Neuhaus E M, Soldati T
Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, D-69120 Heidelberg, Germany.
J Cell Biol. 2000 Sep 4;150(5):1013-26. doi: 10.1083/jcb.150.5.1013.
Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin-myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA(-)/B(-), and myoB(-) cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA(-)/B(-) cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.
基于几何学的机制已被提出用于解释内吞途径中膜和液相的分选，但对于肌动蛋白-肌球蛋白细胞骨架的参与情况知之甚少。在这里，我们证明盘基网柄菌肌球蛋白IB在质膜成分从内体循环回到细胞表面的过程中发挥作用。缺乏肌球蛋白B的细胞（肌球蛋白A缺失/肌球蛋白B缺失细胞，即myoA(-)/B(-)细胞）以及用肌球蛋白抑制剂丁二酮单肟处理的野生型细胞，在细胞内吞泡上积累了质膜标记物和生物素化的表面蛋白。基于质膜蛋白可逆生物素化的分析表明，肌球蛋白A/肌球蛋白B缺失的细胞中膜成分的循环严重受损。此外，在磁珠纯化的早期胞饮体上特异性发现了肌球蛋白B。使用快速冷冻冷冻电子显微镜方法，我们观察到在myoA(-)/B(-)细胞中，与相对早期的内吞泡相连的小泡数量增加，但与晚期内体和溶酶体相连的小泡数量未增加。小泡的这种积累表明膜循环缺陷是由分选区室的形态紊乱导致的。
