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Understanding smooth muscle myosin assembly in vitro and in vivo

了解平滑肌肌球蛋白的体外和体内组装

基本信息

批准号：
2739983
负责人：
金额：
--
依托单位：
University of Leeds
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2022
资助国家：
英国
起止时间：
2022 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=studentship-2739983
关键词：
Understanding smooth muscle myosin assembly

项目摘要

Myosins are molecular motors that interact with actin to generate force and movement. Smooth muscle myosin (SMM), found predominantly in smooth muscle cells, is required for their contraction. It has long been assumed that this myosin is able to exist in either a switched off inactive state or in an active filamentous state, and that it can cycle between these states depending on the level of activation of the muscle. However, it is still unclear how this happens, how much myosin is in these two states, how the contractile cytoskeleton is organised and responds to cell extension, and the role of smoothelin and smoothelin like proteins in this process.To address better understand SMM filament assembly and disassembly we will:1. Investigate SMM filament formation in vitro, using negative stain and Cryo-EM to understand how the myosin assembles and packs into filaments at high resolution.2. Isolate Affimers that recognise shutdown and active forms of SMM , generate GFP-SMM expression constructs, and use these to investigate SMM filament formation, organisation and dynamics in vivo using primary cells and smooth muscle cell lines.3. Develop and use a correlative light and cryo-EM/ET approach, together with the potential for subtomogram image processing to analyse SMM filaments in vivo (in cells).

肌球蛋白是与肌动蛋白相互作用产生力量和运动的分子马达。平滑肌肌球蛋白(SMM)主要存在于平滑肌细胞中，是其收缩所必需的。长期以来，人们一直认为这种肌球蛋白能够以关闭的非活动状态或活动的丝状状态存在，并且它可以根据肌肉的激活程度在这两种状态之间循环。然而，目前还不清楚这是如何发生的，这两种状态下有多少肌球蛋白，收缩的细胞骨架是如何组织起来的，并对细胞延伸做出反应，以及平滑蛋白和平滑蛋白样蛋白在这一过程中的作用。为了更好地了解SMM细丝的组装和拆解，我们将：1.研究SMM细丝的体外形成，使用负染色法和冷冻-EM来了解肌球蛋白如何在高分辨率下组装和包装成细丝。分离识别SMM关闭和活动形式的仿射器，产生GFP-SMM表达构建体，并使用这些构建物使用原代细胞和平滑肌细胞系在体内研究SMM细丝的形成、组织和动力学。开发和使用相关的光和低温EM/ET方法，以及在体内(在细胞内)进行亚断层图像处理以分析SMM细丝的可能性。