MT COBRE: MOLECULAR ANALYSIS OF SIGNALLING ENDOSOMES

MT COBRE：信号内体的分子分析

• 批准号: 7609800
• 负责人: M GRIMES
• 金额: $ 19.38万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7609800
• 关键词: Antibodies; Axon; Binding; Cell membrane; Class; Clathrin; Clathrin-Coated Vesicles; Computer Retrieval of Information on Scientific Projects Database; Electron Microscopy; Elements; Endocytosis; Endosomes; Equilibrium Centrifugation; Funding; Grant; Indium; Institution; Instruction; Intracellular Membranes; Mammals; Mass Spectrum Analysis; Mediating; Membrane Protein Traffic; Methods; Molecular Analysis; Morphology; Nerve Growth Factors; Neurodegenerative Disorders; Neurons; Organelles; Play; Proteins; Research; Research Personnel; Resources; Role; Signal Transduction; Signal Transduction Pathway; Sorting - Cell Movement; Source; United States National Institutes of Health; Vesicle; cell suicide; magnetic beads; nervous system development; neuron apoptosis; neuronal cell body; neurotrophic factor; novel; receptor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During development of the nervous system, more nerve cells are born than are needed and in mammals there is a selection for those which make functional connections. Neurons that do not make proper connections committ cell suicide by activating programmed cell death; neurons that do connect survive and differentiate. When projecting axons reach their target, they receive instructions to survive and differentiate in the form of neurotrophins such as nerve growth factor (NGF), which bind to receptors (Trks) at the axon tip. The axonal conveyance of neurotrophin signals from the axon tip to the cell body (retrograde signalling) is thus a key element in the development of the nervous system, and there is indication that this step may go awry in neurodegenerative disorders. The initial step by which NGF and Trk are internalized together into intracellular, membrane bound organelles that convey the signal is clathrin-mediated endocytosis. Subsequent steps in membrane traffic sort receptors into endocytic organelles. There is good evidence that receptors in endocytic organelles initiate signal transduction pathways that are distinct from those activated by receptors at the plasma membrane or at axon tips. Precisely where signalling in endosomes becomes different from that at the plasma membrane has not been defined. We hypothesize that sorting endosomes compartmentalize receptors into specialized signalling organelles. Using velocity and equilibrium centrifugation, we have isolated several different classes of organelles derived from endocytosis that contain Trk receptors. We have devised an effective method to purify each organelle using immunoisolation with a new type of very tiny magnetic beads. Trk-containing organelles downstream from clathrin-coated vesicles contain the endosome signalling effector, Rap1 and have the morphology of vesicles and tubules by electron microscopy. We propose to determine the contents of these purified organelles. We will use antibodies to proteins known to play a role as signalling effectors and membrane traffic markers, and identify all other components using mass spectroscopy. We hope to identify novel components, which will help elucidate how different signal transduction pathways are compartmentalized following endocytosis and define key elements of retrograde signalling.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 在神经系统的发育过程中，会产生比需要的更多的神经细胞，并且在哺乳动物中，会选择那些进行功能连接的神经细胞。 没有建立适当连接的神经元会通过激活程序性细胞死亡来自杀；确实连接的神经元会存活并分化。当突出的轴突到达目标时，它们会收到神经营养素形式的生存和分化指令，例如神经生长因子（NGF），神经生长因子（NGF）与轴突尖端的受体（Trks）结合。因此，神经营养蛋白信号从轴突尖端到细胞体的轴突传递（逆行信号传导）是神经系统发育的关键要素，并且有迹象表明这一步骤在神经退行性疾病中可能会出错。 NGF 和 Trk 一起内化到传递信号的细胞内膜结合细胞器中的第一步是网格蛋白介导的内吞作用。膜运输的后续步骤将受体分类为内吞细胞器。 有充分的证据表明，内吞细胞器中的受体启动的信号转导途径与质膜或轴突尖端受体激活的信号转导途径不同。内体中的信号传导与质膜上的信号传导不同的确切位置尚未确定。 我们假设内体分选将受体划分为专门的信号细胞器。使用速度和平衡离心，我们分离了几种不同类别的源自内吞作用的含有 Trk 受体的细胞器。我们设计了一种有效的方法，使用新型非常小的磁珠进行免疫分离来纯化每个细胞器。网格蛋白包被的囊泡下游含有 Trk 的细胞器含有内体信号传导效应子 Rap1，并且通过电子显微镜观察具有囊泡和小管的形态。 我们建议确定这些纯化细胞器的含量。我们将使用已知发挥信号效应器和膜交通标记作用的蛋白质的抗体，并使用质谱法鉴定所有其他成分。我们希望识别出新的成分，这将有助于阐明不同的信号转导途径在胞吞作用后如何划分，并定义逆行信号传导的关键要素。