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: 6270778
• 负责人: Sarah F Hamm-Alvarez
• 金额: $ 10万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270778
• 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 微管依赖的运动蛋白、动蛋白和胞浆动力蛋白。 这些马达驱动细胞小泡沿微管运动。 在细胞内。微管依赖的囊泡运输是一种必不可少的 细胞膜运输的组成部分，包括内吞作用和 胞吐。对其调节机制知之甚少。 激动素和细胞质动力蛋白对其的影响或调制 细胞膜运输的活性。无数报道称， 记录了细胞骨架组件的广泛重新排列 (微管、微丝和中间丝) 酸、花盏花素A和微囊藻毒素-LR，属于丝氨酸- 苏氨酸蛋白磷酸酶抑制剂。有几个组织也 据报道，这些毒素在剂量上会抑制囊泡的运输。 这也引发了细胞骨架的重大变化。相当感兴趣 是我们最近关于冈田酸能力的初步数据 在低于以下浓度时刺激囊泡运动 促进显著的细胞骨架变化，表明这些毒素 可能在水泡上引起浓度依赖的双相效应 运输。虽然冈田酸和盏花素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都是不存在的。 它通过膜转运器吸收这种毒素。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- 微囊藻毒素-LR在完整肝细胞中的依赖性囊泡转运。我们的 假设是微囊藻毒素-LR，剂量低于那些扰动 细胞骨架，可以刺激微管依赖的囊泡运输 通过激动素和细胞质动力蛋白的蛋白质磷酸化。 我们的具体目标是： 1.微囊藻毒素-LR对微管依赖性小泡的作用 在完整的肝细胞中运输。 2.微囊藻毒素-LR是否改变了激动素的蛋白磷酸化模式 和细胞质动力蛋白。