Masataka Arai

Biphasic and directed translocation of protein kinase Cα inside cultured endothelial cells before migration

Mechanical wounding of an endothelial monolayer induces an immediate Ca2+ wave. Several hours later, the denuded area is covered by endothelial cells (ECs) that migrate to the wound. This migration process is closely related to protein kinase Cα (PKCα), a Ca2+-dependent protein that translocates from the cytosol to the cell membrane. Because the cells adjacent to the wounded area are the first to migrate into the wound, we investigated whether a mechanical wound immediately induces PKCα translocation in adjacent cells. We monitored Ca2+ dynamics and PKCα translocation simultaneously using fluorescent microscopy. For this simultaneous observation, we used Fura-2–acetoxymethyl ester to visualize Ca2+ and constructed a green fluorescent protein-tagged fusion protein to visualize PKCα. Mechanical wounding of the endothelial monolayer induced an immediate Ca2+ wave in cells adjacent to the wounded cells before their migration. Almost concurrently, PKCα in the neighboring cells translocated to the cell membrane, then accumulated at the periphery near the wounded cell. This report is the first description of this biphasic and directed translocation of PKCα in cells before cell migration. Our results may provide new insights into the directed migration of ECs.

Spatial and temporal translocation of PKCα in single endothelial cell in response to mechanical stimulus

ABSTRACT Endothelial cells (ECs) are exposed to various environmental forces, and a Ca2+ wave is occurred in mechanical stimulated cells. Pharmacological studies reveal that the translocation of protein kinase C&agr; (PKC&agr;) to the membrane is observed simultaneously with intracellular Ca2+ wave. In this study, we investigate whether and how the kinetics of PKC&agr; in ECs is induced in response to mechanical stress. The results show that a mechanical stimulus induced biphasic and directional PKC&agr; translocation; PKC&agr; initially translocated near or at the membrane and then accumulated at the stimulus point. The initial translocation occurred simultaneously with Ca2+ increase. Initial translocation was inhibited in spite of Ca2+ increase when the diacylglycerol (DAG) binding domain of PKC&agr; was inhibited, suggesting that translocation requires intracellular Ca2+ increase and DAG. On the other hand, secondary translocation was delayed, occurring after the Ca2+ wave; however, this translocation occurred even when Ca2+ release from the endoplasmic reticulum was inhibited, while it did not occur when the mechanosensitive (MS) channel was inhibited. These results indicated that at least Ca2+ influx from extracellular space through MS channel is required. Our results support the implication of PKC&agr; in the Ca2+ signaling pathway in response to mechanical stress in ECs. HIGHLIGHTSMechanical stress induced biphasic and directional PKC&agr; translocation.PKC&agr; initially translocated near or at the membrane in parallel with the Ca2+ wave.Thereafter, PKC&agr; accumulated at the stimulus point after the Ca2+ wave.Secondary translocation of PKC&agr; requires Ca2+ influx through the MS channel.

Three-dimensional model of intracellular and intercellular Ca2+ waves propagation in endothelial cells

Intracellular and intercellular Ca2+ waves play key roles in cellular functions, and focal stimulation triggers Ca2+ wave propagation from stimulation points to neighboring cells, involving localized metabolism reactions and specific diffusion processes. Among these, inositol 1,4,5-trisphosphate (IP3) is produced at membranes and diffuses into the cytoplasm to release Ca2+ from endoplasmic reticulum (ER). In this study, we developed a three-dimensional (3D) simulation model for intercellular and intracellular Ca2+ waves in endothelial cells (ECs). 3D model of 2 cells was reconstructed from confocal microscopic images and was connected via gap junctions. Cells have membrane and cytoplasm domains, and metabolic reactions were divided into each domain. Finally, the intracellular and intercellular Ca2+ wave propagations were induced using microscopic stimulation and were compared between numerical simulations and experiments. The experiments showed that initial sharp increases in intracellular Ca2+ occurred approximately 0.3 s after application of stimuli. In addition, Ca2+ wave speeds remained constant in cells, with intracellular and intercellular speeds of approximately 35 and 15 μm/s, respectively. Simulations indicated initial increases in Ca2+ concentrations at points of stimulation, and these were then propagated across stimulated and neighboring cells. In particular, initial rapid increases in intracellular Ca2+ were delayed and subsequent intracellular and intercellular Ca2+ wave speeds were approximately 25 and 12 μm/s, respectively. Simulation results were in agreement with those from cell culture experiments, indicating the utility of our 3D model for investigations of intracellular and intercellular messaging in ECs.