Understanding smooth muscle myosin assembly in vitro and in vivo

肌球蛋白是与肌动蛋白相互作用产生力和运动的分子马达。平滑肌肌球蛋白（SMM）主要存在于平滑肌细胞中，是平滑肌收缩所必需的。长期以来，人们一直认为这种肌球蛋白能够以关闭的非活性状态或活性丝状状态存在，并且它可以根据肌肉的激活水平在这些状态之间循环。然而，目前还不清楚这是如何发生的，有多少肌球蛋白是在这两个国家，如何收缩的细胞骨架是组织和响应细胞的延伸，以及smoothelin和smoothelin样蛋白在这一过程中的作用。采用负染法和冷冻电镜技术研究SMM肌丝的体外形成，以了解肌球蛋白是如何高分辨率地组装成肌丝的.分离识别SMM的关闭和活性形式的Affimers，产生GFP-SMM表达构建体，并使用这些构建体使用原代细胞和平滑肌细胞系在体内研究SMM丝形成、组织和动力学。开发并使用相关的光和冷冻EM/ET方法，以及亚断层图像处理的潜力，以分析体内（细胞中）的SMM细丝。

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).