Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA, United States.
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA, United States.
Cell Calcium. 2022 Nov;107:102654. doi: 10.1016/j.ceca.2022.102654. Epub 2022 Sep 21.
The mitochondrial Ca uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca signaling, is mediated via the calcium uniporter complex (MCUC). In animal cells the MCUC is regulated by the mitochondrial calcium uptake 1 and 2 dimer (MICU1/MICU2), which has been proposed to act as gatekeeper preventing mitochondrial Ca overload at low cytosolic Ca levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca uptake at low extramitochondrial Ca concentrations ([Ca]) and it was though that in the absence of one MICU the other would replace its role. However, previous attempts to knockout both genes were unsuccessful. Here, we designed a strategy to generate TcMICU1/TcMICU2 double knockout cell lines using CRISPR/Cas9 genome editing. Ablation of both genes was confirmed by PCR and Southern blot analyses. The absence of both proteins did not allow Ca uptake at low [Ca], significantly decreased the mitochondrial Ca uptake at different [Ca], without dissipation of the mitochondrial membrane potential, and increased the [Ca] set point needed for Ca uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg was found to be a negative regulator of MCUC-mediated mitochondrial Ca uptake at different [Ca]. Occlusion of the MCUC pore by Mg could partially explain the lack of mitochondrial Ca uptake at low [Ca] in TcMICU1/TcMICU2-KO cells. In addition, TcMICU1/TcMICU2-KO epimastigotes had a lower growth rate, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes.
线粒体 Ca 摄取对于调节生物能量、细胞死亡和细胞质 Ca 信号转导非常重要，它是通过钙单向转运体复合物（MCUC）介导的。在动物细胞中，MCUC 受线粒体钙摄取 1 和 2 二聚体（MICU1/MICU2）的调节，后者被认为是防止低细胞浆 Ca 水平下线粒体 Ca 过载的门卫。与动物细胞不同，在恰加斯病的病原体克氏锥虫中敲除 MICU1 或 MICU2 中的任何一个，都不允许在低细胞外 Ca 浓度 ([Ca]) 下摄取 Ca，并且认为在没有一个 MICU 的情况下，另一个会取代其作用。然而，之前试图敲除这两个基因都没有成功。在这里，我们使用 CRISPR/Cas9 基因组编辑设计了一种生成 TcMICU1/TcMICU2 双敲除细胞系的策略。通过 PCR 和 Southern blot 分析证实了两个基因的缺失。两个基因的缺失都不允许在低 [Ca] 下摄取 Ca，显著降低了不同 [Ca] 下的线粒体 Ca 摄取，而线粒体膜电位没有耗散，并且增加了 Ca 摄取的 [Ca] 设定点，就像我们在 TcMICU1-KO 和 TcMICU2-KO 细胞中看到的那样。Mg 被发现是不同 [Ca] 下 MCUC 介导的线粒体 Ca 摄取的负调节剂。Mg 闭塞 MCUC 孔可以部分解释 TcMICU1/TcMICU2-KO 细胞在低 [Ca] 下缺乏线粒体 Ca 摄取的原因。此外，TcMICU1/TcMICU2-KO 埃普西隆体的生长速度较慢，而感染性的锥鞭毛体侵入宿主细胞并在其中作为无鞭毛体复制的能力降低。
