Ordered recruitment of dynactin to the microtubule plus-end is required for efficient initiation of retrograde axonal transport.
Long-range retrograde axonal transport in neurons is driven exclusively by the microtubule motor cytoplasmic dynein. The efficient initiation of dynein-mediated transport from the distal axon is critical for normal neuronal function, and neurodegenerative disease-associated mutations have been shown to specifically disrupt this process. Here, we examine the role of dynamic microtubules and microtubule plus-end binding proteins (+TIPs) in the initiation of dynein-mediated retrograde axonal transport using live-cell imaging of cargo motility in primary mouse dorsal root ganglion neurons. We show that end-binding (EB)-positive dynamic microtubules are enriched in the distal axon. The +TIPs EB1, EB3, and cytoplasmic linker protein-170 (CLIP-170) interact with these dynamic microtubules, recruiting the dynein activator dynactin in an ordered pathway, leading to the initiation of retrograde transport by the motor dynein. Once transport has initiated, however, neither the EBs nor CLIP-170 are required to maintain transport flux along the mid-axon. In contrast, the +TIP Lis1 activates transport through a distinct mechanism and is required to maintain processive organelle transport along both the distal and mid-axon. Further, we show that the EB/CLIP-170/dynactin-dependent mechanism is required for the efficient initiation of transport from the distal axon for multiple distinct cargos, including mitochondria, Rab5-positive early endosomes, late endosomes/lysosomes, and TrkA-, TrkB-, and APP-positive organelles. Our observations indicate that there is an essential role for +TIPs in the regulation of retrograde transport initiation in the neuron.
Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085, USA.
Armen J
AJ
Moughamian
Gregory E
GE
Osborn
Jacob E
JE
Lazarus
Sandra
S
Maday
Erika L F
EL
Holzbaur
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000378:metabolism; D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D000072159:Dynactin Complex; D004398:Dyneins / Q000235:genetics; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D038761:Photobleaching; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000072159:Dynactin Complex; D004398:Dyneins; D008164:Luminescent Proteins; D051907:Lysosomal Membrane Proteins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D020696:rab5 GTP-Binding Proteins; C491477:Actr1a protein, mouse; C405804:EB1 microtubule binding proteins; C522423:EB3 protein, mouse; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1; C076744:cytoplasmic linker protein 170; C513190:Pafah1b1 protein, mouse