aPKCζ affects directed cell migration through the regulation of myosin light chain phosphorylation

Abstract

Cell motility is an essential cellular process for a variety of biological events. It requires cross-talk between the signaling and the cytoskeletal systems. Despite the recognized importance of aPKCz for cell motility, there is little understanding of the mechanism by which aPKCz mediates extracellular signals to the cytoskeleton. In the present study, we report that aPKCz is required for the cellular organization of acto-non-muscle myosin II (NMII) cytoskeleton, for proper cell adhesion and directed cell migration. We show that aPKCz mediates EGF-dependent RhoA activation and recruitment to the cell membrane. We also show that aPKCz mediates EGFdependent myosin light chain (MRLC) phosphorylation that is carried out by Rho-associated protein kinase (ROCK), and that aPKCz is required for EGF-dependent phosphorylation and inhibition of the myosin phosphatase targeting subunit (MYPT). Finally, we show that aPKCz mediates the spatial organization of the acto-NMII cytoskeleton in response to EGF stimulation. Our data suggest that aPKCz is an essential component regulator of acto-NMII cytoskeleton organization leading to directed cell migration, and is a mediator of the EGF signal to the cytoskeleton.

A ratchet-like apical constriction drives cell ingression during the mouse gastrulation EMT

“…We next investigated the localization of other apical proteins, including the actomyosin components, mono- and di-phosphorylated Myosin Light Chain (pMLC and ppMLC), Rock1, a Rho-associated kinase that phosphorylates and activates myosin light chain, and F-actin. We analyzed the localization of the Crumbs complex proteins PatJ, and aPKC, a regulator of non-muscle Myosin2 contexts (Biehler et al, 2021; Petrov et al, 2017; Roper, 2012; Sidor et al, 2020). Membrane segmentation allowed us to systematically quantify several parameters, including apical surface area, cell elongation, and the intensity of junctional protein accumulation (Figure 4A, Figure 4-figure supplement 1C).…”

Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration

“…Moreover, myosin-11 (MYH11, smooth muscle-specific myosin heavy chain) [ 33 ] was also at the tip of the leading cell edge, which was colocalized with MLC 20 ( Figure 1 B). Because myosin activation by MLC 20 phosphorylation is associated with migration [ 3 , 4 ], we also assessed the spatial localization of phosphorylated MLC 20 , which was found at the leading edge ( Figure 1 C). These results suggest that a fraction of smooth muscle myosin localizes at the leading cell edge during migration.…”

“…Smooth muscle myosin II has been traditionally thought to localize in the cytoplasm solely [ 1 ] and modulates cell migration by affecting stress fiber formation, focal adhesion assembly, and the retraction of the rear [ 3 , 4 ]. In this study, MLC 20 and MYH11 were found at the edge of lamellipodia.…”

“…The phosphorylation of MLC 20 by myosin light chain kinase (MLCK) initiates contractile filament sliding and smooth muscle contraction [ 2 ]. In addition, MLC 20 phosphorylation modulates cell migration by affecting stress fiber formation, focal adhesion assembly, and the retraction of the rear [ 3 , 4 ]. In breast cancer cells, non-muscle myosin IIA localizes at the leading edge, facilitating lamellipodia formation, focal adhesion turnover, and actin polymerization [ 5 ].…”

“…Similar to myosin, MLCK has been thought to localize with myosin in the cytoplasm and catalyze MLC 20 phosphorylation [ 2 , 15 ], which facilitates cell migration [ 3 , 4 ]. In addition, MLCK is believed to interact with F-actin, and affects membrane tension and the migration of mouse intestinal smooth muscle cells [ 16 ].…”