Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide.
Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca(2+)-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quan...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3214045?pdf=render |
| _version_ | 1818691717737480192 |
|---|---|
| author | Lindsay Hewlett Gregor Zupančič Gregory Mashanov Laura Knipe David Ogden Matthew J Hannah Tom Carter |
| author_facet | Lindsay Hewlett Gregor Zupančič Gregory Mashanov Laura Knipe David Ogden Matthew J Hannah Tom Carter |
| author_sort | Lindsay Hewlett |
| collection | DOAJ |
| description | Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca(2+)-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca(2+)-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.Ca(2+)-driven WPB exocytosis occurred at all temperatures studied (7-37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s(-1) (37°C) to 0.80±0.14 s(-1) (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca(2+)-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75-80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60-120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself.We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased. |
| first_indexed | 2024-12-17T12:46:20Z |
| format | Article |
| id | doaj.art-f8f509aeaad34a1f8ebed4440b2573f7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-17T12:46:20Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-f8f509aeaad34a1f8ebed4440b2573f72022-12-21T21:47:44ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-01611e2731410.1371/journal.pone.0027314Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide.Lindsay HewlettGregor ZupančičGregory MashanovLaura KnipeDavid OgdenMatthew J HannahTom CarterWeibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca(2+)-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca(2+)-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.Ca(2+)-driven WPB exocytosis occurred at all temperatures studied (7-37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s(-1) (37°C) to 0.80±0.14 s(-1) (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca(2+)-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75-80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60-120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself.We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.http://europepmc.org/articles/PMC3214045?pdf=render |
| spellingShingle | Lindsay Hewlett Gregor Zupančič Gregory Mashanov Laura Knipe David Ogden Matthew J Hannah Tom Carter Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide. PLoS ONE |
| title | Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide. |
| title_full | Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide. |
| title_fullStr | Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide. |
| title_full_unstemmed | Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide. |
| title_short | Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide. |
| title_sort | temperature dependence of weibel palade body exocytosis and cell surface dispersal of von willebrand factor and its propolypeptide |
| url | http://europepmc.org/articles/PMC3214045?pdf=render |
| work_keys_str_mv | AT lindsayhewlett temperaturedependenceofweibelpaladebodyexocytosisandcellsurfacedispersalofvonwillebrandfactoranditspropolypeptide AT gregorzupancic temperaturedependenceofweibelpaladebodyexocytosisandcellsurfacedispersalofvonwillebrandfactoranditspropolypeptide AT gregorymashanov temperaturedependenceofweibelpaladebodyexocytosisandcellsurfacedispersalofvonwillebrandfactoranditspropolypeptide AT lauraknipe temperaturedependenceofweibelpaladebodyexocytosisandcellsurfacedispersalofvonwillebrandfactoranditspropolypeptide AT davidogden temperaturedependenceofweibelpaladebodyexocytosisandcellsurfacedispersalofvonwillebrandfactoranditspropolypeptide AT matthewjhannah temperaturedependenceofweibelpaladebodyexocytosisandcellsurfacedispersalofvonwillebrandfactoranditspropolypeptide AT tomcarter temperaturedependenceofweibelpaladebodyexocytosisandcellsurfacedispersalofvonwillebrandfactoranditspropolypeptide |