de Graffenried, C. L. | Search bioRxiv

bioRxiv2021

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

Trypanosoma brucei, the causative agent of human African trypanosomiasis, employs a flagellum for dissemination within the parasites mammalian and insect hosts. T. brucei cells are highly motile in culture and must be able to move in all three dimensions for reliable cell division. These characteristics have made long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of a variety of important cell-cycle events. To address this issue, we have devised an imaging approach that confines cells to small volumes that can be imaged continuously for up to 24 h. This system employs cast agarose microwells generated using a PDMS stamp that can be made with different dimensions to maximize cell viability and imaging quality. Using this approach, we have imaged individual T. brucei through multiple rounds of cell division with high spatial and temporal resolution. We have employed this method to study the differential rate of T. brucei daughter cell division and show that the approach is compatible with loss-of-function experiments such as small molecule inhibition and RNAi. We have also developed a strategy that employs in-well "sentinel" cells to monitor potential toxicity due to imaging. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible. ImportanceTrypanosoma brucei causes severe diseases that affect humans and livestock in Sub-Saharan Africa. Efficient strategies for manipulating the T. brucei genome have provided a wealth of information about protein localization and function in diverse cellular processes. However, employing live-cell imaging for phenotypic analysis in T. brucei remains a significant challenge because immobilization of this highly motile parasite rapidly leads to morphologic defects and cell death. While fixed-cell imaging can provide snapshots of cellular events, it cannot provide the direct causal link or precise timing of events that comes from watching a living cell change over time. Our strategy using agarose microwells now allows long-term live cell T. brucei imaging with a simple apparatus that can be adapted for a wide range of experimental conditions.

microbiology↗

bioRxiv2020

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

Microtubules are inherently dynamic cytoskeletal polymers whose length and activity can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle, comprising a single microtubule layer that is crosslinked to each other and the plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins. We characterize the localization and function of the array-associated protein PAVE1, which is a component of the inter-microtubule crosslinking fibrils present within the posterior subdomain. PAVE1 functions to stabilize these microtubules to produce the tapered cell posterior. PAVE1 and the newly identified PAVE2 form a complex that binds directly to the microtubule lattice. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for retaining PAVE1 within the posterior subdomain, and also maintains array-associated proteins in the middle and anterior subdomains of the array. The arrangement of proteins within the array is likely to tune the local properties of the array microtubules and create the asymmetric shape of the cell, which is essential for parasite viability. Author summaryMany parasitic protists use arrays of microtubules that contact the inner leaflet of the plasma membrane, typically known as subpellicular microtubules, to shape their cells into forms that allow them to efficiently infect their hosts. While subpellicular arrays are found in a wide range of parasites, very little is known about how they are assembled and maintained. Trypanosoma brucei, which is the causative agent of human African trypanosomiasis, has an elaborate subpellicular array that produces the helical shape of the parasite, which is essential for its ability to move within crowded and viscous solutions. We have identified a series of proteins that have a range of localization patterns within the array, which suggests that the array is regulated by subdomains of array-associated proteins that may tune the local properties of the microtubules to suit the stresses found at different parts of the cell body. Among these proteins are the first known components of the inter-microtubule crosslinks that are thought to stabilize array microtubules, as well as a potential regulator of the array subdomains. These results establish a foundation to understand how subpellicular arrays are built, shaped, and maintained, which has not previously been appreciated.

cell biology↗