REGULATION OF MICROTUBULE DEPENDENT VESICLE TRANSPORT BY MICROCYSTIN-LR

微囊藻毒素-LR对微管依赖性囊泡运输的调节

• 批准号: 6105634
• 负责人: Sarah F Hamm-Alvarez
• 金额: $ 11.43万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6105634
• 关键词: confocal scanning microscopy; dynein ATPase; intracellular transport; kinesin; liver cells; microtubules; phosphatase inhibitor; phosphorylation; vesicle /vacuole; video microscopy

Our long term objective is to understand the mechanisms of regulation of the microtubule-dependent motor proteins, kinesin and cytoplasmic dynein. These motors drive the movement of cellular vesicles along microtubules within cells. Microtubule-dependent vesicle transport is an essential component of cellular membrane trafficking including endocytosis and exocytosis. Little is known about the mechanisms of regulation of kinesin and cytoplasmic dynein, or of the impact of modulation of their activity on cellular membrane trafficking. Numerous reports have documented the extensive rearrangement of cytoskeletal components (microtubules, microfilaments, and intermediate filaments) by okadaic acid, calyculin A, and microcystin-LR, members of a group of serine- threonine protein phosphatase inhibitors. Several groups have also reported that vesicle transport is inhibited by these toxins at doses that also elicit major cytoskeletal changes. Of considerable interest is our recent preliminary data on the ability of okadaic acid to stimulate vesicle motility at concentrations less than those which promote significant cytoskeletal changes, indicating that these toxins may elicit a concentration-dependent biphasic effect on vesicle transport. While okadaic acid and calyculin A are cell permanent, microcystin-LR is cell impermeant in all cell types except hepatocytes, which take-up this toxin via a membrane transporter. Using video- enhanced DIC microscopy, laser-scanning confocal microscopy, and protein phosphorylation studies, we will examine the regulation of microtubule- dependent vesicle transport by microcystin-LR in intact hepatocytes. Our hypothesis is that microcystin-LR, at doses less than those which perturb the cytoskeleton, can stimulate microtubule-dependent vesicle transport via protein phosphorylation of kinesin and cytoplasmic dynein. Our specific aims are: 1. What is the effect of microcystin-LR on microtubule-dependent vesicle transport in intact hepatocytes. 2. Does microcystin-LR alter protein phosphorylation patterns of kinesin and cytoplasmic dynein.

我们的长期目标是了解 微管依赖性马达蛋白、驱动蛋白和细胞质动力蛋白。 这些马达驱动细胞囊泡沿沿着运动 细胞内。 微管依赖的囊泡运输是一个必不可少的 细胞膜运输的组成部分，包括内吞作用和 胞吐作用 关于调节的机制知之甚少， 驱动蛋白和细胞质动力蛋白，或其调节的影响， 细胞膜运输的活性。 许多报告指出， 证明了细胞骨架成分的广泛重排 （微管，微丝和中间丝）由okadaic 酸，calyculin A和微囊藻毒素-LR，丝氨酸- 苏氨酸蛋白磷酸酶抑制剂。 几个团体还 报道，囊泡运输被这些毒素抑制， 也会引起细胞骨架的变化 关系重大 是我们最近关于冈田酸 刺激囊泡运动的浓度低于 促进显著的细胞骨架变化，表明这些毒素 可能引起对囊泡的浓度依赖性双相效应 运输 而冈田酸和calyculin A是细胞永久性的， 微囊藻毒素-LR在除肝细胞外的所有细胞类型中是细胞不渗透的， 通过膜转运蛋白吸收毒素 使用视频- 增强DIC显微镜、激光扫描共聚焦显微镜和蛋白质 磷酸化研究，我们将研究微管的调节- 完整肝细胞中微囊藻毒素-LR的依赖性囊泡转运。 我们 假设微囊藻毒素-LR，在剂量低于那些干扰 细胞骨架，可以刺激微管依赖的囊泡运输 通过驱动蛋白和细胞质动力蛋白的蛋白磷酸化。 我们的具体目标是： 1.微囊藻毒素-LR对微管依赖性囊泡的影响 在完整肝细胞中转运。 2.微囊藻毒素LR改变驱动蛋白的磷酸化模式 和细胞质动力蛋白。