Mechanisms of Cytokinesis in Yeast

描述（由申请人提供）：肿瘤由不受抑制的细胞分裂引起，包括有丝分裂和胞质分裂。有丝分裂的机制研究已经导致了成功的癌症治疗。胞质分裂的失败导致多倍体和遗传不稳定性，这通常与肿瘤有关。因此，胞质分裂的机制研究可能为设计癌症诊断和/或治疗策略开辟新的途径。动物和真菌细胞的胞质分裂涉及肌动球蛋白环（AMR）收缩和靶向膜沉积。随着遗传模型系统和全基因组筛选的发展，已经鉴定了大量AMR和膜运输的组分，其中大部分是从酵母到人类的保守组分。该领域现在面临的主要挑战是确定这些组件如何组装在一起，以形成高效率和保真度运行的收缩和膜运输“机器”，以及这些机器如何在时间和空间上协调。正是在这种情况下，我们建议解决两个主要问题，在胞质分裂使用的芽殖酵母模型。在第一个目标中，我们将研究胞质分裂过程中II型肌球蛋白的高阶组装的机制和功能，这是一个在任何系统中都知之甚少的基本问题。最近，我们已经证明了旋转阴影EM的第一次，肌球蛋白1，唯一的II型肌球蛋白在芽殖酵母，形成了一个“双头”的结构与“扭结”在其尾巴的中间，轴承的所有主要功能的非肌肉II型肌球蛋白在动物细胞。我们的初步研究还表明，“靶向”的Myo 1的分裂位点和“组装”的Myo 1到更高阶的结构控制通过不同的域在其尾部，在其他系统中，靶向和组装的II型肌球蛋白耦合。因此，芽殖酵母系统提供了一个独特的机会，以解决特定的在胞质分裂过程中的肌球蛋白高阶组装体内功能。在第二个目标中，我们将试图定义一个新的机制，AMR收缩和胞吐介导的ECM重塑胞质分裂过程中的协调。具体来说，我们将测试我们的假设，即C2结构域蛋白Inn 1协调AMR收缩和ECM重塑或初级隔膜（PS）在酵母中的形成，通过与AMR侧的IQGAP和ECM侧的假定转氨酶/蛋白酶Cyk 3相互作用，这反过来又刺激几丁质合成酶Chs 2的活性，以促进PS在分裂位点的形成。我们提出的研究将产生原始的见解，肌球蛋白高阶组装在胞质分裂中的作用，以及AMR收缩和ECM重塑在胞质分裂过程中的协调机制。 公共卫生相关性：胞质分裂是增殖、分化和发育所必需的基本过程。胞质分裂的失败与严重的人类疾病如癌症有关。因此，研究胞质分裂的机制将在基础科学和临床科学中具有深远的意义。

DESCRIPTION (provided by applicant): Tumors arise from uninhibited cell division, which includes mitosis and cytokinesis. Mechanistic study of mitosis has led to successful cancer therapies. Failure in cytokinesis leads to polyploidy and genetic instability that is often associated with tumors. Thus, mechanistic study of cytokinesis may open new avenues for designing strategies for cancer diagnosis and/or treatment. Cytokinesis in animal and fungal cells involves actomyosin ring (AMR) contraction and targeted membrane deposition. With the development of genetic model systems and genome-wide screens, a large number of components of the AMR and membrane trafficking have been identified, most of which are conserved from yeast to humans. The central challenge for the field now is to determine how these components are assembled together to form the contractile and membrane-trafficking "machines" that operate with high efficiency and fidelity, and how these machines are coordinated in time and space. It is within this context, we propose to address two major questions in cytokinesis using the budding yeast model. In the first Aim, we will investigate the mechanism and function of higher-order assembly of type-II myosin during cytokinesis, a fundamental question that remains poorly understood in any system. Recently, we have demonstrated by rotary-shadowing EM for the first time that Myo1, the sole type-II myosin in budding yeast, forms a "two-headed" structure with a "kink" in the middle of its tail, bearing all the major features of non-muscle type-II myosins in animal cells. Our preliminary studies also suggest that the "targeting" of Myo1 to the division site and the "assembly" of Myo1 into higher-order structures are controlled through distinct domains in its tail, in contrast to other systems where targeting and assembly of type-II myosins are coupled. Thus, the budding yeast system provides a unique opportunity to address the specific in vivo function of myosin higher-order assembly during cytokinesis. In the second Aim, we will attempt to define a novel mechanism underlying the coordination between AMR contraction and the exocytosis-mediated ECM remodeling during cytokinesis. Specifically, we will test our hypothesis that the C2-domain protein Inn1 coordinates AMR contraction and ECM remodeling or primary-septum (PS) formation in yeast by interacting with IQGAP on the AMR side and with a putative transglutaminase/protease Cyk3 on the ECM side, which, in turn, stimulates the activity of the chitin synthase Chs2 to promote PS formation at the division site. Our proposed studies will generate original insights into the role of myosin higher-order assembly in cytokinesis as well as the mechanisms underlying the coordination between AMR contraction and ECM remodeling during cytokinesis. PUBLIC HEALTH RELEVANCE: Cytokinesis is a fundamental process essential for proliferation, differentiation, and development. Failure in cytokinesis is associated with serious human diseases such as cancer. Thus, studying the mechanisms of cytokinesis will have profound implications in basic as well as clinical sciences.