A regulatory motif in nom-muscle myosin II-B regulates its assembly and determines its role in plasma membrane protrusion, adhesion dynamics and migratory polarization

Resumen

The actomyosin cytoskeleton is a complex network of intertwined filaments that extends along the cell body, acting as a scaffold that determines the shape of the cell and provides support to the organization of the different cellular components and processes. These filaments are dynamic structures that undergo continuous rearrangements to enable the cell to adapt to different intracellular and extracellular demands, including extracellular matrix contraction, cell shape changes during division, migration, etc. In this context, non-muscle myosin II (NMII) arises as a master controller of the behavior of these filaments. NMII is a molecular motor endowed with actin-binding and ATP-ase mediated contraction activities. The two main paralogs are NMII-A and NMII-B, and they play different roles in the regulation of the maintenance and remodeling of the actomyosin cytoskeleton. NMII-A is more dynamic and underlies de novo assembly of actomyosin bundles. Conversely, NMII-B assembles more slowly and is largely responsible for creating large actomyosin bundles that determine the rear of the cell, allowing the establishment of migratory cell front-back polarity. In this study, we show that the paralog-specific role of NMII-B is controlled by a short stretch of amino acids containing five serines (1935-SFSSSRS-1941), which is absent in NMII-A. This motif resides near the junction between the C-terminus helical and non-helical tail domains of the myosin II heavy chain-B (MHCII-B). Removal of this motif increased NMII-B exchange by reducing its assembly into actomyosin filaments (which is a proxy of stability in this context). On the other hand, its insertion into NMII-A, endowed it with slower dynamics and a NMII-B-like ability to generate thick rear-defining actomyosin bundles. Phosphomimetic mutation of the five serines also decreased NMII-B assembly, rendering it unable to support front-back polarization. Mass spectrometric analysis showed the differential amenability of these residues to become phosphorylated in live cells. Single-site mutagenesis showed that Ser1935 is the major regulatory site of this motif that controls NMII-B dynamics and function. Analysis of focal adhesions behavior, indicated that these structures are regulated by the Ser1935-dependent stability of the filaments. Prevention of the phosphorylation of this residue overcame the signals emanating from the atypical PKCζ signaling pathway.