Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells.
The molecular mechanisms underlying "kiss and run" or "cavicapture" exocytosis of dense core secretory vesicles are presently unclear. Although dynamin-1 has previously been implicated in the recapture process in neurons, the recruitment of this fission protein to a single exocytosing vesicle has not been examined in real time during peptide release from pancreatic beta-cells. Imaged simultaneously in clonal insulin-secreting cells by dual color total internal reflection fluorescence microscopy, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y and green fluorescent protein (GFP)-tagged synaptotagmin-1 or synaptobrevin-2 rapidly diffused from sites of exocytosis, whereas the vesicle membrane protein phogrin and tissue plasminogen activator (tPA) were retained, consistent with fusion pore closure. Vesicle recovery frequently involved the recruitment of enhanced GFP-tagged dynamin-1, and GTPase-defective dynamin-1(K44E) increased the dwell time of tPA-mRFP at the plasma membrane. By contrast, recruitment of GFP chimeras of clathrin, epsin, and amphiphysin was not observed. Expression of dynamin-1(K535A), mutated in the pleckstrin homology domain, caused the apparent full fusion of vesicles, as reported by the additional release of tPA-mRFP (15-nm diameter) and enhanced GFP-tagged phogrin. We conclude that re-uptake of vesicles after peptide release by cavicapture corresponds to a novel form of endocytosis in which dynamin-1 stabilizes and eventually closes the fusion pore, with no requirement for "classical" endocytosis for retreat from the plasma membrane.
Tsuboi
Takashi
T
Henry Wellcome Laboratories for Integrated Cell Signalling and the Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, United Kingdom.
McMahon
Harvey T
HT
Rutter
Guy A
GA
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D034284:Dynamin I / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D004705:Endocytosis; D005089:Exocytosis; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis; D050683:R-SNARE Proteins; D051381:Rats; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D050863:Synaptotagmin I; D050857:Synaptotagmins; D013997:Time Factors; D000097573:Red Fluorescent Protein
D001798:Blood Proteins; D002135:Calcium-Binding Proteins; D034284:Dynamin I; D049452:Green Fluorescent Proteins; D007328:Insulin; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins; D017027:Protein Tyrosine Phosphatases; D050683:R-SNARE Proteins; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D050863:Synaptotagmin I; D050857:Synaptotagmins; D000097573:Red Fluorescent Protein; C494311:Syt1 protein, mouse; C494312:Syt1 protein, rat; C039077:platelet protein P47; C102889:PTPRN2 protein, human