Of the pathogenic trypanosomatids, Trypanosoma cruzi alone retains an ancient feeding apparatus known as the cytostome-cytopharynx complex (SPC) that it uses as its primary mode of endocytosis in a manner akin to its free-living kinetoplastid relatives who capture and eat bacterial prey via this endocytic organelle. In a recent report, we began the process of dissecting how this organelle functions by identifying the first SPC-specific proteins in T. cruzi. Here, we continued these studies and report on the identification of the first enzymatic component of the SPC, a previously identified orphan myosin motor (MyoF) specifically targeted to the SPC. We overexpressed MyoF as a dominant-negative mutant, resulting in parasites that, although viable, were completely deficient in measurable endocytosis in vitro. To our surprise, however, a full deletion of MyoF demonstrated only a decrease in the overall rate of endocytosis, potentially indicative of redundant myosin motors at work. Thereupon, we identified three additional orphan myosin motors, two of which (MyoB and MyoE) were targeted to the preoral ridge region adjacent to the cytostome entrance and another (MyoC) which was targeted to the cytopharynx tubular structure similar to that of MyoF. Additionally, we show that the C-terminal tails of each myosin are sufficient for targeting a fluorescent reporter to SPC subregions. This work highlights a potential mechanism used by the SPC to drive the inward flow of material for digestion and unveils a new level of overlapping complexity in this system with four distinct myosin isoforms targeted to this feeding structure.

中文翻译：

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TcMyoF 的功能特征表明细胞口-细胞咽靶向肌球蛋白家族是克氏锥虫内吞机制的组成部分。


在致病性锥虫中，克氏锥虫保留了一种古老的进食装置，称为细胞口-细胞咽复合体（SPC），它使用该装置作为其主要的内吞作用方式，其方式类似于其自由生活的动质体亲戚，捕获并通过这种内吞细胞器吃细菌猎物。在最近的一份报告中，我们通过鉴定克氏锥虫中第一个 SPC 特异性蛋白质，开始剖析该细胞器的功能。在这里，我们继续这些研究并报告了 SPC 的第一个酶成分的鉴定，这是一种先前鉴定的专门针对 SPC 的孤儿肌球蛋白马达 (MyoF)。我们将 MyoF 过度表达为显性失活突变体，导致寄生虫虽然能存活，但在体外完全缺乏可测量的内吞作用。然而，令我们惊讶的是，MyoF 的完全删除仅表明内吞作用的总体速率下降，这可能表明多余的肌球蛋白马达在工作。随后，我们确定了三个额外的孤儿肌球蛋白运动，其中两个（MyoB和MyoE）靶向邻近细胞口入口的口前脊区域，另一个（MyoC）靶向与MyoF类似的细胞咽管状结构。此外，我们表明每个肌球蛋白的 C 末端尾部足以将荧光报告基因靶向 SPC 亚区。这项工作强调了 SPC 用于驱动消化材料向内流动的潜在机制，并揭示了该系统中针对该喂养结构的四种不同肌球蛋白亚型的重叠复杂性达到了新的水平。