Rock and MlCK Tune Regional Stress Fiber Mechanics via Preferential Myosin Light Chain Phosphorylation

Abstract

Non-muscle myosin II (NMMII) is the key force-generating component of actomyosin stress fibers (SFs), which transmit tension throughout the cytoskeleton and to the extracellular matrix. NMMII motor activity is strongly promoted by phosphorylation of its regulatory light chains (RLCs) at Ser19 (p-RLC) and additionally enhanced by further phosphorylation at Thr18 (pp-RLC). The main RLC regulatory kinases are myosin RLC kinase (MLCK) and Rho-associated kinase (ROCK). While prior work suggests that MLCK and ROCK respectively regulate SFs located in the cellular center and periphery, it is unclear how these kinases differ in their effects on RLC phosphorylation state and how each phosphospecies influences the mechanics of central and peripheral SFs. Here we address these questions by combining graded induction of ROCK or MLCK expression with phospho-RLC immuno-analysis and mechanical measurement of single SFs in living cells. Immunoblots and quantitative immunofluorescence imaging reveal that graded MLCK overexpression preferentially produces graded increases in p-RLC, which localizes to peripheral SFs, while graded ROCK overexpression increases pp-RLC, which localizes to central SFs. Single-SF laser ablation studies revealed that MLCK increases the effective viscosity and stored elastic energy in peripheral SFs but not central SFs, while ROCK preferentially increases these parameters in central SFs. Graded variations in kinase activities revealed surprising and previously unappreciated nonlinearities between phosphorylation levels and SF viscoelasticity. To establish causal relationships between RLC phosphospecies and SF mechanics, we overexpressed RLC mono- and di-phosphomimetic mutants. We found that phosphomimetic p-RLC localized to and selectively influenced the mechanics of peripheral SFs while di-phosphomimetic pp-RLC localized to and selectively influenced the mechanics of central SFs. This work supports a model in which MLCK regulates peripheral SF viscoelasticity via p-RLC while ROCK regulates central SF viscoelasticity via pp-MLC.