Elucidating the Mechanistic Basis for Phagotrophy in the Protozoan Trypanosoma cruzi (equipment supplement)

能够有效地从环境中提取营养物质是任何异养生物的基本活动 从捕食者到寄生虫在自由生活的浮游原生动物中， 食食性捕食者设计了各种方法来捕获和消耗有机物的来源， 它们生长和繁殖所需的碳。最普遍的机械捕食模式之一 这些原生动物所使用的包括纤毛或鞭毛支持的过滤进食。捕获的猎物最终 通过细胞跨越小管内陷被内吞，起源于质膜中的孔，和 最终形成用于溶酶体消化的出芽囊泡。这个孔（细胞口）和它的放射小管 细胞口结构（细胞咽）在此统称为细胞口/细胞咽复合体（SPC）。 尽管它无处不在，但几乎没有人知道SPC是如何产生或运作的。 任何生物体的分子水平。值得注意的是，总的来说，含有原生动物的SPC 在从全球微生物食物网到人类寄生等各种活动中发挥着关键作用。有趣的是， 遗传上易处理的鞭毛虫，克氏锥虫，保留了这种内吞作用的祖先模式， 就像它的自由生活的食菌亲戚（例如Bodo saltans）一样，积极地内吞宿主的物质 也是通过SPC进行的。原生动物如何在其表面捕获食物，内部信号启动 内吞作用并最终将这种物质沿着SPC运输到溶酶体进行消化， 这是一个谜，也是我们在本提案中试图回答的问题的核心。我们发现SPC- 介导的内吞作用对T. cruzi在试管中生长，因此，我们是独一无二的 能够进行广泛的敲除（KO）和互补研究， SPC功能的多个维度首次不影响细胞活力。作为我们 以前发表的工作，这一建议旨在产生一个整体的理解，如何SPC介导 内吞作用基本上起作用。我们开始将把统一的内吞活动分解为 组成过程;通过表面受体（Aim 1）的货物捕获，受体信号转导和 内吞机制（Aim 2）的激活以及最终吞噬的货物沿着SPC的主动运输， 消化（Aim 3）。这些目标中的每一个都将涉及原生动物生物学的重要基本方面， 仍然知之甚少。正如目前正在进行的研究所强调的那样，使用标准的共聚焦活体 显微镜太慢和有毒，无法研究这种高度动态和快速的过程。因此，这项建议 要求资金支持收购一个旋转盘共聚焦显微镜，将能够 在很长一段时间内成像非常快速的原生动物内吞活动， 与光毒性相关，妨碍了分析。这一设备无疑将加强正在进行的研究， 试图阐明原生动物吞噬的神秘过程的机械基础。

Being able to efficiently extract nutrients from one’s environment is an essential activity for any heterotroph ranging from predators to parasites. Among the free-living planktonic protozoans, the extremely diverse class of phagotrophic predators have devised a variety of methods to capture and consume the sources of organic carbon they need to grow and reproduce. One of the most widespread modes of mechanical predation employed by these protozoa involves cilia or flagella supported filter feeding. Captured prey are ultimately endocytosed via a cell spanning tubular invagination, originating at a pore in the plasma membrane, and ending in budding vesicles targeted for lysosomal digestion. This pore (cytostome), and its emanating tubule structure (cytopharynx), are collectively referred to here as the cytostome/cytopharynx complex (SPC). Despite its ubiquitous presence, almost nothing is known about how the SPC is generated or functions at the molecular level in any organism. It is worth noting that, collectively, the SPC containing protozoa play critical roles in diverse activities ranging from the global microbial food web to human parasitism. Intriguingly, the genetically tractable flagellate, Trypanosoma cruzi, has retained this ancestral mode of endocytosis and, much like it’s free-living bacterivorous relatives (e.g. Bodo saltans), actively endocytoses its host’s material sustenance via the SPC as well. How protozoans capture food at their surface, signal internally to initiate endocytosis and ultimately traffic this material down the SPC to the lysosome for digestion remains a mystery and is at the core of the questions we seek to answer in this proposal. We have found that SPC- mediated endocytosis is dispensable for T. cruzi when grown in vitro, and as a result, we are uniquely positioned to be able to conduct extensive knockout (KO) and complementation studies to functionally dissect multiple dimensions of SPC function without impacting cell viability for the first time. As a continuation of our prior published work, this proposal seeks to generate a holistic understanding of how SPC mediated endocytosis fundamentally functions. We will begin by dismantling the unified activity of endocytosis into its constituent processes; cargo capture through surface receptors (Aim1), receptor signal transduction and activation of endocytic machinery (Aim2) and finally active transport of phagocytosed cargo along the SPC for digestion (Aim3). Each of these aims will address important basic aspects of protozoan biology that continue to remain poorly understood. As studies currently underway have highlighted, the use of standard confocal live microscopy is too slow and toxic to study this highly dynamic and rapid process. As a result, this proposal requests funding to support the acquisition of a spinning disk confocal microscope that will be capable of imaging extremely fast protozoan endocytic activities across long periods of time without the concerns associated with phototoxicity hampering analysis. This equipment will no doubt enhance ongoing studies which seeks to elucidate the mechanistic underpinnings of the enigmatic process of protozoan phagotrophy.