Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.
Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.
复制标题
通过直接观察量子点标记的肌球蛋白头和杆在体外平滑肌肌球蛋白丝上移动来证明S2的灵活性。
DOI：
10.1085/jgp.202012751
复制
发表时间：
2021-03-01
期刊：
影响因子：
--
通讯作者：
Cremo CR
中科院分区：
文献类型：
--
作者：
Brizendine RK;Anuganti M;Cremo CR
Brizendine等人在体外直接可视化量子点标记的肌球蛋白头和棒，以了解S2亚结构域的灵活性。他们的结果证实了S2的运动范围及其对肌凝蛋白对肌动蛋白相对运动的影响的理论预测。肌肉中的肌凝蛋白通过c端轻肌凝蛋白（LMM）亚域之间的相互作用组装成细丝。两个头部域通过杆的子片段-2 （S2）子域连接到LMM。我们的混合动力学模型预测，可以从丝上拉出的S2的灵活性和长度影响工作头可以不受肌动蛋白附着头阻碍地移动丝的最大距离。它还表明，当与肌动蛋白结合时，头部相对于丝主干保持静止（驻留）是可能的，随后在脱离后立即有一个可测量的跳跃，以恢复主干轨迹。我们通过使用TIRF显微镜观察纤维在不同ATP下沿着肌动蛋白移动来测试这些预测。我们同时跟踪了两个不同颜色的量子点（QDs），一个附着在杠杆臂上的调节光链上，另一个附着在灯丝主干上的LMM上。我们通过比较量子点的轨迹来确定事件（驻留之后是跳跃）。平均停留时间与已知的肌动球蛋白系统动力学一致，观察到的事件之间等待时间的分布与泊松过程和预期的atp酶速率一致。几何约束表明，最大约26 nm的S2可以从纤维中分离出来，这可能与螺旋状的S2断裂有关，这与其他人观察到的S2从肌肉中的纤维中突出的结果一致。我们提出，从长丝的工作头可以获得足够的力来克服由长丝- s2相互作用施加的刚度。
Brizendine et al. directly visualized quantum dot–labeled myosin heads and rods in vitro to understand the flexibility of the S2 subdomain. Their results confirm theoretical predictions about the range of motion of S2 and its effect on the relative movement of myosin on actin in vitro. Myosins in muscle assemble into filaments by interactions between the C-terminal light meromyosin (LMM) subdomains of the coiled-coil rod domain. The two head domains are connected to LMM by the subfragment-2 (S2) subdomain of the rod. Our mixed kinetic model predicts that the flexibility and length of S2 that can be pulled away from the filament affects the maximum distance working heads can move a filament unimpeded by actin-attached heads. It also suggests that it should be possible to observe a head remain stationary relative to the filament backbone while bound to actin (dwell), followed immediately by a measurable jump upon detachment to regain the backbone trajectory. We tested these predictions by observing filaments moving along actin at varying ATP using TIRF microscopy. We simultaneously tracked two different color quantum dots (QDs), one attached to a regulatory light chain on the lever arm and the other attached to an LMM in the filament backbone. We identified events (dwells followed by jumps) by comparing the trajectories of the QDs. The average dwell times were consistent with known kinetics of the actomyosin system, and the distribution of the waiting time between observed events was consistent with a Poisson process and the expected ATPase rate. Geometric constraints suggest a maximum of ∼26 nm of S2 can be unzipped from the filament, presumably involving disruption in the coiled-coil S2, a result consistent with observations by others of S2 protruding from the filament in muscle. We propose that sufficient force is available from the working heads in the filament to overcome the stiffness imposed by filament-S2 interactions.