A morphological and molecular approach to understanding fine mechanisms of endothelial vesiculation: a novel role for the actin cytoskeleton

Abstract

Endothelial plasma membrane-derived microparticles (MP) actively regulate biological processes and are increased in diseases where vascular damage contributes to pathogenesis. Whilst MP display great clinical potential, field-wide inconsistencies in analytical methodology and an incomplete understanding of their biogenesis limit their application. With advanced flow cytometry and immuno-electron microscopy endothelial MP were characterised by examining phosphatidylserine ligands along with constitutive and inducible markers, in the presence of vesiculation agonist, TNF. MP labelling and detection limitations were assessed, with flow cytometry shown to accurately and reliably measure MP relative expression in this in vitro system. Although implicated in vesiculation, the precise role of the actin cytoskeleton remains unknown. Specifically examining - and -cytoplasmic actins (CYA), TNF induced basal -actin stress fibres via the Rho-kinase pathway, which interacted with unique actin-rich structures, termed CYA-complexes. CYA-complex apically re-positioning enhanced phosphatidylserine-enriched membrane protuberances and MP shedding. -actin regulated CYA-complex spatial distribution, whilst their formation required -actin. CYA-complexes are heterogeneous spheroids that display great structural stability. Comprised of a dense -actin shell and compartmentalised -actin core, they colocalise with specific actin regulatory proteins. This work has identified the CYA-complex, a novel actin-based machinery implicated in the formation and release of membrane MP.