Abstract
Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.
Highlights
Mammalian cell migration in two dimensions (2D) is classically considered to be a multistep process that includes extension and attachment of a leading edge protrusion, coupled to formation of anterior sites of cell-substrate adhesions or focal adhesions
C-terminal Phosphorylation Sites Mediate NMIIA Recruitment to the Leading Edge and Focal Adhesion Maturation during Spreading on Matrix—In view of our earlier evidence for NMIIA C-terminal phosphorylation at Ser-1943 in regulating non-muscle myosin II (NMII) filament localization [23], we asked whether expression of GFP myosin heavy chain (MHC)-IIA constructs mutated at either Ser-1943, Ser-1916, or at both sites in COS-7 cells would affect NMIIA recruitment to the leading edge during active spreading
We have previously demonstrated via mass spectroscopy that there is a transient elevation in phosphorylation on both Ser-1916 and Ser-1943 during spreading on matrix, suggesting that phosphorylation on these sites may affect NMIIA dynamics during spreading [29]
Summary
Mammalian cell migration in two dimensions (2D) is classically considered to be a multistep process that includes extension and attachment of a leading edge protrusion, coupled to formation of anterior sites of cell-substrate adhesions or focal adhesions. COS-7 cell populations expressing wild-type GFP MHC-IIA and the mutant constructs were isolated via transient transfection and fluorescence-activated cell sorting (FACS).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
