Single Exo/Endocytic Events in Neurons & Endocrine Cells

神经元中的单个外切/内吞事件

• 批准号: 7319637
• 负责人: WOLFHARD ALMERS
• 金额: $ 47.66万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7319637
• 关键词: Actins; Address; Biological Assay; Biological Models; Cell membrane; Cell surface; Cells; Cellular Neurobiology; Cisplatin/Doxorubicin/Melphalan/Teniposide; Clathrin; Clathrin-Coated Vesicles; Color; Cytoplasmic Granules; Cytosol; Disease; Docking; Dynamin; Endocrine; Endocytic Vesicle; Endocytosis; Event; Exocytosis; Failure; Fill-It; Fishes; Grant; Hippocampus (Brain); Hormones; Image; Kinetics; Life; Localized; Membrane; Membrane Fusion; Methods; Molecular; Neuroendocrine Cell; Neurologic; Neurons; PC12 Cells; PHluorin; Pineal gland; Plasma; Presynaptic Terminals; Proteins; Purpose; Recruitment Activity; Regulation; Retinal; Retrieval; Role; Running; Secretory Vesicles; Site; Structure; Synapses; Synaptic Vesicles; Testing; Time; Transport Vesicles; Vesicle; amphiphysin; coated pit; human VAPA protein; light microscopy; research study; retinal bipolar neuron; ribbon synapse; seal; sensor; size; vesicle-associated membrane protein; voltage

DESCRIPTION (provided by applicant): Neurons and endocrine cells secrete transmitter or hormones in Ca- triggered bursts of exocytosis. In a broad sense, exocytosis is how neurons and neuroendocrine cells "talk". Secretion involves four steps: (i) Making the secretory vesicle and filling it with cargo, (ii) transporting the vesicle to a site of exocytosis, (iii) membrane fusion or exocytosis, and finally (iv) the retrieval of the vesicle's membrane by mechanisms broadly called endocytosis. In synaptic vesicles, a new synaptic vesicle is made from the membrane thus retrieved, and the four events form a cycle. All four steps involve multiple proteins and multiple layers of regulation. All four can fail in disease, and most or all neurologic diseases ultimately result in the failure of one or more steps in the synaptic vesicle cycle. This grant proposes functional studies of exo- and endocytosis at the level of single secretory vesicles in intact neurons and endocrine cells. Using an unconventional light microscopy we helped to adapt for this purpose, we will specifically and simultaneously mark proteins in exo- and endocytic vesicles in different fluorescent colors, and then observe the recruitment and release of these proteins to vesicles, and the recruitment and release of vesicles from the plasma membrane. Through observations in living cells, we focus on the following questions. (1) How are synaptic vesicles transported and recruited to their docking sites at presynaptic terminals? (2) How completely do secretory vesicles fuse with the plasma membrane? (3) How do neurons and endocrine cells deal with the material that exocytosis has inserted into the plasma membrane, and is such material inserted in the first place? The planned experiments will generate direct evidence in a way not possible with other known methods, and will address fundamental questions in cellular neurobiology.

描述（由申请人提供）： 神经元和内分泌细胞分泌发射机或激素，触发胞吞作用爆发。从广义上讲，胞吐作用是神经元和神经内分泌细胞的“讲话”。分泌涉及四个步骤：（i）制作分泌囊泡并用货物填充，（ii）将囊泡运送到胞吐作用位点，（iii）膜融合或胞吐作用，最后（iv）（iv）通过广泛称为内吞作用的机制检索囊泡的膜。在突触囊泡中，从因此检索的膜制成了一个新的突触小囊泡，而四个事件形成了一个循环。所有四个步骤均涉及多种蛋白质和多层调节。这四个疾病都可能失败，大多数或所有神经系统疾病最终导致突触囊泡周期中一个或多个步骤失败。 该赠款提出了在完整神经元和内分泌细胞中单个分泌囊泡水平上的外吞和内吞作用的功能研究。使用非常规的光学显微镜，我们帮助适应了这个目的，我们将以不同的荧光颜色的外囊和内吞囊泡中的特定和同时标记蛋白质，然后观察这些蛋白质对囊泡的募集和释放，以及从Plasmambrane中募集和募集囊泡。通过对活细胞的观察，我们关注以下问题。 （1）如何将突触囊泡运输和招募到突触前末端的对接位置？ （2）分泌囊泡如何与质膜融合在一起？ （3）神经元和内分泌细胞如何处理胞吞作用已插入质膜的材料，并且首先将这种材料插入？计划的实验将以其他已知方法无法实现的方式产生直接证据，并将解决细胞神经生物学中的基本问题。