背景:在活细胞中经常观察到肌动蛋白丝(AF)和中间丝(IF)之间的关联。这些细胞骨架成分之间的串扰是细胞组织和动力学的基础;然而,丝状相互作用的分子基础尚未完全了解。这里,我们描述了在重组的体外系统中,AFs和DesminIFs(DIFs)之间的相互作用模式。
方法:用荧光染料标记AFs(兔骨骼肌)和DIFs(鸡g)。将DIF固定在重质肌球蛋白(HMM)包被的胶棉表面上。在DIF存在下评估具有ATP水解的HMM驱动的AF。使用荧光显微镜获得单丝的图像。从显微镜图像计算单个AF轨迹的矢量变化。
结果:与DIF接触后,AF速度瞬时降低。在与DIF接触时,移动的AF的入射角和出射角之间的差变宽。较小的入射角倾向于以向列方式导致较小的出射角。移动AF的百分比随着DIF密度的增加而减少,但是移动AF的速度与无desmin控制中的速度相似。大量的DIF倾向于将AF从HMM涂层表面中排除。
结论:DIFs会随着方向的变化而促进AF的运动。DIF可以与HMM结合并削弱肌动蛋白-肌球蛋白相互作用。
结论:研究表明,除了结合强度外,丝状结构的弱相互作用特征的积累可能会影响细胞结构的动态组织。
Associations between actin filaments (AFs) and intermediate filaments (IFs) are frequently observed in living cells. The crosstalk between these cytoskeletal components underpins cellular organization and dynamics; however, the molecular basis of filamentous interactions is not fully understood. Here, we describe the mode of interaction between AFs and desmin IFs (DIFs) in a reconstituted in vitro system.
AFs (rabbit skeletal muscle) and DIFs (chicken gizzard) were labeled with fluorescent dyes. DIFs were immobilized on a heavy meromyosin (HMM)-coated collodion surface. HMM-driven AFs with ATP hydrolysis was assessed in the presence of DIFs. Images of single filaments were obtained using fluorescence microscopy. Vector changes in the trajectories of single AFs were calculated from microscopy images.
AF speed transiently decreased upon contact with DIF. The difference between the incoming and outgoing angles of a moving AF broadened upon contact with a DIF. A smaller incoming angle tended to result in a smaller outgoing angle in a nematic manner. The percentage of moving AFs decreased with an increasing DIF density, but the speed of the moving AFs was similar to that in the no-desmin control. An abundance of DIFs tended to exclude AFs from the HMM-coated surfaces.
DIFs agitate the movement of AFs with the orientation. DIFs can bind to HMMs and weaken actin-myosin interactions.
The study indicates that apart from the binding strength, the accumulation of weak interactions characteristic of filamentous structures may affect the dynamic organization of cell architecture.