Department of Biology, Missouri State University, 901 South National Ave., Springfield, MO 65897, USA.
Eur J Cell Biol. 2010 Jul;89(7):499-508. doi: 10.1016/j.ejcb.2010.02.002. Epub 2010 Mar 2.
Mammalian dynamin is responsible for scission of endocytic vesicles from the plasma membrane. A previous study showed that Vps1, a yeast dynamin-like protein, plays an important role in pheromone receptor internalization (Yu and Cai, 2004; J. Cell Sci. 117, 3839-3853). However, the details of how Vps1 acts in various phases of endocytosis including early internalization of the endocytic vesicle are poorly understood. To investigate the potential roles of Vps1 in both endocytic vesicle formation/maturation on the plasma membrane and endocytic vesicle internalization, time-lapse fluorescent images of GFP-tagged endocytic markers in live cells were analyzed using a particle tracking software. The loss of Vps1 leads to a robust increase in the lifespan of newly forming cortical endocytic vesicles carrying Las17-GFP, Ede1-GFP, Sla1-GFP, and Abp1-GFP, indicating that Vps1 is required for the proper assembly and maturation of endocytic vesicles. Particle track analysis revealed that Abp1-GFP vesicles in vps1 null cells moved a relatively short distance away from the cell membrane due to their non-directional movement. Furthermore, we found that the GTPase and the GED domains of Vps1 are required for the proper endocytic function of Vps1. Our tracking analysis data also revealed that the post-internalized vesicle motility en route to the vacuole was decreased significantly, perhaps due to severe disruption of the actin cables in Vps1 mutant cells.
哺乳动物的动力蛋白负责从质膜上切割内吞小泡。之前的研究表明，酵母动力蛋白样蛋白 Vps1 在激素受体内化中发挥着重要作用（Yu 和 Cai，2004；J. Cell Sci. 117, 3839-3853）。然而，Vps1 在包括内吞小泡早期内化在内的各个内吞阶段中发挥作用的细节仍知之甚少。为了研究 Vps1 在质膜上形成/成熟内吞小泡以及内吞小泡内化的各个阶段中的潜在作用，使用粒子跟踪软件对活细胞中 GFP 标记的内吞标记物的延时荧光图像进行了分析。Vps1 的缺失导致新形成的携带 Las17-GFP、Ede1-GFP、Sla1-GFP 和 Abp1-GFP 的皮质内吞小泡的寿命显著延长，表明 Vps1 是内吞小泡正确组装和成熟所必需的。粒子轨迹分析显示，由于 Abp1-GFP 小泡的非定向运动，它们在 vps1 缺失细胞中远离质膜的移动距离相对较短。此外，我们发现 Vps1 的 GTP 酶和 GED 结构域对于 Vps1 的正常内吞功能是必需的。我们的跟踪分析数据还表明，内吞后囊泡向液泡的运动能力显著降低，这可能是由于 Vps1 突变细胞中肌动蛋白纤维严重破坏所致。
