Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.

ABSTRACT

Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.