Peptide hormone release monitored from single vesicles in "membrane lawns" of differentiated male pituitary cells: SNAREs and fusion pore widening.
In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 μm(2), which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.
Celica Biomedical Center, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
Matjaž
M
Stenovec
Paula P
PP
Gonçalves
Robert
R
Zorec
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008561:Membrane Fusion / Q000502:physiology; D018613:Microscopy, Confocal; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000502:physiology*; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D013574:Synaptosomes / Q000502:physiology; D050827:Syntaxin 1 / Q000502:physiology; D050988:Vesicle-Associated Membrane Protein 2 / Q000502:physiology
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; D050827:Syntaxin 1; D050988:Vesicle-Associated Membrane Protein 2; D011388:Prolactin; D002118:Calcium