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.
Live-cell imaging is a vital tool for understanding the spatio-temporal relationships between different cellular events. This approach has been difficult to establish in motile parasites such as T. brucei because these organisms do not tolerate immobilization. We have developed a method for T. brucei live-cell imaging that employs agarose microchambers that allows us to image parasites for multiple cell divisions, which will provide important new insights into cellular processes such as cell division and life-cycle transitions that have not been feasible before.
