Zhang Wenwu, Gunst Susan J
Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA.
J Physiol. 2017 Jul 1;595(13):4279-4300. doi: 10.1113/JP273906. Epub 2017 May 8.
Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.
The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.
非肌肉（NM）肌球蛋白II和平滑肌（SM）肌球蛋白II均在平滑肌组织中表达，但NM肌球蛋白在SM收缩中的作用尚不清楚。气管平滑肌组织的收缩刺激会促使NM肌球蛋白重链在Ser1943位点发生磷酸化，并导致NM肌球蛋白丝在SM细胞皮质组装。在SM组织中表达不可磷酸化的NM肌球蛋白突变体NM肌球蛋白S1943A，可抑制乙酰胆碱（ACh）诱导的NM肌球蛋白丝组装和SM收缩，还可抑制收缩刺激过程中膜黏附体复合物的组装。在ACh刺激过程中，RhoA GTP酶调节NM肌球蛋白调节轻链（RLC）的磷酸化，而不调节SM肌球蛋白RLC的磷酸化，且NM RLC磷酸化是NM肌球蛋白丝组装和SM收缩所必需的。NM肌球蛋白II在气道SM收缩中起关键作用，这一作用独立于且有别于SM肌球蛋白的功能。
肌球蛋白II的非肌肉（NM）异构体在平滑肌（SM）组织中的分子功能及其在收缩中可能发挥的作用在很大程度上尚不清楚。我们评估了犬气管SM组织收缩刺激过程中NM肌球蛋白的功能。通过Triton溶解性分析和旨在测量NM肌球蛋白单体之间相互作用的邻近连接分析评估，ACh刺激导致NM肌球蛋白丝组装。ACh刺激气管SM组织中NM肌球蛋白重链在Ser1943位点的磷酸化，这在体外可调节NM肌球蛋白IIA丝的组装。在SM组织中表达不可磷酸化的突变体NM肌球蛋白S1943A，可抑制ACh诱导的内源性NM肌球蛋白Ser1943磷酸化、NM肌球蛋白丝形成、膜黏附体复合物组装和张力产生。NM肌球蛋白横桥循环抑制剂blebbistatin可抑制黏附体复合物组装和SM收缩，但不抑制NM肌球蛋白Ser1943磷酸化或NM肌球蛋白丝组装。RhoA失活选择性抑制NM肌球蛋白调节轻链（RLC）的磷酸化、NM肌球蛋白丝组装和收缩，尽管它不抑制SM RLC磷酸化。我们得出结论，在气道SM组织收缩刺激过程中，RhoA介导的NM肌球蛋白RLC磷酸化和NM肌球蛋白重链Ser1943磷酸化调节NM肌球蛋白II的组装和激活。NM肌球蛋白II肌动球蛋白横桥循环调节膜黏附体复合物的组装，这些复合物介导产生张力所需的细胞骨架过程。NM肌球蛋白II在气道SM收缩中起关键作用，这一作用独立于且有别于SM肌球蛋白的功能。
