Rho Kinase Differentially Regulates Phosphorylation of Nonmuscle Myosin II Isoforms A and B during Cell Rounding and Migration

Kris A. DeMali,Anthony R. Means,Joshua C. Sandquist,Katherine I. Swenson,Keith Burridge

doi:10.1074/jbc.m605343200

Kris A. DeMali, Anthony R. Means + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m605343200

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Abstract

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.

Highlights

Distinct Functions of Nonmuscle Myosin II Isoforms thought to be regulated in a similar fashion; that is, through phosphorylation of the regulatory light chains (RLCs) [2]
We demonstrate that phosphorylation of the RLC associated with IIA or IIB is differentially regulated by Rho kinase, suggesting that at least part of how IIA and IIB perform discrete cellular functions is via selective regulation downstream from different signaling pathways
To examine the distinct roles of IIA and IIB in epithelial sheet migration, we developed isoform-specific Small-inhibitory RNAs (siRNA) to characterize the loss-of-function phenotypes associated with depletion of each isoform

Summary

Introduction

IIA and IIB Distinctly Regulate Cellular Protrusions—In examining videos of control and IIA- and IIB-depleted cells migrating into a wound, we observed marked differences in membrane ruffling and lamellipodial protrusions between the cell types (Fig. 2 and supplemental Videos 1–3). The distinct lamellipodia exhibited by the control and IIAand IIB-depleted cells were examined further by fixation of cells migrating into a wound followed by visualization of the actin cytoskeleton via staining with rhodamine-conjugated phalloidin.

Results

Conclusion