Regulation and Function of a Bacterial Cytoskeleton

描述(由申请人提供)： 我的实验室的一个主要目标是了解细胞骨架聚合物如何在细胞质中建立长程有序，并帮助将一群吵闹的大分子转化为活细胞。最初，我们专注于驱动真核细胞迁移的肌动蛋白细丝网络，但在2004年，我们的兴趣扩大到包括细菌聚合物(Garner，2004)。直到最近，像肌动蛋白细丝和微管这样的细胞骨架聚合物还被认为是真核生物的创新，在细菌中并不存在。然而，最近的工作已经确定了细菌中的一些细胞骨架系统，包括维持细胞形状所需的肌动蛋白样蛋白(Jones，2001)；通过细胞质运输货物(Moller-Jensen，2003；Kruse，2003)；以及组织细胞内间隔(Komeili，2006)。我的实验室特别关注肌动蛋白样蛋白Parm，它形成动态的细丝，将货物推向杆状细菌的相反两极。Parm基因是在许多低拷贝质粒(如临床上重要的R1和R100耐药质粒)上发现的一个分割基因(PAR操纵子)的一部分。为了确保质粒遗传，PAR操纵子由三个组分构成一个DNA分离纺锤体：(1)一段称为ParC的着丝粒DNA(Dam，1994)；(2)与Parc位点结合的阻遏蛋白PARR(van den Ent，2002)；以及(3)肌动蛋白样蛋白Parm。PARR/PARC复合体利用PARM聚合的能量来产生力，推动成对的质粒向相反的方向穿过细胞质(Moller-Jensen，2002；Moller-Jensen，2003)。我们先前描述了Parm细丝的组装动力学(Garner，2004)，并使用纯化的成分在体外重新构建了基于Parm的DNA分离(Garner，2007)。本提案的目的是从分子细节上了解Parm介导的质粒运动。我们进行这些研究有几个原因，包括：(1)了解依赖于Parm的DNA分离有助于深入了解组织原核细胞质的相关系统。(2)PARM纤维具有比微管更简单的结构，是研究动态不稳定性分子基础的最佳系统。(3)了解细菌群体中维持耐药质粒的机制有助于我们应对临床耐药的出现。我们将在三个尺度上进行定量研究：(I)单分子和整体 生物化学研究；(Ii)重组的Parm纺锤体的生物物理和显微研究；以及(Iii)活细胞中Parm的细胞生物学研究。

DESCRIPTION (provided by applicant): A major goal of my laboratory is to understand how cytoskeletal polymers establish long-range order in the cytoplasm and help convert a rowdy mob of macromolecules into a living cell. Initially, we focused on actin filament networks that drive migration of eukaryotic cells but, in 2004, our interests expanded to include bacterial polymers (Garner, 2004). Until recently, cytoskeletal polymers like actin filaments and microtubules were thought to be eukaryotic innovations, not present in bacteria. Recent work, however, has identified a number of cytoskeletal systems in bacteria, including actin-like proteins required to maintain cell shape (Jones, 2001); transport cargo through cytoplasm (Moller-Jensen, 2003; Kruse, 2003); and organize intracellular compartments (Komeili, 2006). My laboratory has focused particular attention on the actin-like protein, ParM, which forms dynamic filaments that push cargo to opposite poles of rod-shaped bacteria. The ParM gene is part of a partitioning locus (the par operon) found on many low-copy plasmids (e.g. clinically important R1 and R100 drug-resistance plasmids). To ensure plasmid inheritance the par operon constructs a DNA-segregating spindle from three components: (1) a stretch of centromeric DNA called parC (Dam, 1994); (2) a repressor protein, ParR, that binds the parC locus (van den Ent, 2002); and (3) the actin-like protein ParM. The ParR/parC complex harnesses the energy of ParM polymerization to produce forces that push pairs of plasmids in opposite directions through the cytoplasm (Moller-Jensen, 2002; Moller-Jensen, 2003). We previously characterized the assembly dynamics of ParM filaments (Garner, 2004) and reconstituted ParM-based DNA segregation in vitro using purified components (Garner, 2007). The goal of the present proposal is to understand ParM-mediated plasmid movement in molecular detail. We pursue these studies for several reasons, including: (1) understanding ParM-dependent DNA segregation provides insight into related systems that work to organize prokaryotic cytoplasm. (2) ParM filaments have a simpler architecture than microtubules, making them the best system for studying the molecular basis of dynamic instability. (3) Understanding mechanisms that maintain drug resistance plasmids in bacterial populations can help us deal with the emergence of clinical drug resistance. We will perform quantitative studies at three size scales: (i) single molecule and bulk biochemical studies; (ii) biophysical and microscopical studies of reconstituted ParM spindles; and (iii) cell biological studies of ParM in living cells.