Detection of the centriole tyr- or acet-tubulin changes in endothelial cells treated with thrombin using microscopic immunocytochemistry

We used electron microscopic immunocytochemistry to examine the pattern of centriolar staining for tyrosinated or acetylated alpha-tubulin in endothelial cells during short-term incubation with thrombin. Endothelial cells isolated from human aorta (HAEC) and those isolated from umbilical vein (HUVEC) displayed an increase in the intensity of centriolar staining for acet-tubulin within 1 min after thrombin addition. A decrease in the intensity of centriolar staining for tyr-tubulin was detected in HUVEC within 1 min after thrombin addition, while in HAEC centriolar staining for tyr-tubulin became less intense only 5 min later. Mother and daughter centrioles of HUVEC cells displayed different intensity of immunostaining for acet-tubulin and showed no significant variation in the number of subdistal appendages after thrombin addition. Differently, HAEC cells had the same staining pattern of mother and daughter centrioles in both thrombin-untreated and thrombin-treated cultures. A sharp increase in the number of subdistal appendages of mother centriole occurred in HAEC within 5 min of incubation with thrombin. Our findings provided the direct evidence for centrosome involvement in the ligand-mediated signaling events and showed for the first time that ligand-dependent centrosome reorganization includes the centriole per se. Furthermore, based on our observations we would like to propose that MT-nucleating/anchoring properties of the centrosome are subject to rapid regulation by external signals such as thrombin.