Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells

Authors:
Breckenridge MT, Dulyaninova NG, Egelhoff TT
In:
Source: Mol Biol Cell
Publication Date: (2008)
Issue: epub: online
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
HeLa
Species: human
Tissue Origin: cervix
Platform:
Nucleofector® I/II/2b
Abstract
To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (DeltaIQ2) destabilizes the 10 S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Deltatailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Deltatailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6 S/10 S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.