Macroendocytosis and endosome processing in snake motor boutons.
We have examined the processing of endosomes formed by macroendocytosis (ME), or bulk membrane retrieval, in active motor terminal boutons at the snake nerve-muscle synapse. Endocytic probes were imaged at light (FM1-43) and electron (horseradish peroxidase (HRP)) levels over stimulus frequencies representing low, intermediate and high levels of use. Endosomes formed rapidly (1-2 s) at all frequencies, concomitant with clathrin-mediated vesicular endocytosis (CME). Endosomes dissipated rapidly into vesicles (approximately 10 s). The dissipation rate was not influenced by activity. Many endosomes split into clusters of 2-20 smaller endosomes of varying size. Vesicles budded from these smaller endosomes, from large endosomes that had not undergone fission into smaller ones, and from precursor membrane infoldings that had not yet internalized. In snake, exocytosed vesicular membrane is not competent for reuse until after a delay (> 3 min). We found that time required for endosome processing is not responsible for this delay. Endosome processing might, however, limit availability of some vesicles for release at very high levels of use. Generally, endosome processing paralleled that of vesicles internalized directly from the plasma membrane via CME, regardless of stimulus frequency. There was no evidence for differential recruitment of ME versus CME depending upon level of use.
Teng
Haibing
H
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Av., Box 8228, St Louis, MO 63110, USA.
Lin
Michael Y
MY
Wilkinson
Robert S
RS
D000009:Abdominal Muscles / Q000294:innervation*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D017812:Colubridae; D004558:Electric Stimulation / Q000379:methods; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase; D066298:In Vitro Techniques; D008561:Membrane Fusion; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D009046:Motor Neurons / Q000378:metabolism* / Q000648:ultrastructure; D009469:Neuromuscular Junction / Q000166:cytology / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D013997:Time Factors
C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D006735:Horseradish Peroxidase