Biophysical Society Thematic Meeting | Singapore

Mechanobiology of Disease

Poster Abstracts

69-POS Board 69 Studying Nonmuscle Myosin-2 Function in Cells: Not as Easy as It Seems! James Sellers , Sarah Heissler, Neil Billington. National Heart, Lung and Blood Institute, NIH, Bethesda, MD, USA.

We investigate nonmuscle myosin-2 (NM2) filaments using in-vitro approaches to inform studies of myosin function in cells. Here we detail some complexities of studying the function of NM2 in cells. There are three NM2 paralogs which form short bipolar filaments. These myosins co-assemble with each other to form heteropolymeric filaments. A structurally related myosin, myosin-18A, is an enzymatically inactive pseudo-enzyme which also co-polymerizes with NM2. NM2B and NM2C heavy chains are alternatively spliced, giving rise to molecules with altered enzymatic and regulatory features. There are three regulatory light chains (RLC) and two essential light chains that can associate with these myosins. The RLCs are phosphorylated at two the myosin may interact with beta- or gamma-actin which might affect its enzymatic porperties and these actin filaments might be complexed with a variety of tropomyosin isoforms which also differentially affect the activity. Thus one myosin filament may have very different mechanical properties from another seemingly identical filament in the same cell. Overexpression of GFP- RLC which dynamically exchanges into myosin is used for live cell imaging. However RLC binds to at least four myosin classes and fusion of GFP to RLC lowers the enzymatic activity by 50%. Use of phospho-mimetic RLC is not recommended since these mutants do not mimic phosphorylation of S19. Use of disease-associated myosin mutations for "rescue" experiments is also not advised since some of the defects may be related to protein folding which could vary with differing cell types.

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