Investigating Trypanosomatid life-cycle transitions using live-cell imaging

Brown University, Providence RI

Investigators

Abstract

PROJECT SUMMARY Trypanosomatids are important human pathogens that cause severe diseases around the world. These parasites feature free living life-cycle stages that have previously been intractable to rigorous study using long term live- cell imaging because most confinement strategies disrupt cellular processes or cause death. This has left significant gaps in knowledge about the rates of cellular events such as cell division and if states seen in fixed- cell imaging represent bona fide intermediates of cellular processes or are strictly endpoints or fixation artefacts. We recently devised a strategy using agarose microwells cast from PDMS stamps to confine trypanosomatids in nanoliter volumes of media without impacting their motility or viability. These volumes are sufficiently small that 10 to 25 microwells fit within the imaging volume of an inverted microscope, allowing us to observe multiple cells during one imaging run, which can last as long as 3 days using white light and fluorescence. The method is also compatible with small molecule and RNAi treatments, which allows novel investigation of loss-of-function experiments. We have used the agarose microwell method to study the cell division mechanism of Trypanosoma brucei and Trypanosoma cruzi, showing that the daughter cells produced by the parasites subsequently divide at different rates, which suggests that the inherent asymmetry in the daughter cells takes different amounts of time to resolve before they can divide again. We also showed that an intermediate that appears in fixed samples during RNAi that was proposed to function as an alternate cell division mechanism could be observed during live cell imaging, but that it did not lead to productive cell divisions, which emphasizes the value of our approach. We have updated our microscope to increase the number of microwells that we can observe simultaneously and decrease the amount of light we need to image cells. This update has now made it possible to observe rare or asynchronous events, such as life-cycle transitions, that also appear to be more sensitive to light exposure. We will use our updated imaging approach to study a variety of life-cycle transitions in both T. brucei and T. cruzi. In Aim 1a, we will be studying the mechanism of metacyclogenesis T. brucei, which produces parasites that are ready for transmission into the mammalian host, to determine how the parasite replaces cell surface proteins and how it reorganizes the cell body. In Aim 1b, we will study the T. brucei slender to stumpy transition, which preadapts bloodstream form parasites for transmission back into the insect host, using recent scRNA-seq data to identify intermediate states in the process. In Aim 1c, we will determine if the switch from the insect-resident epimastigote form to the metacyclic trypomastigote form in T. cruzi, which requires a significant repositioning of the flagellum and all its associated structures, requires a cell division to occur and how attachment to a surface affects the transition. Our work will provide unprecedented details about the mechanisms of trypanosomatid life- cycle transitions, which are poorly understood and could yield new avenues for blocking parasite transmission.