Uncovering how the dynamic architecture of a layered contractile ring induces furrow ingression

揭示分层收缩环的动态结构如何引起沟槽侵入

• 批准号: 10004677
• 负责人: Caroline Laplante
• 金额: $ 30.82万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004677
• 关键词: Abnormal Cell; Actin-Binding Protein; Actins; Actomyosin; Address; Affect; Alleles; Animal Model; Architecture; Biological Models; Biology; Bundling; Cell division; Cell membrane; Cells; Complex; Congenital Abnormality; Contractile Proteins; Cytokinesis; Cytoplasm; Data; Defect; Development; Disease; Event; Fission Yeast; Fluorescence; Foundations; Generations; Genes; Genetic Materials; Human; Immune System Diseases; Individual; Kinetics; Knowledge; Lasers; Lead; Life; Light; Malignant Neoplasms; Measures; Mechanics; Membrane; Methodology; Microfilaments; Microscopy; Microsurgery; Modeling; Molecular; Mothers; Motor; Mutation; Myosin ATPase; Organism; Outcome; Pathogenesis; Plus End of the Actin Filament; Process; Production; Proteins; Publishing; Research; Research Proposals; Role; Speed; Structure; System; Testing; Work; Yeasts; base; cell cortex; confocal imaging; constriction; daughter cell; frontier; fungus; imaging genetics; innovation; molecular dynamics; nanometer resolution; nervous system disorder; novel; photoactivation; public health relevance; transmission process

Project description: 30 lines Cytokinesis, the separation of a mother cell into two daughter cells, is one of life's most fundamental processes; it is central to the making of multicellular organisms and the transmission of genetic material across generations. The core machinery of cytokinesis is a contractile ring of actin, myosin motors and other proteins and is conserved from fungi to human. The contractile ring is connected to the inside of the cell cortex and its constriction leads the ingression of the plasma membrane into a furrow between the two cytoplasmic compartments that will become individual cells after the completion of cell division. Furrow ingression requires the cooperation between two main mechanisms: 1) constriction of actin filaments by the action of myosin motors and 2) the transmission of this contractile force to the plasma membrane via anchor proteins that connect the actomyosin bundle to the plasma membrane. We found that the proteins of the contractile ring occupy distinct layers with plasma membrane-interacting proteins in the outer layer adjacent to the cortex and the force- producing myosin motors in the inner layer. The next frontier in understanding the mechanism of cytokinesis is to determine how these proteins are organized into complex structures, how these structures move within the contractile ring and are removed from the ring during constriction, and how this dynamic architecture governs the force-generation function of the contractile rings. The objective of this application is to determine the anchoring role of the outer ring and the tension-force producing role of the inner ring by uncovering their dynamic architecture and effects on mechanics of constriction. Our hypothesis is that furrow ingression results from contractile forces produced in the inner layer of the contractile ring, conveyed to the plasma membrane via anchoring achieved by proteins in the outer layer. We plan to test our central hypothesis with the following Specific Aims: 1) determine how cytokinetic node proteins anchor the contractile ring and transmit contractile forces to the plasma membrane during furrow ingression and 2) determine how the molecular architecture of the inner ring governs the constriction of the contractile ring during furrow ingression. The proposed research in this application is innovative because we will use a unique combination of high-speed Fluorescence Photoactivation Localization Microscopy (hsFPALM) in live cells to determine protein organization and its dynamics with laser microsurgery to probe the mechanics of this tension-force producing machine in live cells. The proposed research in this application is significant as it will result in the identification of previously unknown parameters for new molecular and functional models of the contractile ring. As the proteins of the contractile ring are conserved from yeast to human, we expect that the core mechanism of cytokinesis elucidated in fission yeast will act as a template for understanding the mechanism of cytokinesis and even perhaps other non-musclecontractile cellular machines in other species.

项目描述：30线 胞质分裂，即母细胞分裂为两个子细胞，是生命最基本的过程之一; 它对多细胞生物的形成和遗传物质的世代传递至关重要。 胞质分裂的核心机制是肌动蛋白、肌球蛋白马达和其他蛋白质的收缩环， 从真菌到人类都是保守的。收缩环连接到细胞皮层的内部， 收缩导致质膜进入两个细胞质之间的沟 细胞分裂完成后将成为单个细胞的隔室。犁沟侵入需要 两种主要机制之间的合作：1）肌球蛋白马达作用下肌动蛋白丝的收缩 和2）这种收缩力通过连接细胞膜的锚蛋白传递到质膜。 肌动球蛋白束到质膜。我们发现，收缩环的蛋白质占据了不同的位置， 在邻近皮质的外层中具有质膜相互作用蛋白的层和力- 在内层产生肌球蛋白马达。理解胞质分裂机制的下一个前沿是 为了确定这些蛋白质是如何组织成复杂结构的，这些结构是如何在细胞内移动的， 收缩环，并在收缩期间从环中移除，以及这种动态结构如何控制 收缩环的力量产生功能。本申请的目的是确定 外环的锚固作用和内环的张力产生作用， 结构和对收缩力学的影响。我们的假设是犁沟内移是由 在收缩环的内层产生的收缩力，通过 锚定是由外层的蛋白质实现的。我们计划用以下方法来检验我们的中心假设 具体目的：1）确定细胞动力学结蛋白如何锚定收缩环并传递收缩信号。 力的质膜在犁沟侵入和2）确定如何分子结构的膜， 内环控制沟内陷过程中收缩环的收缩。这项研究中提出的 应用是创新的，因为我们将使用高速荧光光活化的独特组合 活细胞中的定位显微镜（hsFPALM），用于确定蛋白质组织及其激光动力学 显微手术来探测活细胞中产生张力的机制。拟议 在这种应用中的研究是重要的，因为它将导致识别先前未知的参数， 收缩环的新分子和功能模型。由于收缩环的蛋白质是保守的 从酵母到人类，我们期望在裂殖酵母中阐明的胞质分裂的核心机制将作为一个 这是理解胞质分裂机制的模板，甚至可能是其他非肌肉收缩细胞的模板。 其他物种的机器