没有收缩肌动蛋白环（CAR）的情况下形成分裂沟的机制，CAR是一种由丝状肌动蛋白、II型肌球蛋白和其他蛋白质组成的结构，长期以来被认为是动物和真菌细胞中形成沟的必要条件。然而，现在很清楚的是，许多通常具有CAR的细胞可以在没有CAR的情况下分裂，而其他系统发育群中的大多数真核细胞通过形成沟而分裂，但没有肌球蛋白- ii，因此这一过程必须有另一个驱动因素。为了阐明衣藻沟槽的机制和相关的细胞骨架功能，将追求三个相互关联的目标。(1)将开发用于表达感兴趣的标记蛋白以及细胞分裂过程和细胞骨架动力学的显微可视化的改进方法，并用于阐明这些过程的细节和在分子水平上要提出的问题。(2)结合显微镜，生物化学，遗传学和转录组分析将用于研究f -肌动蛋白在卵裂沟中的可能作用，以及最近发现的肌动蛋白解聚的戏剧性转录调节反应的机制和作用。类似的方法将用于研究单个衣藻septin蛋白的功能，该蛋白也有望（从与其他生物的比较中）参与细胞分裂。(3)基于成像的方法和高通量平台将用于识别细胞分裂和/或细胞骨架动力学缺陷的突变体；这些突变体的特征将有助于揭示细胞周期中沟槽形成和细胞骨架行为的潜在机制。

Cytokinesis is the final stage in cell division in which the single mother cell is physically separated into two daughter cells, and is essential for the normal proliferation and differentiation of cells of every form of life. Historically, studies of cytokinesis have focused on just two groups of organisms: the animals, fungi and their relatives, whose cells divide by forming an inward-growing "cleavage furrow", and the land plants, whose cells divide by forming an outward-growing "cell-plate". Thus, there is currently an unresolved paradox at the heart of cell biology: by what mechanism did the common ancestor of modern cells divide, and how did that mechanism evolve into the seemingly very different mechanisms seen today? This project will exploit the experimental advantages and favorable evolutionary position of the green alga Chlamydomonas reinhardtii (which is closely related to land plants but divides by forming a cleavage furrow like animals) to help elucidate the common core mechanisms that underlay cytokinesis in ancestral cells and are still shared in modern organisms despite their superficial differences. Understanding these core mechanism will provide a better understanding not only of animal, fungal, and plant cells, but also of a broad range of other cell types that have been largely ignored by cell biologists but are of enormous ecological and economic importance. In addition to basic knowledge, this project will provide new experimental tools that should be valuable both in basic research and in applied studies (particularly in biofuels development), and it will provide opportunities for postdoctoral, graduate, undergraduate, and high-school students (including students from underrepresented groups) to learn genetics, molecular biology, and cell biology as a prelude to continuing in careers in science and engineering.This project focuses on elucidation of the mechanisms by which Chlamydomonas cells form cleavage furrows for cell division without a contractile actomyosin ring (CAR), a structure formed of filamentous (F) actin, type II myosin, and other proteins, which has long been thought to be necessary for furrow formation in animal and fungal cells. However,it is now clear both that many cells that normally have a CAR can divide without it and that most eukaryotic cells in other phylogenetic groups divide by forming furrows but have no myosin-II, so there must another driver(s) of this process. To elucidate the mechanisms of furrowing in Chlamydomonas and the associated cytoskeletal functions, three interrelated aims will be pursued. (1) Improved methods for expressing tagged proteins of interest and for microscopic visualization of cytokinesis processes and cytoskeletal dynamics will be developed and used to clarify the details of these processes and the questions to be asked at the molecular level. (2) A combination of microscopy, biochemistry, genetics, and transcriptome analyses will be used to investigate the possible role of F-actin in the cleavage furrow and the mechanisms and roles of the recently discovered dramatic transcriptional regulatory response to actin depolymerization. Similar approaches will be used to investigate the function(s) of the single Chlamydomonas septin protein, which is also expected (from comparisons to other organisms) to be involved in cytokinesis. (3) Imaging-based methods and high-throughput platforms will be used to identify mutants defective in cytokinesis and/or cytoskeletal dynamics; characterization of these mutants should help to reveal the underlying mechanisms of furrow formation and cytoskeletal behavior through the cell cycle.