title: Cooperation between β- and γ-cytoplasmic actins in the mechanical regulation of endothelial microparticle formation.
abstract: Elevated endothelial microparticle (MP) levels are observed in numerous diseases, increasingly supporting roles as effectors and valuable markers of vascular dysfunction. While a contractile role for the actin cytoskeleton has been implicated in vesiculation, i.e., MP production, the precise interactions and mechanisms of its constituents, β- and γ-cytoplasmic actins, is unknown. Human cerebral microvascular endothelial cells were stimulated with known agonists, and vesiculation development was monitored by scanning electron microscopy (SEM) and flow cytometry. These data in combination provide new insight into the kinetics, patterns of vesiculating cell recruitment, and degrees of response specific to stimuli. Reorganization of β- and γ-actins, F-actin, vinculin, and talin accompanied significant MP release. β-Actin redistribution into basal stress fibers following stimulation was associated with increased apically situated actin-rich particulate structures, which in turn directly correlated with electron-lucent membrane protrusions observed by SEM. Y-27632 Rho-kinase inhibition abolished basal β-actin fiber formation, minimizing apically associated actin-rich structures, significantly reducing membrane protrusions and MP release to near basal levels. Cytoskeletal protein expression and distribution varied between MPs and mother cells, as determined by Western blot. These data strongly suggest that β-actin plays an active facilitative role in agonist-induced protuberance formation, through mechanical interactions with newly described actin-rich structures.
authors.affiliation: Vascular Immunology Unit, Department of Pathology, University of Sydney, 92-94 Parramatta Rd., Camperdown, NSW 2050, Australia. sharissa.latham@sydney.edu.au
authors.forename: Sharissa L
authors.initials: SL
authors.lastname: Latham
authors.forename: Christine
authors.initials: C
authors.lastname: Chaponnier
authors.forename: Vera
authors.initials: V
authors.lastname: Dugina
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Couraud
authors.forename: Georges E R
authors.initials: GE
authors.lastname: Grau
authors.forename: Valery
authors.initials: V
authors.lastname: Combes
mesh_terms: D000199:Actins / Q000502:physiology* / Q000648:ultrastructure; D000577:Amides / Q000494:pharmacology; D001696:Biomechanical Phenomena; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D055252:Cell-Derived Microparticles / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning; D008954:Models, Biological; D010446:Peptide Fragments / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
chemical_list: D000199:Actins; D000577:Amides; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; C082835:thrombin receptor peptide (42-47); C108830:Y 27632; D000001:Calcimycin; D007371:Interferon-gamma; D054460:rho-Associated Kinases