结蛋白N端结构域决定其在分化的肌母细胞中的负向运输

摘要

Objective:To investigate the transportation of an intermediate filament protein desmin in differentiating myotubes.Methods:Desmin-EGFP transportation in the wild type myotubes was monitored by fluorescence recovery after photobleaching(FRAP)assay.Desmin localization in Kif5b knockout cells was examined under the overexpression of dynamitin which inhibits cytoplasmic dynein activity.Different desmin truncations with Flag tags were expressed in the wild type or Kif5b knockout cells and the effects of domain truncations on the desmin localization were examined by immunofluorescence.Results:The fluorescent signals of EGFP-desmin came into a photobleached region from both directions in wild type myotubes.Desmin centrally aggregates in the Kif5b knockout cells; overexpression of dynamitin abolished its aggregation in these cells.Deletion of the N-terminal domain of desmin did not change its distribution pattern in the wild type cells,but alleviated its central aggregation in Kif5b knockout cells.Conclusion:The localization of desmin is the result of anterograde and retrograde transportation balance.Cytoplasmic dynein may play a role in the retrograde transportation of desmin,which is dependent on the N-terminus of desmin.%目的:在分化的肌母细胞或肌管中,研究结蛋白的运输机制.方法:利用荧光漂白恢复的方法,在野生型肌管细胞中直接观察绿色荧光蛋白标记的结蛋白desmin-EGFP的运动;通过过表达dynamitin,抑制胞浆动力蛋白cytoplasmic dynein介导的沿微管向负极的运输,观察结蛋白在微管驱动蛋白Kinesin-1重链Kif5b敲除细胞中定位的改变;构建Flag tag标记的不同结构域缺失的结蛋白表达载体,转染野生型及Kif5b敲除细胞,通过免疫荧光染色检测不同结构域缺失对结蛋白定位的影响.结果:在野生型肌管细胞内,desmin-EGFP沿正向和负向同时进入被漂白的区域.在Kif5b敲除细胞内,结蛋白聚集于细胞中心；抑制动力蛋白的活性使得结蛋白在该细胞中的聚集程度减轻.结蛋白N端蛋白结构域缺失对其在野生型细胞中的定位没有影响,但使其在Kif5b敲除细胞中心的聚集明显减弱.结论:结蛋白在分化的肌母细胞或肌管中的定位是正向运输和负向运输双重作用的结果,其中负向运输可能由动力蛋白负责,并依赖于结蛋白N端蛋白结构域的表达.