Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analysed by total internal reflection fluorescence microscopy.
OBJECTIVE
The interaction of syntaxin-1 and SNAP-25 with insulin exocytosis was examined using the diabetic Goto-Kakizaki (GK) rat and a total internal reflection fluorescence (TIRF) imaging system.


METHODS
Primary rat pancreatic beta cells were immunostained with anti-syntaxin-1A, anti-SNAP-25 and anti-insulin antibodies, and then observed by TIRF microscopy. The real-time image of GFP-labelled insulin granules motion was monitored by TIRF.


RESULTS
The number of syntaxin-1A and SNAP-25 clusters, and the number of docked insulin granules on the plasma membrane were reduced in GK beta cells. When GK rats were treated with daily insulin injection for 2 weeks, the number of syntaxin-1 and SNAP-25 clusters was restored, along with the number of docked insulin granules. The infection of GK beta cells with Adex1CA SNAP-25 increased the number of docked insulin granules. TIRF imaging analysis demonstrated that the decreased number of fusion events from previously docked insulin granules in GK beta cells was restored when the number of docked insulin granules increased by insulin treatment or Adex1CA SNAP-25 infection.


CONCLUSIONS
There was a close correlation between the number of syntaxin-1 and SNAP-25 clusters and the number of docked insulin granules, which is associated with the fusion of insulin granules.
Ohara-Imaizumi
M
M
Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Nishiwaki
C
C
Nakamichi
Y
Y
Kikuta
T
T
Nagai
S
S
Nagamatsu
S
S
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000648:ultrastructure*; D003921:Diabetes Mellitus, Experimental / Q000473:pathology; D007328:Insulin / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000473:pathology*; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D017208:Rats, Wistar; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins / Q000378:metabolism
D000954:Antigens, Surface; D007328:Insulin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins