REGULATION OF VESICLE TRAFFIC IN NEURONS & RELATED CELLS

神经元中囊泡运输的调节

• 批准号: 2268439
• 负责人: George S Bloom
• 金额: $ 11.4万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2268439
• 关键词: G protein; SDS polyacrylamide gel electrophoresis; adrenal medulla; animal tissue; cytoskeleton; interference microscopy; intracellular transport; kinesin; laboratory mouse; microfilaments; microtubule associated protein; microtubules; molecular cloning; monoclonal antibody; neuroendocrine system; neuronal transport; neurons; phosphorylation; posttranslational modifications; protein isoforms; protein purification; protein sequence; tissue /cell culture; tubulin; vesicle /vacuole; video recording system; western blottings

Neurons and neuroendocrine cells possess an intricate cytoplasmic machinery for the transport and release of membrane-bounded organelles. This organelle trafficking serves a multitude of purposes: to supply proteins to the axon, which cannot synthesize its own resident proteins; to deliver secretory products, such as neurotransmitters and catecholamines, to the cell surface; and for retrograde transport of endocytosed trophic factors. Collectively, these types of organelle movements are essential for the development and maintenance of the neuronal phenotype, and for the specialized secretory functions of neuroendocrine cells. Two types of cytoskeletal polymers play pivotal roles in organelle trafficking. Microbutules serve as the tracks for long range organelle motility, while dense meshworks of microfilaments must be traversed by motile organelles near the cell surface and in cytoplasmic regions immediately surrounding microtubules. The broad purpose of the experiments outlined in this proposal is to characterize mechanisms for regulating organelle transport in neuronal and neuroendocrine cells. It is evident that regulation must occur because organelles capable of being transported along microbutules are not always in transit, and often are capable of moving in either direction relative to microtubule polarity. How, then, does an organelle know when to move along a microtubule and which way to travel? Moreover, do changes in the number and organization of microfilaments affect the ability of organelles to travel through the cell, as evidence suggests, or do microfilaments serve different roles in organelle trafficking? Questions such as these will be addressed in the proposed study. Research will focus on three distinct and complementary levels at which regulation is likely to occur. The first Specific Aim concerns the role of phosphorylation in regulating the functions of kinesin. This microtubule- stimulated ATPase is evidently responsible for anterograde transport of membranous organelles in the axon, and for related organelle motility elsewhere in neurons and in other cell types. Recent evidence from the laboratories of the PI and Co-PI has indicated that the kinesin heavy and light chains become phosphorylated in vivo. The effects of phosphorylation on the ATPase, microtubule binding and organelle binding activities of kinesin will be investigated. The second Specific Aim is focused on the role of MAPs in regulating access of kinesin to the microtubule surface, and on the differential abilities of various brain tubulin isoforms to interact with kinesin. The final Specific Aim will deal with a small G protein recently discovered by the PI's laboratory to interact with the cytoskeleton. This G protein will be cloned and sequenced, and its biological role will be characterized by a combination of cell biological and biochemical approaches.

神经元和神经内分泌细胞具有复杂的细胞质机制 用于膜结合细胞器的运输和释放。 这 细胞器的运输有多种用途： 轴突不能合成自身的蛋白质， 分泌产物，如神经递质和儿茶酚胺， 细胞表面;以及用于内吞营养因子的逆行转运。 总的来说，这些类型的细胞器运动是必不可少的， 神经元表型的发育和维持，以及 神经内分泌细胞的特殊分泌功能。 两种类型的 细胞骨架聚合物在细胞器运输中起关键作用。 微管作为长距离细胞器运动的轨道， 运动的细胞器必须穿过致密的微丝网 在细胞表面附近和细胞质区域中， 微管 本提案中概述的实验的主要目的是 表征调节神经元细胞器运输的机制， 神经内分泌细胞 显然，必须进行监管，因为 能够沿着微管运输的细胞器并不总是 在运输中，并且通常能够相对于 微管极性 那么，一个细胞器是如何知道什么时候该沿着的呢 一个微管和它的行进路线 此外，数字的变化 和组织影响细胞器的能力， 有证据表明，微丝在细胞中穿行， 在细胞器贩卖中扮演不同的角色 像这样的问题将是 在拟议的研究中。 研究将集中在三个不同和互补的层面， 监管可能会发生。 第一个具体目标涉及的作用 磷酸化在调节驱动蛋白功能中的作用。 这个微管- 受刺激的ATP酶显然负责 轴突中的膜性细胞器，以及相关的细胞器运动 在神经元和其他细胞类型中。 的最新证据 PI和Co-PI的实验室已经表明，驱动蛋白重和 轻链在体内被磷酸化。 磷酸化的影响 对ATP酶、微管结合和细胞器结合活性的影响 将研究驱动蛋白。 第二个具体目标的重点是 MAP在调节驱动蛋白进入微管表面中的作用， 以及各种脑微管蛋白亚型的不同能力， 与驱动蛋白相互作用。 最后的具体目标将处理一个小G PI实验室最近发现的蛋白质与 细胞骨架 这种G蛋白将被克隆和测序，其 生物学作用的特征将是细胞生物学 和生物化学方法。