Biochemical characterisation of membrane bound proteins of the division machinery in bacteria

细胞分裂（胞质分裂）是原核细胞的关键生物学过程之一。细胞分裂的位点通常由所谓的Z-环的组装标记，所述Z-环由材料微管蛋白同源物FtsZ组成。参与分裂的蛋白质以确定的层次顺序定位于收缩Z环，形成多亚基复合物，称为分裂体或隔体。大多数分裂蛋白含有至少一个跨膜跨度或者是整合的膜蛋白。由于复杂的荧光显微镜方法，最近的研究这些蛋白质给出了详细的图片的本地化和时间组织的分裂蛋白质。遗传学方法已经使人们对不同蛋白质的某些特性有了深入的了解。然而，大多数分裂蛋白的详细生物化学表征仍然缺乏。这种广泛而深刻的知识缺乏在某种程度上是由于大多数分裂蛋白是膜结合的，因此不容易进行过表达、纯化和生化表征。本研究提案旨在回答其中一些问题。蛋白质RodA和FtsW被认为促进细胞壁前体（脂质II）的移位。包括重组在内的生物化学技术将被用来解开拟议中的“翻转酶”的特性。分裂蛋白FtsL和DivIC可能作为SNARE样蛋白，以稳定细胞分裂过程中的膜内陷。这两种蛋白质将使用体外和体内技术进行检查，以提供其生物学作用的证据。此外，膨压效应对细胞分裂和形态发生的影响将被检查。将使用GFP融合蛋白监测渗透变化对细胞骨架和分裂的影响。

Cell division (cytokinesis) is one of the pivotal biological processes in prokaryotic cells. The site of cell division is usually marked by the assembly of a so called Z-ring which is composed out of the Material tubulin homologue FtsZ. Proteins involved in division localise in a defined hierarchical order to the contractile Z-ring, forming a multisubunit complex, named divisome or septosome. Most of the division proteins contain at least one transmembrane span or are integral membrane proteins. Due to sophisticated fluorescence microscopy methods, recent studies of these proteins have given a detailed picture of the localisation and temporal organisation of the division proteins. Genetic approaches have given insights into some of the properties of the different proteins. However a detailed biochemical characterisation of most of the division proteins is still lacking. This broad and profound lack of knowledge is in some part due to the fact that most division proteins are membrane bound and therefore not easily amenable to overexpression, purification and biochemical characterisation. This research proposal is aimed at answering some of these questions. The proteins RodA and FtsW are thought to facilitate the translocation of cell wall precursors (Lipid II). Biochemical techniques including reconstitution will be used to unravel properties of the proposed "flippase". The division proteins FtsL and DivIC may act as SNARE-like proteins in order to stabilise membrane invagination during cell division. Both proteins will be examined using in vitro and in vivo techniques to provide evidence for their biological role. Furthermore, the influence of turgor effects on cell division and morphogenesis will be examined. The influence of osmotic changes on cytoskeleton and division will be monitored using GFP fusion proteins.