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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
Cell Calcium ( IF 4.3 ) Pub Date : 2020-10-09 , DOI: 10.1016/j.ceca.2020.102291
Rhiannon E Roberts 1 , Tim Vervliet 2 , Geert Bultynck 2 , Jan B Parys 2 , Maurice B Hallett 1
Affiliation  

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.



中文翻译:

EPIC3 是一种位于细胞皮层和微脊中的新型 Ca2+ 指示剂,可在 Ca2+ 流入和吞噬过程中检测高 Ca2+ 亚结构域

位于细胞皮层和细胞表面皱纹的低亲和力 Ca 2+探针的构建被称为。EPIC3(É zrin- p rotein的ndicator Ç一个2+)。新型探针是 CEPIA3 与 ezrin 的融合,并与 Ca 2+ 不敏感探针 ezrin-mCherry结合使用,两者均位于细胞皮层。EPIC3 用于监测 Ca 2+流入对巨噬细胞系 RAW 264.7 中皱纹内 Ca 2+的影响。在实验诱导的 Ca 2+流入期间,EPIC3 报告了 Ca 2+细胞皮层的浓度在 30-50 μM 范围内,皱纹尖端的峰值位置达到 80 μM。这些浓度与 ezrin(Ca 2+活化蛋白酶钙蛋白酶-1的底物)的裂解有关,并释放 C 端荧光。皮质Ca 2+水平仅限于吞噬过程中吞噬杯形成和伪足延伸的部位附近,在吞噬过程中也达到高水平(50-80 μM)。随着吞噬作用的完成,还观察到吞噬体附近的Ca 2+热点。

更新日期:2020-10-30
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