Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event.
The classical model of secretory vesicle recycling after exocytosis involves the retrieval of membrane (the omega figure) at a different site. An alternative model involves secretory vesicles transiently fusing with the plasma membrane (the 'kiss and run' mechanism) [1,2]. No continuous observation of the fate of a single secretory vesicle after exocytosis has been made to date. To study the dynamics of fusion immediately following exocytosis of insulin-containing vesicles, enhanced green fluorescent protein (EGFP) fused to the vesicle membrane protein phogrin [3] was delivered to the secretory vesicle membrane of INS-1 beta-cells using an adenoviral vector. The behaviour of the vesicle membrane during single exocytotic events was then examined using evanescent wave microscopy [4-6]. In unstimulated cells, secretory vesicles showed only slow Brownian movement. After a depolarizing pulse, most vesicles showed a small decrease in phogrin-EGFP fluorescence, and some moved laterally over the plasma membrane for approximately 1 microm. In contrast, secretory vesicles loaded with acridine orange all showed a transient (33-100 ms) increase in fluorescence intensity followed by rapid disappearance. Simultaneous observations of phogrin-EGFP and acridine orange indicated that the decrease in EGFP fluorescence occurred at the time of the acridine orange release, and that the lateral movement of EGFP-expressing vesicles occurred after this. Post-exocytotic retrieval of the vesicle membrane in INS-1 cells is thus slow, and can involve the movement of empty vesicles under the plasma membrane ('kiss and glide').
Tsuboi
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Photon Medical Research Center, Hamamatsu University School of Medicine, Japan.
Zhao
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C
Terakawa
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Rutter
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GA
D000165:Acridine Orange / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D004558:Electric Stimulation; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins; D007328:Insulin / Q000502:physiology*; D008164:Luminescent Proteins / Q000032:analysis; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D017027:Protein Tyrosine Phosphatases / Q000032:analysis; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D022142:Secretory Vesicles / Q000502:physiology* / Q000648:ultrastructure
D007328:Insulin; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins; C102889:PTPRN2 protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D000165:Acridine Orange