Assembly and function of cytoskeletal systems in eukaryotic and prokaryotic cells

 描述(申请人提供)：我的实验室的一个主要重点是了解细胞骨架聚合物如何帮助一系列大分子协同工作，建立一个共同的身份：成为一个活细胞。NIGMS对我的实验室的资助资助了真核生物和原核生物中几个基本细胞生物学过程的工作，包括：(1)真核细胞中驱动膜运动的产生力量的“片脂”肌动蛋白网络的组装和功能(GM061010和NDC路线图赠款)；(2)螺旋蛋白家族蛋白的肌动蛋白成核活性和生物学功能(GM075287)；(3)细胞核中基于肌动蛋白的结构和功能(GM061010)；以及(4)由细胞骨架聚合物(GM095263和GM079556)组装驱动的细菌细胞中的DNA分离。米拉拨款资助所有这些项目，将使我们能够将以前致力于维持多个R0-1拨款的精力重新投入到更多原创工作中。为了了解分子特性如何控制活细胞的结构和功能，我们在多个尺度上进行了定量研究：(I)细胞骨架组件的单分子和整体生化研究；(Ii)在体外重建的复杂细胞结构的生物物理和显微研究；以及(Iii)活细胞中细胞骨架系统的细胞生物学和高分辨率显微镜研究。一般说来，这笔赠款支持的正在进行的工作可以分为四个部分：1.原核生物细胞骨架系统的研究。原核生物基因组编码40多类肌动蛋白样蛋白(Alp)。我们已经研究了三个真细菌Alps(Parm、Alfa和Alp7A)的组装和功能，目前我们正在努力了解Alps在古生菌中的作用。2.树突状肌动蛋白网络的研究包括：Arp2/3复合体、WASP/WAVE家族蛋白、Ena/Vasp、Forins、Caping蛋白、Cofilin和Profilin。除了为某些类型的细胞运动提供动力外，这 “树突状网络马达”也有助于吞噬、内吞、细胞内病原体的移动和膜破裂的愈合。除其他外，我们目前正在努力了解这些网络产生和适应机械力的机制。3.阿米巴快速迁徙的研究。我们将“进化细胞生物学”的方法与3D“贝塞尔光束”显微镜相结合，以了解细胞如何产生复杂的膜动力学，然后利用它们进行快速运动。简而言之，我们使用比较基因组学和广泛差异(非模式)生物的细胞生物学研究来揭示细胞运动的分子和生物物理机制。4.真核细胞中肌动蛋白微丝的组装和功能研究。我们最近发现了Fmn2和Spire家族分子在DNA损伤时产生的核肌动蛋白细丝。这些细丝有助于快速清除双链DNA断裂(Belin和Mullins，提交)，我们正在努力了解它们的功能，并找出产生它们的信号通路。

 DESCRIPTION (provided by applicant): A major focus of my laboratory is to understand how cytoskeletal polymers help a collection of macromolecules work together to establish a common identity: to become a living cell. Grants to my laboratory from NIGMS have funded work on several basic cell biological processes, in both eukaryotes and prokaryotes, including: (i) the assembly and function of force-generating `lamellipodial' actin networks that drive membrane movements in eukaryotic cells (GM061010 and an NDC Roadmap Grant); (ii) the actin nucleating activity and biological function of Spire-family proteins (GM075287); (iii) the architecture and function of actin-based structures in the nucleus (GM061010); and (iv) DNA segregation in bacterial cells, driven by assembly of cytoskeletal polymers (GM095263 and GM079556). A MIRA grant funding all these projects would enable us redirect energy previously devoted to maintaining multiple R0-1 grants into doing more original work. To understand how molecular properties govern the architecture and function of living cells, we perform quantitative studies at multiple size scales: (i) single-molecule and bulk biochemical studies of cytoskeletal components; (ii) biophysical and microscopical studies of complex cellular structures reconstituted in vitro; and (iii) cell biological and high-resolution microscopy studies of cytoskeletal systems in living cells. Broadly speaking, the ongoing work that would be supported by this grant can be divided into four parts: 1. Studies of prokaryotic cytoskeletal systems. Prokaryotic genomes encode more than forty classes of actin-like proteins (ALPs). We have studied the assembly and function of three eubacterial ALPs (ParM, AlfA, and Alp7A) and we are currently working to understand the role of ALPs in archaea. 2. Studies of dendritic actin networks assembled by: the Arp2/3 complex, WASP/WAVE-family proteins, Ena/VASP, formins, capping protein, cofilin, and profilin. In addition to powering some types of cell locomotion, this `dendritic network motor' also contributes to phagocytosis, endocytosis, movement of intracellular pathogens, and healing of membrane ruptures. Among other things, we are currently working to understand the mechanisms by which these networks generate and adapt to mechanical forces. 3. Studies of fast amoeboid migration. We combine `Evolutionary Cell Biology' approaches with 3D "Bessel Beam" microscopy to understand how cells generate complex membrane dynamics and then harness them for rapid movement. Briefly, we employ comparative genomics and cell biological studies of widely divergent (non-model) organisms to uncover molecular and biophysical mechanisms of cell movement. 4. Studies of the assembly and function of actin filaments in eukaryotic nuclei. We recently discovered nuclear actin filaments created by Fmn2 and Spire-family molecules in response to DNA damage. These filaments contribute to rapid clearance of double-strand DNA breaks (Belin and Mullins, submitted) and we are working to understand their functions and to work out the signaling pathways that create them.