Cytoskeletal disruption induces T cell apoptosis by a caspase-3 mediated mechanism.

T cell apoptosis can be triggered by different mechanisms that lead to distinctive features such as cell shrinkage, membrane blebbing, phosphatidylserine externalization, and internucleosomal DNA fragmentation. Prevailing models for the induction of apoptosis place the cytoskeleton as a distal target of the death effector molecules ('executioners'). However, the cytoskeleton can also play a role in the induction of apoptosis as suggested by the finding that cytoskeletal disruption can induce apoptosis. The mechanism by which this occurs is unknown. Here, we report that T cell apoptosis by cytoskeletal disruption involves a protein synthesis-independent mechanism leading to up-regulation of caspase-3 protease activity and increased accessibility of active caspase-3 to its substrate. Thus, cytoskeleton integrity may regulate the subcellular compartmentalization of death effector molecules.

Transendothelial migration of monocytes in rat aorta: distribution of F-actin, alpha-catnin, LFA-1, and PECAM-1.

To determine changes in the distribution of cell adhesion molecules during diapedesis of monocytes in situ, we labeled aortic whole mounts from hypercholesterolemic rats with Texas red-phalloidin and antibodies to LFA-1, PECAM-1, or alpha-catenin, and analyzed them by laser scanning confocal microscopy. Monocytes transmigrated through circular openings (transmigration passages) formed by pseudopodia that penetrated between adjacent endothelial cells. Transmigrating monocytes remained spherical above the endothelium, while spreading beneath it. The transmigration passage was lined by F-actin and partially by alpha-catenin, suggesting cadherin-mediated heterotypic interactions. LFA-1 was present in clusters at the monocyte cell surface throughout diapedesis, but was concentrated at the margin of the transmigration passage. PECAM-1 was enriched in the endothelial contact regions where the monocytes transmigrated. PECAM-1 was barely detectable in monocytes before and after diapedesis, but appeared during diapedesis at the cell surface in the parts of the monocyte located above the endothelium. PECAM-1 was enriched near the endothelial cell-cell junctions, but was not detected in parts that spread beneath the endothelium. Our results suggest a major role for LFA-1 during diapedesis and reveal dynamic changes in the distribution of PECAM-1, the actin cytoskeleton, and alpha-catenin during monocyte diapedesis in situ.

Changes in the distribution of LFA-1, catenins, and F-actin during transendothelial migration of monocytes in culture.

To determine changes in the spatial and temporal distribution of cell-cell adhesion molecules during transendothelial migration of monocytes, we examined an in vitro model system of diapedesis using high resolution laser scanning confocal microscopy. Human arterial endothelial cells were cultured to confluence on coverslips coated with Matrigel and activated with IL-1beta before the addition of monocytic THP-1 cells. Seventy per cent of monocytes transmigrated through the endothelium within one hour. Diapedesis, but not adhesion and spreading, was inhibited 8-fold in co-cultures that contained endothelial cell conditioned medium, suggesting the release of an endothelial derived inhibitor. Double immunofluorescence labeling with antibodies to LFA-1, alpha- and beta-catenin, VE-cadherin and with Texas Red phalloidin, identified a circular transmigration passage in endothelial cell-cell contact regions. This passage was formed by an LFA-1-containing pseudopodium that penetrated between endothelial cells. Apical to the transmigration passage, monocytes remained round in shape, while underneath the endothelium, they spread along the Matrigel. The margins of the transmigration passage contained high levels of LFA-1 and F-actin, suggesting a major role of these molecules during the transmigration process itself. Endothelial adherens junctions, as judged by the presence of VE-cadherin and alpha-catenin adjacent to the passage, remained intact during diapedesis. The presence of catenins at heterotypic contact regions between monocytes and endothelial cells during diapedesis suggested cadherin-mediated interactions between the two cell types. These results reveal dynamic changes in the distribution of adhesion molecules and the actin cytoskeleton during monocyte transendothelial migration in culture.