Probing the Role of FtsQLB in Regulating Septal Cell Wall Synthesis Activity

探讨 FtsQLB 在调节隔膜细胞壁合成活性中的作用

• 批准号: 10633108
• 负责人: Brooke Marie Britton
• 金额: $ 7.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10633108
• 关键词: 3-Dimensional; Antibiotic Therapy; Bacteria; Biochemical; Cell Shape; Cell Wall; Cell division; Cell model; Cells; Complex; Coupled; Cytolysis; Dependence; Development; Dominant-Negative Mutation; Ensure; Enzymes; Escherichia coli; Exhibits; Failure; Genetic; Genetic study; Growth; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Learning; Macromolecular Complexes; Molecular; Movement; Mutation; Nature; Peptidoglycan; Polymers; Population; Process; Proteins; Regulation; Repression; Resolution; Role; Series; Structure; Time; Visualization; alpha Tubulin; antimicrobial drug; cell type; constriction; daughter cell; experimental study; genetic approach; imaging approach; insight; live cell imaging; molecular imaging; mutant; polymerization; protein protein interaction; response; scaffold; single molecule; spatiotemporal; superresolution imaging; temporal measurement; treadmill; z-ring

Project Summary Cell wall constriction, the synthesis of new cell wall and the splitting of the old cell wall, is an additional step during cell division that occurs in walled bacteria. The synthesis and degradation of the cell wall, made of peptidoglycan (PG), must be highly coordinated because mis-regulation can result in fatal lysis of the cell. The divisome, a macromolecular complex of over 30 proteins, is responsible for this extremely coordinated process. FtsZ, a tubulin homolog and GTPase, is an essential divisome protein that begins cell division by polymerizing into a ring-like structure (Z-ring) at the midcell. This Z-ring acts as a scaffold (known as the Z-track) for other divisome proteins including the septal PG (sPG) synthase complex, FtsWI, and regulators FtsN and FtsQLB. Divisome proteins can exit the Z-track onto a second track (sPG-track) where sPG synthesis occurs. This application proposes to study the role of the FtsQLB complex in regulating the activity of FtsWI in with high spatiotemporal resolution during cell wall constriction in E. coli. Aim 1 uses single molecule tracking (SMT) and three-dimensional (3D) superresolution imaging to investigate how FtsQLB is coupled to the Z-track. I will examine how FtsQLB responds to altered FtsZ dynamics. Aim 2 focuses on the modulation of the sPG synthesis activity of FtsWI. Using the same SMT and imaging approaches I will determine if FtsQLB is coupled with FtsWI on the sPG track. Finally, Aim 3 uses superfission and dominant negative mutants to dissect the roles of key protein-protein interactions of FtsQLB, FtsN, and FtsWI. The use of single-molecule live cell imaging combined with perturbational analysis will enable me to identify molecular determinants responsible for cell wall constriction, providing insight into the spatial and temporal regulation of bacterial cell division. Due to the highly conserved nature of the bacterial cell wall, mechanistic insights learned from this study can be applicable to a wide range of bacterial species to facilitate the development of antimicrobial drugs.

项目摘要 细胞壁收缩，新细胞壁的合成和旧细胞壁的分裂，是另一项 在细胞中发生的细胞分裂过程中的一步。细胞壁的合成和降解，由 肽聚糖（PG）必须高度协调，因为错误调节会导致细胞致命的裂解。这 Divisome是一种超过30种蛋白质的大分子复合物，负责这一极为协调的过程。 FTSZ是一种微管蛋白同源物和GTPase，是一种必不可少的分裂蛋白，通过聚合开始细胞分裂 在中间细胞中进入类似环的结构（Z形）。这种Z形是其他的脚手架（称为Z-Track） 分裂体蛋白质包括中间PG（SPG）合酶复合物，FTSWI和调节剂FTSN和FTSQLB。 Divisome蛋白可以将Z轨道退出到第二个轨道（SPG-TRACK）上，其中SPG合成发生。这 应用建议研究FTSQLB复合物在调节FTSWI的活性中的作用 大肠杆菌中细胞壁收缩期间的时空分辨率。 AIM 1使用单分子跟踪（SMT）和 三维（3D）上分辨率成像，以研究FTSQLB如何耦合到Z轨道。我会 检查FTSQLB如何响应更改的FTSZ动力学。 AIM 2专注于SPG合成的调节 ftswi的活动。使用相同的SMT和成像方法，我将确定FTSQLB是否与FTSWI结合 在SPG轨道上。最后，AIM 3使用SuperFossion和Pridens负突变体来剖析密钥的作用 FTSQLB，FTSN和FTSWI的蛋白质蛋白质相互作用。单分子活细胞成像的使用合并 通过扰动分析将使我能够确定负责细胞壁的分子决定因素 收缩，提供对细菌细胞分裂的空间和时间调节的见解。由于高度 细菌细胞壁的保守性质，从这项研究中学到的机械见解可以适用于 广泛的细菌种类，以促进抗菌药物的发展。