MT COBRE: MOLECULAR ANALYSIS OF SIGNALLING ENDOSOMES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在神经系统的发育过程中，产生的神经细胞比需要的要多，在哺乳动物中，对那些建立功能联系的神经细胞进行了选择。不能正常连接的神经元通过激活程序性细胞死亡来自杀；连接的神经元存活并分化。当突出的轴突到达它们的目标时，它们接受神经营养因子（如神经生长因子（NGF））形式的生存和分化指令，神经生长因子（NGF）与轴突尖端的受体（Trks）结合。神经营养因子信号从轴突尖端向细胞体的轴突传递（逆行信号）因此是神经系统发育的关键因素，有迹象表明这一步骤可能在神经退行性疾病中出错。NGF和Trk一起内化到传递信号的胞内膜结合细胞器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进入内吞细胞器。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的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究竟在哪里不同还没有明确的定义。我们假设分选核内体将受体分隔成专门的信号细胞器。使用速度和平衡离心，我们已经分离出几种不同类别的细胞器来源于含有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免疫分离来纯化每个细胞器。在网格蛋白包覆的囊泡下游含有trk的细胞器含有核内体信号效应物Rap1，并且在电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。我们将使用已知的蛋白质抗体作为信号效应物和膜交通标记物，并使用质谱识别所有其他成分。我们希望发现新的成分，这将有助于阐明不同的信号转导途径是如何在胞吞作用后被划分的，并定义逆行信号传导的关键要素。

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.