Microtubule-dependent movement of late endocytic vesicles in vitro: Requirements for dynein and kinesin
Microtubule-dependent movement of late endocytic vesicles in vitro: Requirements for dynein and kinesin
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DOI：
10.1091/mbc.e04-04-0278
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发表时间：
2004-08-01
影响因子：
3.3
通讯作者：
Wolkoff, AW
中科院分区：
文献类型：
--
作者：
Bananis, E;Nath, S;Wolkoff, AW
我们先前的研究表明，在注射德克萨斯红脱唾液酸类粘蛋白后从大鼠肝脏制备的荧光早期内吞囊泡含有脱唾液酸糖蛋白及其受体，并使用驱动蛋白I和KIFC 2沿着微管移动和经历沿着分裂，Rab4调节KIFC 2活性（J.Cell Sci. 116，2749，2003）。在目前的研究中，程序来制备荧光晚期内吞囊泡的设计。此外，流式细胞术用于制备高度纯化的荧光内吞囊泡，允许验证基于显微镜的实验以及直接的生化分析。这些研究表明，晚期囊泡结合并沿着微管移动，但与早期囊泡相反，不进行分裂。与早期囊泡相比，晚期囊泡与受体Rab4和驱动蛋白I的关联减少，但与动力蛋白Rab7、动力蛋白和KIF3A高度相关。动力蛋白和KIF3A抗体抑制晚期囊泡运动，而驱动蛋白I和KIFC 2抗体没有影响。动力素抗体阻止了晚期囊泡与微管的结合。这些结果表明，收购和交换特定的电机和调节蛋白的特点，并可能调节早期到晚期的内吞囊泡的过渡。流式细胞仪纯化最终应有助于详细的蛋白质组学分析和内吞囊泡相关蛋白质的绘图。
Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.