REGULATION OF VESICLE TRAFFIC IN NEURONS & RELATED CELLS

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 细胞器运输有多种目的：提供蛋白质 到轴突，它不能合成自己的驻留蛋白质；传递 分泌性产品，如神经递质和儿茶酚胺， 细胞表面；以及内吞营养因子的逆行运输。 总的来说，这些类型的细胞器运动对于 神经元表型的发展和维持，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着关键作用。 微珠柄是细胞器远距离运动的轨迹，而 致密的微丝网络必须由可移动的细胞器穿过 靠近细胞表面和紧邻其周围的细胞质区域 微管。 本提案中概述的实验的主要目的是 表征神经元和神经细胞中细胞器运输的调节机制 神经内分泌细胞。显然，监管必须发生，因为 能够沿着微球运输的细胞器并不总是 在运输过程中，并且通常能够相对于 微管的极性。那么，细胞器如何知道何时前进呢？ 一个微管，该往哪个方向走？此外，在数量上做一些变化 和微丝的组织影响细胞器 如证据所示，在细胞中穿行，或者微丝起作用 在细胞器贩卖中扮演不同的角色？像这样的问题将会是 在拟议的研究中解决了这些问题。 研究将集中在三个截然不同和互补的水平上 监管很可能会发生。第一个具体目标涉及到 肌动蛋白功能调节中的磷酸化。这个微管- 受刺激的ATPase明显负责顺行转运 轴突内的膜性细胞器及其相关细胞器的运动 在神经元和其他细胞类型的其他地方。最近的证据来自 PI和Co-PI的实验室已经表明，运动蛋白重和 轻链在体内被磷酸化。磷酸化的影响 三磷酸腺苷酶、微管结合和细胞器结合活性的研究 将对激动素进行调查。第二个具体目标集中在 MAP在调节动蛋白进入微管表面中的作用， 以及不同脑微管蛋白亚型的分化能力 与激动素相互作用。最终的具体目标将处理一个小G PI的实验室最近发现的与细胞相互作用的蛋白质 细胞骨架。该G蛋白将被克隆和测序，其 生物角色的特征将由细胞生物学结合而成 以及生化方法。

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.