3 years ago

Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation.

Sanjay Kumar, Jasmine H Hughes, Elena Kassianidou
The assembly and mechanics of actomyosin stress fibers (SFs) depend on regulatory light chain (RLC) phosphorylation, which is driven by Myosin Light Chain Kinase (MLCK) and Rho-Associated Kinase (ROCK). While previous work suggests that MLCK and ROCK regulate distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC mono-phosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC di-phosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and di-phosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and di-phosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation and SF viscoelasticity.

Publisher URL: http://doi.org/10.1091/mbc.E17-06-0401

DOI: 10.1091/mbc.E17-06-0401

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