MT COBRE: MOLECULAR ANALYSIS OF SIGNALLING ENDOSOMES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在神经系统的发育过程中，出生的神经细胞比所需的要多，在哺乳动物中，可以选择那些建立功能连接的神经细胞。没有建立适当连接的神经元通过激活程序性细胞死亡而导致细胞自杀；确实连接的神经元存活并分化。当投射的轴突到达它们的目标时，它们会收到指令，以神经营养因子的形式生存和分化，例如神经生长因子(NGF)，它与轴突顶端的受体(Trks)结合。因此，神经营养因子信号从轴突尖端到细胞体的轴突运输(逆行信号传递)是神经系统发育的关键因素，有迹象表明，在神经退行性疾病中，这一步骤可能会出错。NGF和Trk一起内化到细胞内传递信号的膜结合细胞器的第一步是笼蛋白介导的内吞作用。膜运输的后续步骤是将受体分类到内吞细胞器中。有很好的证据表明，内吞细胞器中的受体启动的信号转导通路不同于质膜或轴突顶端的受体激活的信号转导通路。内体中的信号与质膜中的信号究竟在哪里不同还没有定义。我们假设，分类内小体将受体划分成专门的信号细胞器。使用速度和平衡离心法，我们分离了几种不同类型的细胞器，这些细胞器来自于含有Trk受体的内吞作用。我们设计了一种有效的方法，利用一种新型的非常微小的磁珠进行免疫隔离来纯化每个细胞器。笼蛋白包裹的囊泡下游含有TrK的细胞器含有内体信号传递效应器Rap1，在电子显微镜下具有囊泡和小管的形态。我们建议测定这些纯化细胞器的含量。我们将使用已知起信号效应器和膜交通标志作用的蛋白质的抗体，并使用质谱学识别所有其他成分。我们希望识别新的成分，这将有助于阐明不同的信号转导通路在内吞作用后是如何划分的，并确定逆行信号的关键成分。

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.