Chang Ching-Wei, Kumar Sanjay
Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720.
Sci Rep. 2015 Sep 4;5:13736. doi: 10.1038/srep13736.
While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces.
虽然人们普遍认识到非肌肉肌球蛋白II（NMMII）能使应力纤维（SFs）对细胞外基质产生牵引力，但对于特定的NMMII亚型和功能域如何影响SF力学却知之甚少。在这里，我们结合生物光子学和遗传学方法来解决这些悬而未决的问题。首先，我们抑制NMMII亚型MIIA和MIIB，并应用飞秒激光纳米手术切除并研究单个SFs的粘弹性回缩。与MIIA和MIIB抑制相关的SF回缩动力学分别定性地模拟了我们早期在Rho激酶（ROCK）和肌球蛋白轻链激酶（MLCK）抑制情况下的测量结果。此外，荧光成像和光漂白恢复显示，MIIA和MIIB分别在ROCK和MLCK控制的中央和外周SFs中富集且定位更稳定。额外的结构域映射研究令人惊讶地发现，头部结构域的缺失会加速SF回缩，我们将其归因于肌动球蛋白交联产生的阻力和摩擦损失的减少。我们提出了一个模型，其中ROCK/MIIA和MLCK/MIIB在功能上调节SFs的共同池，MIIA的交联和运动功能共同影响SF回缩动力学和细胞牵引力。
