Molecular requirements for bi-directional movement of phagosomes along microtubules.
Molecular requirements for bi-directional movement of phagosomes along microtubules.
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DOI：
10.1083/jcb.137.1.113
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发表时间：
1997-04-07
期刊：
影响因子：
--
通讯作者：
Griffiths G
中科院分区：
文献类型：
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作者：
Blocker A;Severin FF;Burkhardt JK;Bingham JB;Yu H;Olivo JC;Schroer TA;Hyman AA;Griffiths G
微管通过有利于吞噬体与内吞区室的相互作用来促进吞噬体的成熟。在这里，我们发现吞噬体在细胞内沿着几微米的轨道以 pH 值和微管依赖性方式离心和向心移动。吞噬体运动在体外重建，需要该细胞器的能量、细胞质和膜蛋白。这些吞噬体蛋白的活性或存在随着细胞器的成熟而受到调节，“晚期”吞噬体的移动频率是“早期”吞噬体的三倍。大多数移动的吞噬体是负端定向的；其余的向微管正端移动，一小部分则双向移动。负端运动显示出动力蛋白预期的药理学特征，被细胞质动力蛋白的免疫耗竭所抑制，并且可以通过添加细胞质动力蛋白来恢复。正端运动表现出驱动蛋白的药理学特性，通过驱动蛋白的免疫耗竭部分抑制，并通过添加抗驱动蛋白 IgG 完全抑制。 dynactin（一种动力蛋白激活复合物）的免疫耗竭仅抑制负端定向运动。提供了动力蛋白介导的囊泡运输所需的动力蛋白相关激酶的证据。两个方向的运动受到来自运动阻断抗体结合区域的运动蛋白（一种推定的运动蛋白膜受体）的肽片段的抑制。通过免疫印迹或免疫电子显微镜在吞噬体上检测到来自这些基于微管的运动因子的多肽亚基。这是第一项使用单一体外系统的研究，该系统描述了驱动蛋白、驱动蛋白、细胞质动力蛋白和动力蛋白在纯化膜细胞器的微管介导的运动中所发挥的作用。
Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubuledependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with “late” phagosomes moving threefold more frequently than “early” ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.