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

项目总结 布鲁氏锥虫和相关的锥虫寄生虫会引起人和牲畜的疾病，这些疾病 人类遭受巨大苦难的根源。而RNAi和CRISPR/Cas9等方法介导的细胞丢失 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 锥虫，对不同的表型如何表现或活体中蛋白质的动态知之甚少 细胞。这是因为布氏毛滴虫的活体细胞成像是困难的，因为这种寄生虫活动能力很强。 当它们固定在表面时，不会经历许多细胞过程，如细胞分裂。至 为了解决这一局限，我们设计了一种活细胞成像方法，使用琼脂糖微孔来限制 从单个细胞到小体积。我们定制了一种用于长期布氏锥虫成像的显微镜，可以捕捉到 多个通道同时进行，这使我们能够观察寄生虫分裂中的事件长达36小时 以最小的扰动。 在目标1中，我们将使用我们的琼脂糖微孔方法来鉴定一系列荧光蛋白标记，这些标记将 使我们能够想象布鲁氏毛滴虫活细胞中的一系列细胞器。然后我们将调查这一事件的后果 布氏毛滴虫细胞分裂机制不对称对子细胞分裂率有影响。我们还将确定是否 “备用”胞质分裂，这被认为是一种替代的细胞分裂途径，发生在 常规机制失败了，实际上能够产生可存活的后代。 在目标2中，我们将首次使用我们的活细胞方法来直接观察布氏锥虫的生活史转变。 我们将使用携带荧光蛋白标记物的寄生虫进行不同细胞器和生命阶段的转录 标记，这样我们就可以将细胞形态的变化与不同转录的激活联系起来 程序。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 明确定义过渡过程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 可以克服我们目前技术中的一个关键缺陷。该微孔井成本低且易于生产， 这将允许其他人雇用他们研究广泛的锥虫生物学。

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.