Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Instituto Nacional de Biologia Estrutural e Bioimagem and Centro Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Brazil.
Department of Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, UK.
Parasitol Int. 2022 Feb;86:102444. doi: 10.1016/j.parint.2021.102444. Epub 2021 Aug 28.
Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cytostome-cytopharynx complex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.
克氏锥虫增殖形式通过一种名为胞口-胞咽复合体（SPC）的专门结构进行内吞作用。SPC 是细胞膜的一种专门内陷，通过细胞体延伸到后区，其开口靠近鞭毛囊。最近，在胞咽中发现了多种蛋白质，包括两种肌球蛋白马达。其中之一是孤儿肌球蛋白 MyoF，它被证明对滋养体的内吞作用是必不可少的。然而，MyoF 在沿着内吞途径和通过 T. cruzi 生命周期的定位动力学仍然不清楚。使用 CRISPR-Cas9 基因组编辑，我们从其内源基因座产生了表达与 mNeonGreen 融合的 MyoF 的滋养体。使用这些细胞，我们观察到在滋养体细胞周期中，MyoF 信号在 G2 期间消失，在早期胞质分裂时重新出现。此外，我们表明 MyoF 在循环体发生期间的定位与 SPC 的逐渐消失兼容，在循环体动基体中不存在。去污剂分级显示 MyoF 主要存在于不溶性部分，并在整个细胞骨架准备的胞口微管中免疫定位。此外，在通过 SPC 摄取示踪剂时，MyoF 沿着内吞途径跟随示踪剂，在 30 分钟后在后部隔室中发现。总之，数据表明 MyoF 可能不仅在货物进入部位起作用，而且在整个内吞途径中起作用。
