Assembly of Cytoskeletal Proteins into the Cleavage Furrow

该项目的长期目标是确定细胞用于组装和调节形成卵裂沟的肌动蛋白和肌球蛋白丝的相互作用的机制。许多显微成像方法将用于用各种探针微注射的活细胞，以分析裂解环主要成分分子的分布。第一个目标将是确定肌动蛋白丝是如何组装到皮质膜区域的，在那里未来的收缩环将形成。构成收缩环的肌动蛋白的来源是不确定的。考虑到这一分裂过程对细胞的重要性，可能有两种独立的方法来引导肌动蛋白进入分裂沟。实验计划通过确定丝是否以及何时沿着皮质表面移动到卵裂沟，以及在沟区是否存在先前存在的肌动蛋白丝的伸长，以及确定肌动蛋白单体是否添加到沟区现有肌动蛋白丝的一端或两端来验证这一假设。还计划进行实验，利用aequorin来测量这些组织培养细胞分裂环组装和收缩过程中钙水平的波动。细胞内的钙水平被认为是由内质网调节的。活细胞的共聚焦显微镜计划确定内质网和肌动蛋白丝是否在分裂环的组装和收缩中共同分布。所有提出的实验都旨在确定所有动物细胞中存在的基本过程的机制：细胞分裂。繁殖能力是生命最基本、最重要的属性。细胞分裂是所有细胞生物这种现象的内在组成部分。对于单细胞生物来说，繁殖就是简单的细胞分裂。对于多细胞生物来说，细胞分裂是繁殖的关键，通过产生生殖系细胞（配子、孢子）和从孢子或受精卵再生整个生物体（发育）。一个细胞分裂成两个细胞是一个非常受控制的过程；切割的精确几何形状对子细胞和/或生物体的存活至关重要。动物细胞的分裂机制通常涉及一个以肌动球蛋白为基础的收缩装置，即环绕在分裂沟内部的细胞的收缩环。该项目将使用实验操作和最先进的显微镜观察直接对活细胞，以更好地了解裂解过程。

The longterm goal of this project is to determine the mechanisms that cells use for assembling and regulating the interactions of the actin and myosin filaments that form the cleavage furrow. A number of microscopic imaging methods will be used with living cells that have been microinjected with various probes, to analyze the distribution of the major component molecules of the cleavage ring. The first aim will be to determine how actin filaments are assembled into the region of the cortical membrane where the future contractile ring will form. The source of the actin that composes the contractile ring is uncertain. Considering the importance of this division process for the cells, there may be two independent methods for directing actin to the cleavage furrow. Experiments are planned to test this hypothesis by determining if and when the filaments move along the cortical surface to the cleavage furrow and if there is elongation of the pre-existing actin filaments in the furrow region, and to determine if actin monomers add to one or both ends of the existing actin filaments in the furrow regions. Experiments are also planned that will utilize aequorin to measure fluctuations in calcium levels during the assembly and contraction of the cleavage ring in these dividing tissue culture cells. The calcium levels inside cells are thought to be regulated by the endoplasmic reticulum. Confocal microscopy of living cells is planned to determine if there is any co-distribution of the endoplasmic reticulum and actin filaments in the assembling and contracting cleavage ring. All of the proposed experiments are designed to determine the mechanisms responsible for a basic process present in all animal cells: cytokinesis. %%% The ability to reproduce is the most fundamental, defining property of life. Cell division is an intrinsic part of this phenomenon for all cellular organisms. For single-celled organisms, reproduction is, simply, cell division. For multicellular organisms, cell division is key to reproduction, by generating germ-line cells (gametes, spores) and for regenerating whole organisms from spores or zygotes (development). The division of one cell into two is a remarkably controlled process; the precise geometry of cleavage is critical to the survival of the daughter cells and/or the organism. The cleavage mechanism in animal cells generally involves an actomyosin based contractile apparatus, the contractile ring, which girdles the cell at the inside of the cleavage furrow. This project will use experimental manipulations and state-of-the-art microscopic observations directly on living cells to better understand the cleavage process.