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.

项目摘要 布鲁氏锥虫和相关的锥虫寄生虫引起人类和牲畜的疾病 巨大的人类苦难的根源。诸如RNAi-和CRISPR/CAS9介导的方法的损失 功能实验和基因标记方法已经产生了有关蛋白质功能的大量数据 锥虫剂，对不同表型的表现或蛋白质的动力学在现场表现如何 细胞。这是由于T. Brucei中的活细胞成像很难 并且，当将它们固定在表面上时，请勿经历许多细胞过程，例如细胞分裂。到 解决此限制，我们设计了一种活细胞成像方法 单个细胞至少量。我们定制了一个可以捕获的长期T. brucei成像的显微镜 简而 扰动最小。 在AIM 1中，我们将使用琼脂糖微孔方法来识别一系列荧光蛋白标记物 允许我们在活的T. brucei细胞中成像一系列细胞器。然后，我们将研究 不对称的T. Brucei细胞分裂机制对子细胞分裂的速率具有。我们还将确定是否 “备份”细胞动物，已提出是一种替代细胞分裂途径，发生在 常规机制失败，实际上能够产生可行的后代。 在AIM 2中，我们将使用活细胞的方法第一次直接观察Brucei生命周期过渡。 我们将采用携带荧光蛋白标记的寄生虫进行不同的细胞器和生命阶段转录 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 程序。我们将确定这些生命周期过渡是否需要细胞分裂，这目前未知，并且 明确定义了过渡过程的不同部分的顺序。 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 可以克服当前技术中的关键缺点。微区成本低，易于产生， 这将使其他人能够雇用他们研究广泛的锥形生物学。