Biology subjects

Campbell, P. C.

Publications and source records attributed to Campbell, P. C..

4 recordsLinked to original sources

bioRxiv2023

Morphogenesis in Trypanosoma cruzi epimastigotes proceeds via a highly asymmetric cell division

Trypanosoma cruzi is a protist parasite that is the causative agent of Chagas disease, a neglected tropical disease endemic to the Americas. T. cruzi cells are highly polarized and undergo morphological changes as they cycle within their insect and mammalian hosts. Work on related trypanosomatids has described cell division mechanisms in several life-cycle stages and identified a set of essential morphogenic proteins that serve as markers for key events during trypanosomatid division. Here, we use Cas9-based tagging of morphogenic genes, live-cell imaging, and expansion microscopy to study the cell division mechanism of the insect-resident epimastigote form of T. cruzi, which represents an understudied trypanosomatid morphotype. We find that T. cruzi epimastigote cell division is highly asymmetric, producing one daughter cell that is significantly smaller than the other. Daughter cell division rates differ by 4.9 h, which may be a consequence of this size disparity. Many of the morphogenic proteins identified in T. brucei have altered localization patterns in T. cruzi epimastigoes, which may reflect fundamental differences in the cell division mechanism of this life cycle stage, which widens and shortens the cell body to accommodate the duplicated organelles and cleavage furrow rather than elongating the cell body along the long axis of the cell, as is the case in life-cycle stages that have been studied in T. brucei. This work provides a foundation for further investigations of T. cruzi cell division and shows that subtle differences in trypansomatid cell morphology can alter how these parasites divide. Author SummaryTrypanosoma cruzi causes Chagas disease, which is among the most neglected of tropical diseases, affecting millions of people in South and Central America along with immigrant populations around the world. T. cruzi is related to other important pathogens such as Trypanosoma brucei and Leishmania spp, which have been the subject of molecular and cellular characterizations that have provided an understanding of how these organisms shape their cells and undergo division. Work in T. cruzi has lagged due to an absence of molecular tools for manipulating the parasite and the complexity of the original published genome; these issues have recently been resolved. Building on work in T. brucei, we have studied the localization of key cell cycle proteins and quantified changes in cell shape during division in an insect-resident form of T. cruzi. This work has uncovered unique adaptations to the cell division process in T. cruzi and provides insight into the range of mechanisms this family of important pathogens can employ to colonize their hosts.

microbiology↗

bioRxiv2022

Endocytosis in Trypanosoma cruzi Depends on Proper Recruitment and Regulation of Functionally Redundant Myosin Motors

Utilized by the free-living kinetoplastid Bodo saltans to feed on bacterial prey, the cytostome-cytopharynx complex (SPC) is an endocytic organelle absent from all human trypanosomatid pathogens save Trypanosoma cruzi. Building upon our previous work identifying the myosin motor MyoF as the first enzymatic component of the T. cruzi SPC, we sought to expand our understanding of this distinct organelle by identifying additional protein machinery which contribute to the endocytic process. While deletion of MyoF alone did not fully ablate endocytosis, we found that deletion of both MyoF and the similarly localized MyoC produced an endocytic-null phenotype that was rescued upon complementation. To identify potential regulatory components of this motor complex, we pulled down MyoF and identified an SPC-targeted protein that contained an annotated EF-hand calcium-binding motif that was conserved across a wide range of protozoan lineages. Surprisingly, deletion of this myosin associated protein (MyAP) alone was sufficient to produce an endocytic-null phenotype, which we were able to fully rescue via complementation. The deletion of MyAP also caused the mis-localization of both cytopharynx myosins to the cytosol. While MyAP lacking the EF-hand domain was unable to complement endocytosis, it was sufficient to restore proper myosin localization. This suggested that MyAP plays two distinct roles, one in targeting myosins to the SPC and a second in regulating myosin motor activity. Transmission electron microscopy also revealed that endocytic-null mutants lacked the electron lucent lipid inclusions typically seen in the pre-lysosomal reservosomes of T. cruzi epimastigotes. Mass spectrometry based lipidomic analysis subsequently revealed a dramatic reduction in the scavenged cholesterol content in the endocytic-null mutants, which can be attributed to an inability to endocytose exogenous lipid-protein complexes for storage in the reservosomes. Overall, this work showcases the first viable endocytic-null mutants generated in T. cruzi through specific gene deletion and highlights the feasibility of leveraging this strategy towards a full dissection of the endocytic machinery and biogenesis of the SPC. ImportanceTrypanosoma cruzi chronically infects over 7 million people in the Americas and current therapeutics are insufficient to effectively cure infection. The lack of progress in developing effective vaccines or drug treatments is due, in part, to longstanding technical limitations in studying this parasite and a lack of resources committed to support research and eradication efforts. As part of its parasitic lifestyle, T. cruzi is forced to obtain basic nutrients directly from its host environment, making the development of methods to block nutrient uptake an attractive strategy to control parasite growth and transmission. While the bulk uptake of complex nutrients by T. cruzi occurs via an endocytic structure, often referred to as the cytostome-cytopharynx complex (SPC), how exactly this tubular endocytic organelle functions at a mechanistic level has remained a mystery. In this work, we investigated the contribution of several SPC targeted myosin motors and an associated protein factor to endocytic activity. By identifying and characterizing the molecular machinery responsible for nutrient uptake, we hope to both expand our basic understanding of how this deadly pathogen acquires essential nutrients from its host, while also revealing new potential therapeutic targets to impede nutrient uptake.

microbiology↗

bioRxiv2021

A Trypanosoma brucei orphan kinesin employs a convergent microtubule organization strategy to complete cytokinesis

Many single-celled eukaryotes have complex cell morphologies defined by cytoskeletal elements comprising microtubules arranged into higher-order structures. Trypanosoma brucei (T. brucei) cell polarity is mediated by a parallel array of microtubules that underlie the plasma membrane and define the auger-like shape of the parasite. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the division plane and is essential for the completion of cytokinesis. To gain mechanistic insight into how this novel kinesin functions to complete cleavage furrow ingression, we characterized the biophysical properties of the KLIF motor domain in vitro. We found that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked in an antiparallel orientation are translocated relative to one another by KLIF, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we found that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new posterior, which is a unique requirement for the completion of cytokinesis in T. brucei.

biophysics↗

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↗