Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India.
Institute for Molecular Biosciences, University of Queensland, St Lucia, Australia.
PLoS Pathog. 2020 May 4;16(5):e1008489. doi: 10.1371/journal.ppat.1008489. eCollection 2020 May.
Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.
细胞外刺激引起的肌动蛋白细胞骨架的重构是阿米巴细胞中许多细胞过程所必需的。在吞噬作用、转噬作用、迁移和侵袭过程中，细胞突起的发育需要肌动蛋白细胞骨架的快速和局部重排。在这里，我们证明了 EhC2B，一种含有 C2 结构域的蛋白质，是一种肌动蛋白调节剂。EhC2B 最初被鉴定为 EhRab21 的效应物，从 E. histolytica 中鉴定出来。包括 GST 下拉、荧光测定和 ITC 的体外相互作用研究也证实了我们的观察结果。在阿米巴滋养体中，EhC2B 聚集在伪足和吞噬杯的尖端。基于 FRAP 的研究证实了 EhC2B 在吞噬杯中募集和动态变化。此外，我们还展示了 EhC2B 在红细胞吞噬中的作用。众所周知，在阿米巴寄生虫的吞噬作用中，钙离子依赖的信号转导对于细胞骨架动力学至关重要。使用脂质体沉淀测定，我们证明了 EhC2B 在钙离子存在的情况下优先结合磷脂酰丝氨酸。EhC2B 突变体在钙或脂质结合方面存在缺陷，无法定位于质膜下方。这些突变体过表达的细胞在红细胞吞噬作用中也显著减少。通过 siRNA 基因敲低方法验证了 EhC2B 在红细胞吞噬和伪足形成中的作用。最后，通过使用荧光光谱和全内反射荧光显微镜的体外成核测定，我们确定 EhC2B 是一种肌动蛋白成核因子。综上所述，根据研究结果，我们提出 EhC2B 作为一种分子桥，通过其肌动蛋白成核活性，在钙离子依赖的方式下促进膜变形，从而促进吞噬杯的进展。
