Understanding smooth muscle myosin assembly in vitro and in vivo

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).