In Vitro Motility of Liver Connexin Vesicles along Microtubules Utilizes Kinesin Motors
In Vitro Motility of Liver Connexin Vesicles along Microtubules Utilizes Kinesin Motors
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DOI：
10.1074/jbc.m111.219709
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发表时间：
2011-07-01
影响因子：
4.8
通讯作者：
Spray, David C.
中科院分区：
文献类型：
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作者：
Fort, Alfredo G.;Murray, John W.;Spray, David C.
形成间隙连接（连接蛋白）的蛋白质从合成位点运输到连接结构域似乎需要细胞骨架递送机制。尽管许多细胞类型表现出将连接蛋白特异性递送至极化细胞位点，例如特异性定位于肝细胞基底外侧膜域的连接蛋白32 (Cx32)间隙连接，但基于肌动蛋白和微管蛋白的系统的确切作用仍不清楚。我们在极化肝细胞系 (WIF-B9) 中观察到荧光标记的 Cx32 以平均 0.25 μ m/s 的速度线性运输，50 μM 的微管破坏剂诺考达唑可消除这种运输。为了探索细胞骨架成分在连接蛋白传递中的参与，我们使用了从大鼠肝细胞中分离出的含有 Cx32 的囊泡的制剂，并在体外测定了它们沿着稳定的罗丹明标记的微管的 ATP 驱动的运动性。这些测定揭示了同一囊泡中存在 Cx32 和驱动蛋白运动蛋白。添加 50 μM ATP 刺激囊泡沿线性微管轨迹运动，速度为 0.4-0.5 μ m/s，该运动被 1 mM 驱动蛋白抑制剂 AMP-PNP（腺苷酰亚胺二磷酸）和抗驱动蛋白抗体抑制，但仅受 5 μM 钒酸盐（一种动力蛋白抑制剂）或抗动力蛋白抗体的最小影响。这些研究提供的证据表明，Cx32 可以在细胞内沿着微管转运，并可能转运至细胞中的连接域，并强调了驱动蛋白马达蛋白在 Cx32 微管依赖性运动中的重要作用。
Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin-and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 mu m/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 mu M of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 mu M ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 mu m/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 mu M vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.