Targeting and assembly of E. coli cell division proteins

描述（由申请人提供）：在任何细胞分裂产生可存活的子细胞之前，它必须首先分离其复制的染色体并在它们之间分裂其细胞质。细胞质分裂这一基本的重要事件，必须在染色体分离后的正确时间和地点，在分离的染色体之间发生。在细菌中，细胞质分裂是由一种重要且高度保守的微管蛋白样蛋白FtsZ精心安排的，FtsZ在细胞中点的内膜上组装成一个圆周环结构，称为z环。一旦组装好，大肠杆菌的z环将至少10个额外的必需分裂蛋白招募到正在发育的分裂位点的膜上，之后，环在生长的间隔壁的前缘收缩，将细胞分裂成两个。令人惊讶的是，大多数这些蛋白质在细胞分裂机器的功能中的分子作用是未知的。目前还不清楚机器中的各种蛋白质是如何相互招募和稳定的，或者一旦机器组装好，Z环是如何被触发收缩的。我们之前的工作表明，通过改变其他蛋白质的活性，可以以很少的成本消除其中一些蛋白质，这表明细胞分裂机器可能过度构建。我们试图通过区分核心成分和调节成分来了解机器中蛋白质的功能。我们的方法利用遗传学、蛋白质生物化学和全细胞成像。具体来说，我们建议(i)了解FtsZ组装如何受到细胞分裂蛋白（如动作蛋白样FtsA）的调节；（ii）定义FtsZ和FtsA如何通过蛋白质-蛋白质相互作用的合作网络招募和构建机器的其余部分；（三）利用基因技术剥离机器的其余部分，只保留其核心部件。研究细菌细胞分裂不仅因为它是一个需要理解的基本细胞过程，而且因为细胞分裂是抗菌剂的一个重要潜在靶点。

DESCRIPTION (provided by applicant): Before any cell divides to yield viable daughters, it must first separate its duplicated chromosomes and split its cytoplasm between them. This fundamentally important event, cytokinesis, must occur at the correct time, after chromosome segregation, and place, between the segregated chromosomes. In bacteria, cytokinesis is orchestrated by an essential and highly conserved tubulin-like protein, FtsZ, which assembles into a circumferential ring structure, called the Z-ring, on the inner membrane at the cell midpoint. Once assembled, the Z-ring of E. coli then recruits at least 10 additional essential division proteins to the membrane at the developing division site, after which the ring contracts at the leading edge of the growing septal wall to split the cell into two. Surprisingly, the molecular roles of most of these proteins in the functioning of the cell division machine are unknown. It is also unclear how the various proteins in the machine recruit and stabilize each other, or how the Z ring is triggered to contract once the machine is assembled. Our previous work has shown that some of these proteins can be eliminated with little cost by changing the activities of other proteins, indicating that the cell division machine may be overbuilt. We seek to understand the function of the proteins in the machine by distinguishing the core components from the regulatory components. Our approach utilizes genetics, protein biochemistry, and imaging of whole cells. Specifically, we propose to (i) understand how FtsZ assembly is regulated by cell division proteins such as the actin-like FtsA; (ii) define how FtsZ and FtsA recruit and build the rest of the machine via a cooperative network of protein-protein interactions; and (iii) strip down the rest of the machine to its core components using genetics. The study of bacterial cell division is important not only because it is a basic cellular process that needs to be understood, but also because cytokinesis is an important potential target of antimicrobials.