Cytokinesis without an actomyosin ring: studies in Chlamydomonas

这个项目是关于细胞分裂的进化，将在几个方面有更广泛的意义。首先，它将有助于对抗细胞和分子生物学研究对动物、真菌和陆地植物的过度关注。关于其他有趣的细胞类型的巨大多样性，有大量的东西需要学习，其中许多在生态和/或经济上也很重要。其次，它不仅将为衣藻研究人员提供新的生物学见解，而且还将为衣藻研究人员提供有价值的研究工具，例如改进转基因调控表达和细胞分裂同步的方法。这些工具不仅对基础研究有价值，而且对生物技术，特别是生物燃料工业也有价值，而生物燃料工业反过来又有利于燃料供应和环境保护。第三，它将支持一个非常有才华的年轻科学家的研究事业，帮助他进入一个独立的学术职位。最后，它将有助于支持本科生、高中生和公立学校教师参与研究，特别强调教师，因为他们自己要教的学生数量很大。几乎所有以前接待的学生（包括许多来自代表性不足的群体）都在STEM领域继续发展，老师们都把实验室的想法和材料带回了教室。这些模式应该在当前项目下继续。本项目将利用绿藻衣藻的进化地位和实验优势，阐明细胞质分裂的核心机制，这是细胞分裂的最后一步。研究的重点是细胞如何在没有“可收缩的肌动球蛋白环”（CAR）的情况下形成分裂沟。CAR存在于动物和真菌中，由肌动蛋白丝、II型肌球蛋白和其他蛋白质组成。长期以来，人们一直认为它通过肌动蛋白-肌球蛋白的相互作用提供了沟侵入的力量，就像在肌肉中一样。然而，许多通常有CAR的细胞在没有CAR的情况下也能分裂，就像大量形成沟但没有II型肌球蛋白的细胞（如衣单胞菌）一样；因此，一定有另一个驱动犁沟的因素。为了阐明这些机制，将探讨几个相关的目标。首先，结合显微镜、生物化学、药理学和遗传学的方法来分析肌动蛋白和微管细胞骨架的可能作用。其次，将使用类似的方法组合来探索沟形成的主要驱动因素是质膜和细胞外基质的局部生长的可能性。第三，无偏见和有针对性的基因筛选将寻找具有特定细胞分裂缺陷的突变体；这些突变的特征应该有助于揭示驱动沟形成的潜在机制。这些研究应该有助于阐明现代生物中看似不同的细胞分裂模式的共同机制，以及这些现代机制从祖先细胞的机制进化而来。一些证据表明，细胞质分裂的起源和核心机制是新细胞膜和细胞外基质的局部沉积，肌动蛋白和微管细胞骨架在这一过程中起辅助作用。衣藻是研究这些问题的一个有吸引力的系统。它在进化上与细胞质分裂研究以前所关注的有机体相距甚远；通过沟的形成分裂，但没有II型肌凝蛋白；具有一个常规肌动蛋白，一个非常规肌动蛋白和一个发育良好的微管细胞骨架；在卵裂沟周围显示肌动蛋白和微管的富集；并且已经有很多基因工具可用。其细胞质分裂的机制几乎没有研究，阐明这些机制应该有助于细胞质分裂领域的必要范式转变。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project is about the evolution of cell division and will have broader significance in several ways. First, it will help to counter the excessive focus of research in cell and molecular biology on the animals, fungi, and land plants. There is an enormous amount to be learned about the huge diversity of other interesting cell types, many of which are also ecologically and/or economically important. Second, it will provide Chlamydomonas researchers not only with new biological insights but also with valuable research tools, such as improved methods for regulated transgene expression and synchronization of cell division. These tools should be of value not only for basic research but also for biotechnology, particularly in the biofuels industry, which in turn could have benefits for fuel supplies and environmental conservation. Third, it will support the research career of a very talented young scientist and help him to progress into an independent academic position. Finally, it will help to support the involvement of undergraduates, high-school students, and public-school teachers in research, with special emphasis on the teachers because of the large numbers of students that they themselves then go on to teach. Nearly all previously hosted students (including many from underrepresented groups) have gone on in STEM fields, and the teachers have all carried ideas and materials from the laboratory back to their classrooms. These patterns should continue under the current project.This project will exploit the evolutionary position and experimental advantages of the green alga Chlamydomonas to elucidate the still-obscure core mechanisms of cytokinesis, the final step in cell division. The focus is on how cells can form division furrows in the absence of a "contractile actomyosin ring" (CAR). The CAR is found in animals and fungi and is composed of actin filaments, type II myosin, and other proteins. It has long been thought to provide the force for furrow ingression via an actin-myosin interaction like that in muscle. However, many cells that normally have a CAR can divide without it, as can the vast number of cell types (such as Chlamydomonas) that form furrows but have no type II myosin; thus, there must be another driver(s) of furrowing. To elucidate these mechanisms, several related aims will be pursued. First, a combination of microscopic, biochemical, pharmacological, and genetic methods will be used to analyze the possible roles of the actin and microtubule cytoskeletons. Second, a similar combination of methods will be used to explore the possibility that the primary driver of furrow formation is localized growth of the plasma membrane and extracellular matrix. Third, unbiased and targeted genetic screens will seek mutants with specific defects in cytokinesis; characterization of these mutants should help to reveal the underlying mechanisms that drive furrow formation. These studies should help to elucidate both the shared mechanisms underlying the seemingly diverse modes of cytokinesis in modern organisms and the evolution of these modern mechanisms from those of ancestral cells. Several lines of evidence suggest that the ancestral and still-core mechanism of cytokinesis is the localized deposition of new cell membrane and extracellular matrix, and that the actin and microtubule cytoskeletons play auxiliary roles in this process. Chlamydomonas is an attractive system in which to investigate these issues. It is evolutionarily distant from the organisms on which cytokinesis studies have previously focused; divides by furrow formation but does not have a type II myosin; has one conventional actin, one unconventional actin, and a well developed microtubule cytoskeleton; displays an enrichment of both actin and microtubules around the cleavage furrow; and has many genetic tools already available. Its mechanisms of cytokinesis have hardly been studied, and elucidating them should contribute to a needed paradigm shift in the cytokinesis field.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.