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http://hdl.handle.net/10773/24315
Title: | Peptide hormone release monitored from single vesicles in 'membrane lawns' of differentiated male pituitary cells: SNAREs and fusion pore widening |
Author: | Stenovec, Matjaz Gonçalves, Paula P. Zorec, Robert |
Issue Date: | 2013 |
Publisher: | Endocrine Society |
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 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. |
Peer review: | yes |
URI: | http://hdl.handle.net/10773/24315 |
DOI: | 10.1210/en.2012-2022 |
ISSN: | 0013-7227 |
Appears in Collections: | CESAM - Artigos DBio - Artigos |
Files in This Item:
File | Description | Size | Format | |
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Stenovec et al. - 2013 - Peptide Hormone Release Monitored From Single Vesi.pdf | 1.4 MB | Adobe PDF |
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