Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event

Curr Biol. 2000 Oct 19;10(20):1307-10. doi: 10.1016/s0960-9822(00)00756-9.


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').

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acridine Orange / analysis
  • Animals
  • Cell Line
  • Cell Membrane / physiology
  • Cell Membrane / ultrastructure
  • Electric Stimulation
  • Exocytosis / physiology*
  • Green Fluorescent Proteins
  • Insulin / physiology*
  • Luminescent Proteins / analysis
  • Membrane Fusion
  • Membrane Glycoproteins / analysis
  • Membrane Proteins*
  • Microscopy, Fluorescence / methods
  • Microscopy, Video / methods
  • Protein Tyrosine Phosphatases / analysis
  • Receptor-Like Protein Tyrosine Phosphatases, Class 8
  • Secretory Vesicles / physiology*
  • Secretory Vesicles / ultrastructure


  • Insulin
  • Luminescent Proteins
  • Membrane Glycoproteins
  • Membrane Proteins
  • Green Fluorescent Proteins
  • PTPRN2 protein, human
  • Protein Tyrosine Phosphatases
  • Receptor-Like Protein Tyrosine Phosphatases, Class 8
  • Acridine Orange