Author/Editor     Stenovec, Matjaž; Goncalves, Paula P; Zorec, Robert
Title     Peptide hormone release monitored from single vesicles in 'membrane lawns' of differentiated male pituitary cells: SNAREs and fusion pore widening
Type     članek
Source     Endocrinology
Vol. and No.     Letnik 154, št. 3
Publication year     2013
Volume     str. 1235-46
Language     eng
Abstract     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 calciumdependent 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 PRLpositive 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 m2, 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.