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.
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