Malfara, M. F. | bioRxiv Science

bioRxiv2026

The trypanosomatid dynamin-like protein associates with glycosomes

Subcellular organelles must undergo periodic fission to be evenly distributed during cell division. These division events are mediated by protein members of the dynamin family, including dynamin-related proteins. Protozoan parasites, including trypanosomatids such as Trypanosoma brucei, have several single-copy organelles, suggesting tightly regulated systems for organelle fission and segregation. However, trypanosomatid genomes typically encode only one dynamin-like protein (DLP), which in T. brucei has multiple roles including endocytosis and mitochondrial fission. How DLPs are recruited to different membranes, and how their fission activity is regulated, are unknown. We used tandem-affinity purification in the related trypanosomatid Crithidia fasciculata to identify interacting partners of DLP. Surprisingly, we found that CfDLP co-purified with multiple proteins predicted to localize to glycosomes, peroxisome-related glycolytic organelles. Using expansion microscopy, we confirmed the localization of CfDLP to glycosomes, specifically those that appear to be undergoing division. To see if changes in the levels of DLP could alter glycosome morphology, we conducted RNAi-mediated knockdown and inducible overexpression experiments in T. brucei. TbDLP knockdown causes subtle changes in glycosome size, while overexpression of TbDLP1 causes an increase cytoplasmic vesicles and altered permeability of glycosomal membranes. These results suggest that the multifunctional DLP of trypanosomatids plays a role in glycosome maintenance. Author SummaryTrypanosomatids are eukaryotic parasites that cause devastating diseases in humans and animals. Like all eukaryotic cells, they must maintain their subcellular compartments through organelle division and other membrane remodeling events. Dynamin-like proteins are enzymes that work with other proteins to apply mechanical force to membranes. The dynamin-like proteins of Trypanosoma brucei, the causative agent of human African trypanosomiasis, have been implicated in endocytosis and mitochondrial division, although how these activities are regulated is not known. We have used a model trypanosomatid, the mosquito parasite Crithidia fasciculata, to look for dynamin-interacting proteins. In addition to proteins of unknown function, we show that dynamin-like protein associates with proteins found on glycosomes, trypanosomatid-specific organelles that contain enzymes required for breakdown of sugars. Knockdown and overexpression of dynamin-like proteins in T. brucei causes changes in glycosomes, supporting a role in organelle maintenance. Dynamin-like proteins likely regulate organelle structure and function, allowing parasites to adapt to different energetic requirements during their life cycle.

cell biology↗

bioRxiv2022

Adhesion of Crithidia fasciculata promotes a rapid change in developmental fate driven by cAMP signaling

Kinetoplastids are single-celled parasites responsible for human and animal disease. For the vast majority of kinetoplastids, colonization of an insect host is required for transmission. Stable attachment to insect tissues via the single flagellum coincides with differentiation and morphological changes. Although this process is essential for the generation of infectious forms, the molecular mechanism driving differentiation following adherence is not well understood. To study this process, we elaborate upon an in vitro model in which flagellated swimming cells of the kinetoplastid Crithidia fasciculata rapidly differentiate following adhesion to artificial substrates. Manipulation of culture parameters revealed that growth phase and time had a strong influence on the proportion of adherent cells. Live imaging of cells transitioning from swimming to an attached cell fate show parasites undergoing a defined sequence of events including an initial adhesion near the base of the flagellum, immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like structure between the tip of the shortened flagellum and the substrate. We have also assayed the role of the cyclic AMP (cAMP) signaling pathway in differentiation of C. fasciculata. Pharmacological inhibition of cAMP phosphodiesterases eliminated the ability of swimming cells to attach without affecting their growth rate. Further, treatment with inhibitor did not affect the growth rate of established attached cells, indicating its effect is limited to a critical window of time during the early stages of adhesion. Finally, in swimming parasites we have shown that a receptor adenylate cyclase localizes to the distal portion of the flagellum. In attached cells it is absent from the shortened flagellum and instead localizes to the cell body. Similarly, a putative phosphodiesterase, found along the length of the flagellum, also relocalizes to the cell body in attached parasites. These data suggest that in C. fasciculata cAMP signaling is required for adherence, that cAMP flux in the flagellum of swimming cells is spatially restricted, and that signaling domains may be reorganized during differentiation and attachment. These studies contribute to our understanding of the flagellum as a multi-functional organelle integrating processes related to motility, signaling, attachment, and differentiation and further develop C. fasciculata as a model kinetoplastid. Author SummaryParasites from the order Kinetoplastida are transmitted by insects and cause diseases such as Leishmaniasis, Chagas disease, and Human African Trypanosomiasis. A key aspect of the life cycle of these parasites is their ability to adhere to surfaces within the insect and subsequently differentiate into forms that are transmissable to their next host. Here, we explore the molecular mechanisms underpinning both adherence and differentiation using the mosquito parasite Crithidia fasciculata. This organism is closely related to pathogenic species but grows to high densities in culture and adheres robustly in vitro. We identified factors affecting the rate of adherence and defined morphological stages of this process including rapid shortening of the cells single flagellum. Using electron microscopy, we confirmed that adhesion to artificial substrates results in the formation of a filament-rich adhesive plaque at the tip of the flagellum that resembles the attachment to insect tissue. We also demonstrate the key role played by the cyclic AMP signaling pathway during adherence and differentiation. Specifically, pharmacological inhibition of enzymes that degrade cyclic AMP completely blocks adherence, and tagging of several proteins in this pathway show that their localization along the flagellum changes following differentiation to the attached form. These data provide insights into processes critical for all kinetoplastid life cycles.

microbiology↗