Mechanisms of Cytokinesis in Yeast

描述（由申请人提供）：肿瘤产生于不受抑制的细胞分裂，包括有丝分裂和细胞分裂。有丝分裂的机制研究导致了成功的癌症治疗。细胞分裂失败导致多倍体和遗传不稳定，这通常与肿瘤有关。因此，细胞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。动物和真菌细胞的细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，大量的AMR和膜转运成分已经被鉴定出来，其中大部分是从酵母到人类的保守成分。该领域目前面临的核心挑战是确定这些组件如何组装在一起，形成高效、保真地运行的可收缩和膜运输“机器”，以及这些机器如何在时间和空间上协调。正是在这种背景下，我们建议解决两个主要问题在细胞质分裂使用出芽酵母模型。在第一个目标中，我们将研究细胞分裂过程中ii型肌球蛋白高阶组装的机制和功能，这是一个在任何系统中仍然知之甚少的基本问题。最近，我们首次通过旋转阴影电镜证明，出芽酵母中唯一的ii型肌球蛋白Myo1形成了一个“双头”结构，其尾部中间有一个“扭结”，具有动物细胞中非肌肉型ii型肌球蛋白的所有主要特征。我们的初步研究还表明，Myo1的“靶向”到分裂位点和Myo1的“组装”到高阶结构是通过其尾部的不同结构域控制的，与其他系统相比，ii型肌球蛋白的靶向和组装是耦合的。因此，出芽酵母系统提供了一个独特的机会来解决细胞分裂过程中肌球蛋白高阶组装的特定体内功能。在第二个目标中，我们将尝试定义一个新的机制，在细胞分裂过程中，AMR收缩和胞外分泌介导的ECM重塑之间的协调。具体来说，我们将验证我们的假设，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细胞质分裂中的作用以及细胞质分裂过程中AMR收缩和ECM重塑之间协调的机制产生独到的见解。

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.