Targeting and assembly of E. coli cell division proteins

描述(申请人提供)：在任何细胞分裂以产生可存活的子代之前，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。这一根本上重要的事件，胞质分裂，必须发生在正确的时间，在染色体分离之后，以及在分离的染色体之间的位置。在细菌中，胞质分裂是由一种基本的和高度保守的微管蛋白样蛋白FtsZ协调的，它在细胞中点的内膜上组装成一个环状结构，称为Z环。一旦组装完成，大肠杆菌的Z环就会在发育中的分裂部位将至少10个额外的必需分裂蛋白招募到细胞膜上，之后环在不断增长的隔壁的前沿收缩，将细胞一分为二。令人惊讶的是，这些蛋白质中的大多数在细胞分裂机器功能中的分子作用尚不清楚。目前也不清楚机器中的各种蛋白质是如何相互招募和稳定的，也不清楚机器组装后Z环是如何触发收缩的。我们之前的工作表明，其中一些蛋白质可以通过改变其他蛋白质的活性来消除，而成本很低，这表明细胞分裂机器可能被过度建造了。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。我们的方法利用了遗传学、蛋白质生物化学和整个细胞的成像。具体地说，我们建议(I)了解FtsZ组装是如何由细胞分裂蛋白(如肌动蛋白样FTSA)调控的；(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；以及(Iii)利用遗传学将机器的其余部分剥离到其核心组件。细菌细胞分裂的研究很重要，不仅因为这是一个需要了解的基本细胞过程，还因为胞质分裂是抗菌药物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.