Hammell Megan Jean, Kachmar Linda, Balassy Zsombor, IJpma Gijs, Lauzon Anne-Marie
Department of Biological and Biomedical Engineering, McGill University, Montreal, Quebec, Canada.
Research Institute of the McGill University Health Centre, Meakins-Christie Laboratories, Montreal, Quebec, Canada.
J Gen Physiol. 2022 Oct 3;154(10). doi: 10.1085/jgp.202213117. Epub 2022 Aug 24.
Smooth muscle (SM) is found in most hollow organs of the body. Phasic SM, as found in the gut, contracts to propel content, whereas tonic SM, as found in most blood vessels, maintains tension. This force maintenance is referred to as the latch state and occurs at low levels of myosin activation (myosin light chain [LC20] phosphorylation). Molecular mechanisms have been proposed to explain the latch state but have been studied only at the whole-muscle level because of technological limitations. In the current study, an assay chamber was devised to allow injection of myosin light chain phosphatase (MLCP) during laser trap and in vitro motility assays, without creating bulk flow, to reproduce latch state conditions at the molecular level. Using the laser trap in a single-beam mode, an actin filament was brought in contact with several myosin molecules on a pedestal. Myosin pulled on the actin filament until a plateau force was reached, at which point, MLCP was injected. Force maintenance was observed during LC20 dephosphorylation, the level of which was assessed in a parallel in vitro motility assay performed in the same conditions. Force was maintained longer for myosin purified from tonic SM than from phasic SM. These data support the longstanding dogma of strong bonds caused by dephosphorylated, noncycling cross-bridges. Furthermore, MLCP injection in an in vitro motility mixture assay performed with SM and skeletal muscle myosin suggests that the maintenance of these strong bonds is possible only if no energy is provided by surrounding actively cycling myosin molecules.
平滑肌（SM）存在于人体大多数中空器官中。在肠道中发现的阶段性平滑肌会收缩以推动内容物，而在大多数血管中发现的紧张性平滑肌则维持张力。这种力的维持被称为闩锁状态，发生在肌球蛋白激活水平较低时（肌球蛋白轻链[LC20]磷酸化）。虽然已经提出了分子机制来解释闩锁状态，但由于技术限制，此前仅在全肌肉水平上进行过研究。在本研究中，设计了一种测定室，以便在激光阱和体外运动测定过程中注射肌球蛋白轻链磷酸酶（MLCP），同时不产生大量流动，从而在分子水平上重现闩锁状态条件。使用单光束模式的激光阱，使一根肌动蛋白丝与基座上的几个肌球蛋白分子接触。肌球蛋白拉动肌动蛋白丝，直到达到平台力，此时注射MLCP。在LC20去磷酸化过程中观察到力的维持，其水平在相同条件下进行的平行体外运动测定中进行评估。从紧张性平滑肌中纯化的肌球蛋白比从阶段性平滑肌中纯化的肌球蛋白维持力的时间更长。这些数据支持了由去磷酸化、非循环横桥导致强键形成这一长期存在的观点。此外，在使用平滑肌和骨骼肌肌球蛋白进行的体外运动混合测定中注射MLCP表明，只有在周围没有活跃循环的肌球蛋白分子提供能量的情况下，这些强键才有可能维持。
