Mechanisms of Cytokinesis in Yeast

描述(申请人提供)：肿瘤起源于不受抑制的细胞分裂，包括有丝分裂和胞质分裂。有丝分裂的机制研究导致了癌症治疗的成功。胞质分裂失败会导致多倍体和遗传不稳定，这通常与肿瘤有关。因此，细胞质分裂的机制研究可能会为癌症诊断和/或治疗策略的设计开辟新的途径。动物和真菌细胞的胞质分裂包括肌动球蛋白环(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，人们已经鉴定出大量的AMR组分和膜转运，其中大部分是从酵母到人类的保守。目前该领域的中心挑战是确定这些部件如何组装在一起，形成高效率和高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。正是在这一背景下，我们建议使用发芽酵母模型来解决细胞质分裂中的两个主要问题。在第一个目标中，我们将研究胞质分裂过程中II型肌球蛋白的高级组装的机制和功能，这是一个在任何系统中仍然知之甚少的基本问题。最近，我们通过旋转跟踪EM首次证明了发芽酵母中唯一的II型肌球蛋白Myo1形成了一个在尾巴中间有一个“扭结”的“双头”结构，具有动物细胞中非肌肉II型肌球蛋白的所有主要特征。我们的初步研究还表明，Myo1对分裂位点的靶向和Myo1向高阶结构的“组装”是通过其尾部不同的结构域来控制的，而不是其他系统中的II型肌球蛋白的靶向和组装是耦合的。因此，发芽酵母系统提供了一个独特的机会来解决肌球蛋白在胞质分裂过程中的高级组装的体内特定功能。在第二个目标中，我们将尝试定义一种新的机制，在胞质分裂过程中AMR收缩和胞吐介导的ECM重塑之间的协调。具体地说，我们将测试我们的假设，即C2结构域蛋白Inn1通过与AMR侧的IQGAP和ECM侧的转谷氨酰胺酶/蛋白酶Cyk3相互作用，协调酵母中AMR收缩和ECM重塑或初级隔膜(PS)的形成，进而刺激几丁质合成酶Chs2的活性，促进分裂部位PS的形成。我们提出的研究将对肌球蛋白高阶组装在胞质分裂中的作用以及胞质分裂过程中AMR收缩和ECM重塑之间的协调机制产生独到的见解。 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。细胞质分裂失败与癌症等严重的人类疾病有关。因此，研究胞质分裂的机制在基础科学和临床科学中都具有深远的意义。

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.