Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

通过长期锥虫活细胞成像揭示时空动态

• 批准号: 10307600
• 负责人: Christopher Luis de Graffenried
• 金额: $ 23.27万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307600
• 关键词: Address; Affect; African Trypanosomiasis; Back; Biology; Blood Circulation; CRISPR/Cas technology; Cell Death; Cell Division Process; Cell Survival; Cell division; Cell physiology; Cells; Cellular Morphology; Cellular biology; Custom; Cytokinesis; Data; Defect; Dimensions; Disease; Event; Fluorescence; Genes; Genetic Transcription; Hour; Human; Image; Immobilization; Individual; Insecta; Knowledge; Left; Leishmania; Life; Life Cycle Stages; Link; Livestock; Measurement; Mediating; Methods; Microscope; Monitor; Morphologic artifacts; Morphology; Organelles; Organism; Parasites; Pathway interactions; Pharmaceutical Preparations; Phenotype; Process; Production; Protein Dynamics; Proteins; RNA Interference; Running; Sentinel; Sepharose; Series; Source; Surface; System; Techniques; Time; Trypanosoma brucei brucei; Trypanosoma cruzi; Work; cell motility; cell transformation; cellular imaging; cost; daughter cell; drug discovery; experimental study; flagellum motility; homologous recombination; imaging approach; insight; internal control; live cell imaging; loss of function; novel; programs; protein biomarkers; protein function; small molecule; spatiotemporal; tool

PROJECT SUMMARY Trypanosoma brucei and the related trypanosomatid parasites cause diseases in humans and livestock that are the source of tremendous human suffering. While methods such as RNAi- and CRISPR/Cas9-mediated loss of function experiments and gene tagging approaches have yielded a wealth of data on protein function in trypanosomatids, very little is known about how different phenotypes manifest or the dynamics of proteins in live cells. This is due to the fact that live-cell imaging in T. brucei is difficult because the parasites are highly motile and do not undergo many cellular processes, such as cell division, when they are immobilized on surfaces. To address this limitation, we have devised a live-cell imaging approach that employs agarose microwells to confine individual cells to small volumes. We customized a microscope for long-term T. brucei imaging that can capture multiple channels simultaneously, which has allowed us to observe events in dividing parasites for up to 36 h with minimal perturbations. In Aim 1, we will use our agarose microwell approach to identify a series of fluorescent protein markers that will allow us to image a range of organelles in live T. brucei cells. We will then investigate the consequence of the asymmetric T. brucei cell division mechanism has on the rate of daughter cell division. We will also determine if “back-up” cytokinesis, which has been proposed as an alternate cell division pathway that occurs when the conventional mechanism fails, is actually capable of generating viable progeny. In Aim 2, we will use our live-cell approach to directly observe T. brucei life-cycle transitions for the first time. We will employ parasites carrying fluorescent protein markers for different organelles and life-stage transcription markers so that we can correlate changes in cell morphology with the activation of different transcription programs. We will determine if these life cycle transitions require cell divisions, which is currently unknown, and unambiguously define the order in which different parts of the transition process occur. This work will establish our live-cell imaging approach as an important tool for trypanosomatid cell biology that can overcome a key shortcoming in our current techniques. The microwells are low cost and easy to generate, which will allow others to employ them to study a wide range of trypanosomatid biology.

项目概要 布氏锥虫和相关的锥虫寄生虫会引起人类和牲畜的疾病，这些疾病 而 RNAi 和 CRISPR/Cas9 等方法则介导了人类巨大痛苦的根源。 功能实验和基因标记方法已经产生了大量关于蛋白质功能的数据 锥体虫，对于不同表型如何表现或活体中蛋白质的动态知之甚少 这是因为 T. brucei 的活细胞成像很困难，因为寄生虫具有高度活动性。 当它们固定在表面上时，不会经历许多细胞过程，例如细胞分裂。 为了解决这个限制，我们设计了一种活细胞成像方法，利用琼脂糖微孔来限制 我们定制了一种可以捕获布氏锥虫长期成像的显微镜。 同时多个通道，这使我们能够观察寄生虫分裂事件长达 36 小时 以最小的扰动。 在目标 1 中，我们将使用琼脂糖微孔方法来鉴定一系列荧光蛋白标记物，这些标记物将 让我们能够对活体布氏锥虫细胞中的一系列细胞器进行成像，然后我们将研究该结果。 不对称布氏锥虫细胞分裂机制对子细胞分裂率是否有影响。 “备用”胞质分裂，被认为是一种替代细胞分裂途径，发生在 传统机制失败了，实际上能够产生可存活的后代。 在目标 2 中，我们将首次使用活细胞方法直接观察布氏锥虫生命周期的转变。 我们将使用携带不同细胞器和生命阶段转录荧光蛋白标记的寄生虫 标记，以便我们可以将细胞形态的变化与不同转录的激活相关联 我们将确定这些生命周期转变是否需要细胞分裂（目前尚不清楚），以及 明确定义转换过程的不同部分发生的顺序。 这项工作将建立我们的活细胞成像方法作为锥虫细胞生物学的重要工具 可以克服我们当前技术的一个关键缺点，即成本低且易于生成， 这将使其他人能够利用它们来研究广泛的锥虫生物学。