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，极其多样化的纲 食性捕食者设计了各种方法来捕获和消耗有机资源 它们生长和繁殖所需的碳。最普遍的机械捕食模式之一 这些原生动物利用纤毛或鞭毛支持滤食。捕获的猎物最终是 通过跨越小管内陷的细胞内吞，起源于质膜上的一个孔，以及 最终形成针对溶酶体消化的萌芽小泡。这个毛孔(细胞气孔)和它的发射小管 结构(细胞咽部)，在这里统称为细胞孔/细胞咽部复合体(SPC)。 尽管SPC无处不在，但关于SPC是如何生成或运行的，几乎一无所知 任何生物体中的分子水平。值得注意的是，总的来说，含有原生动物的SPC发挥了 在从全球微生物食物网到人类寄生等各种活动中发挥关键作用。有趣的是， 遗传上易驯化的鞭毛虫克氏锥虫保留了这种祖先的内吞模式， 就像它的自由生活的食菌近亲(例如，博多盐滩)一样，它积极地内吞宿主的物质 也可以通过最高人民法院维持生计。原生动物如何在它们的表面捕捉食物，内部发出信号启动 内吞作用，并最终将这种物质沿着SPC输送到溶酶体进行消化 这是我们在这项提案中试图回答的问题的核心。我们发现SPC- 对于体外培养的克鲁兹毛滴虫来说，介导的内吞作用是必不可少的，因此，我们是独一无二的 能够进行广泛的击倒(KO)和互补研究，从功能上进行剖析 首次在不影响细胞活力的情况下实现多维度的SPC功能。作为我们的延续 之前发表的工作，这项建议试图产生一个全面的理解如何SPC调解 内吞作用从根本上起作用。我们将首先将内吞作用的统一活动分解为其 组成过程：通过表面受体捕获货物(Aim1)，受体信号转导和 激活内吞机械(AIM2)，最终将吞噬的货物沿着SPC主动运输 消化(Aim3)。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，这些方面继续 人们对此仍然知之甚少。正如目前正在进行的研究强调的那样，使用标准的共焦LIVE 显微镜太慢，太有毒，无法研究这种高度动态和快速的过程。因此，这项提议 申请资金支持购买旋转圆盘共聚焦显微镜，该显微镜能够 在没有担心的情况下，在很长一段时间内成像极快的原生动物内吞活动 与光毒性相关，阻碍了分析。这种设备无疑将加强正在进行的研究 旨在阐明原生动物吞噬的神秘过程的机制基础。

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.