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

阐明原生动物克氏锥虫吞噬作用的机制基础（设备补充）

• 批准号: 10799091
• 负责人: RONALD DREW ETHERIDGE
• 金额: $ 20万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799091
• 关键词: Active Biological Transport; Biology; Carbon; Cell Survival; Cell membrane; Cells; Cilia; Complement; Complex; Consumption; Digestion; Dimensions; Endocytosis; Environment; Equipment; Flagella; Food; Food Webs; Funding; Human; In Vitro; Knock-out; Lysosomes; Mastigophora; Mechanics; Mediating; Methods; Microscope; Microscopy; Molecular; Nutrient; Organism; Parasites; Phototoxicity; Play; Positioning Attribute; Predatory Behavior; Process; Protozoa; Publishing; Receptor Signaling; Request for Proposals; Role; Signal Transduction; Source; Structure; Surface; Time; Trypanosoma cruzi; Tubular formation; Uncertainty; Vesicle; Work; feeding; imaging capabilities; microbial; parasitism; receptor

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.

能够从一个人的环境中有效提取养分是任何异育的重要活动 从捕食者到寄生虫。在自由生活的浮游原生动物中，非常多样化的班级 人类嗜生物捕食者已经设计了多种捕获和消费有机物来源的方法 他们需要生长和繁殖需要的碳。机械捕食的最广泛模式之一 这些原生动物使用的涉及纤毛或鞭毛支撑的过滤器摄食。捕获的猎物最终是 内吞通过细胞跨越管状的无知，起源于质膜中的孔， 以针对溶酶体消化为目标的新蔬菜结束。这个孔（细胞击败）及其发射的小管 结构（细胞咽部）在这里统称为细胞抑制剂/细胞咽部复合物（SPC）。 尽管存在着无处不在的存在，但几乎一无所知，关于SPC的生成或功能 任何组织的分子水平。值得注意的是，总体而言，含有原生动物的SPC 从全球微生物食品网到人类寄生虫的潜水活动中的关键作用。有趣的是， 一般可牵引的旗杆锥虫cruzi保留了这种祖先的内吞作用模式，并保留 就像它是自由生活的细菌亲戚（例如Bodo saltans）一样，积极地将其宿主的材料内吞 也通过SPC寄托。原生动物如何在其表面捕获食物，内部信号以启动 内吞作用并最终使该材料向下流入SPC到溶酶体以进行消化 神秘，是我们在此提案中寻求回答的问题的核心。我们发现SPC- 在体外生长时，介导的内吞作用对于t. cruzi是可分配的，结果我们是独特的 有能力进行广泛的淘汰（KO）和完成研究以在功能上进行剖析 SPC功能的多个维度首次不影响细胞活力。作为我们的延续 该提案事先发表的工作，旨在对SPC介导的如何产生整体理解 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拆除为 构成过程；通过表面受体捕获货物（AIM1），受体信号转导和 内吞机械的激活（AIM2），最后是沿SPC的吞噬货物的主动运输 消化（AIM3）。这些目标中的每一个都将解决原生动物生物学的重要基本方面 保持不当理解。正如目前正在进行的研究所强调的那样，使用标准共聚焦实时 显微镜太慢且有毒，无法研究这种高度动态和快速的过程。结果，这个建议 请求资金支持获得旋转磁盘共聚焦显微镜的获取 在长时间内成像非常快的原生动物内吞作用，而无需担心 与光毒性阻碍分析相关。毫无疑问，该设备将增强正在进行的研究 旨在阐明原生动物疗法的神秘过程的机械基础。