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EPIC3, a novel Ca2+ indicator located at the cell cortex and in microridges, detects high Ca2+ subdomains during Ca2+ influx and phagocytosis

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The construction of a low affinity Ca2+-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 (ezrin-protein indicator of Ca2+). The novel probe is a fusion of CEPIA3 with ezrin, and is used in combination with a Ca2+-insensitive probe, ezrin-mCherry, both of which locate at the cell cortex. EPIC3 was used to monitor the effect of Ca2+ influx on intra-wrinkle Ca2+ in the macrophage cell line, RAW 264.7. During experimentally-induced Ca2+influx, EPIC3 reported Ca2+ concentrations at the cell cortex in the region of 30-50 μM, with peak locations towards the tips of wrinkles reaching 80 μM. These concentrations were associated with cleavage of ezrin (a substrate for the Ca2+ activated protease calpain-1) and released the C-terminal fluors. The cortical Ca2+ levels, restricted to near the site of phagocytic cup formation and pseudopodia extension during phagocytosis also reached high levels (50-80 μM) during phagocytosis. As phagocytosis was completed, hotspots of Ca2+ near the phagosome were also observed.
Tijdschrift: Cell Calcium
ISSN: 0143-4160
Volume: 92
Aantal pagina's: 9
Jaar van publicatie:2020
Trefwoorden:Celbiologie
Toegankelijkheid:Open