title: Dynein-dynactin complex mediates protein kinase A-dependent clustering of Weibel-Palade bodies in endothelial cells.
abstract: OBJECTIVE
  Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell-specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail.
  
  
  RESULTS
  Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein-dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering.
  
  
  CONCLUSIONS
  These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex.
authors.affiliation: Department of Plasma Proteins, Sanquin Research, University of Amsterdam, The Netherlands.
authors.forename: Mariska G
authors.initials: MG
authors.lastname: Rondaij
authors.forename: Ruben
authors.initials: R
authors.lastname: Bierings
authors.forename: Astrid
authors.initials: A
authors.lastname: Kragt
authors.forename: Karina A
authors.initials: KA
authors.lastname: Gijzen
authors.forename: Erica
authors.initials: E
authors.lastname: Sellink
authors.forename: Jan A
authors.initials: JA
authors.lastname: van Mourik
authors.forename: Mar
authors.initials: M
authors.lastname: Fernandez-Borja
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004837:Epinephrine / Q000494:pharmacology; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D022101:Microtubule-Organizing Center / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D014471:Umbilical Veins / Q000166:cytology; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D020302:Weibel-Palade Bodies / Q000378:metabolism*
chemical_list: C000606361:DCTN2 protein, human; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D010726:Phosphodiesterase Inhibitors; D014662:Vasoconstrictor Agents; D005576:Colforsin; D003994:Bucladesine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004398:Dyneins; D015056:1-Methyl-3-isobutylxanthine; D004837:Epinephrine