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.

能够有效地从环境中提取营养物质是任何异养生物的一项基本活动 在自由生活的浮游原生动物中，从捕食者到寄生虫，种类极其多样化。 吞噬性捕食者设计了多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是最普遍的机械捕食方式之一。 这些原生动物利用纤毛或鞭毛支持滤食，最终捕获猎物。 通过跨越管状内陷的细胞内吞，起源于质膜的孔，并且 终止于针对溶酶体消化的出芽囊泡，该孔（细胞口）及其发出的小管。 结构（细胞咽），这里统称为细胞口/细胞咽复合体（SPC）。 尽管 SPC 无处不在，但人们对 SPC 的生成方式或功能几乎一无所知。 值得注意的是，SPC 在任何生物体中都发挥着分子水平的作用。 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。 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亲戚（例如 Bodo saltans）一样，积极地内吞宿主的物质 原生动物如何在其表面捕获食物，并通过内部信号启动。 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仍然是一个 这是我们在本提案中寻求回答的问题的核心，我们发现 SPC- 当体外生长时，克氏锥虫介导的内吞作用是可有可无的，因此，我们是独一无二的 能够广泛进行敲除（KO）和互补研究以进行功能剖析 首次实现 SPC 功能的多个维度而不影响细胞活力。 该提案旨在对 SPC 如何调解产生全面的理解 我们首先将内吞作用的统一活动拆解为它的基本功能。 组成过程；通过表面受体捕获货物（Aim1）、受体信号转导和 内吞机器 (Aim2) 的激活，最后沿着 SPC 主动运输吞噬的货物 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，这些方面将继续发展。 正如目前正在进行的研究所强调的，标准共聚焦实时技术的使用仍然知之甚少。 显微镜太慢且有毒，无法研究这种高度动态和快速的过程。因此，该建议。 请求资金支持购买转盘共焦显微镜，该显微镜能够 长时间内对极快的原生动物内吞活动进行成像，无需担心 该设备无疑将增强正在进行的与光毒性阻碍分析相关的研究。 旨在阐明原生动物吞噬神秘过程的机制基础。