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Mechanobiology of Disease
Poster Abstracts
121
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.