Biology subjects

Zelena, M.

Publications and source records attributed to Zelena, M..

4 recordsLinked to original sources

bioRxiv2025

Flagellar pocket collar biogenesis: Cytoskeletal organization and novel structures in a unicellular parasite

Understanding how cells build and organize their internal structures is a fundamental question in biology, with important implications for human health and disease. Trypanosomes are single-celled flagellated parasites that cause life-threatening diseases in human and animals. Their survival relies on a specialized compartment called the flagellar pocket (FP), which serves as a gateway for nutrient uptake, and immune evasion. The formation and function of the FP are supported by an intricate cytoskeletal structure known as the flagellar pocket collar (FPC). However, the mechanisms underlying its assembly remain poorly understood. In this study, we used cutting-edge ultrastructure expansion microscopy (U-ExM) to investigate FPC biogenesis in Trypanosoma brucei. We mapped the formation of the new microtubule quartet (nMtQ) alongside flagellum growth, providing new insights into its assembly. Additionally, we tracked the localization dynamics of key structural proteins - BILBO1, MORN1, and BILBO2 - during the biogenesis of the FPC and the hook complex (HC). Notably, we identified two previously undetected structures: the proFPC and the transient FPC-interconnecting fibre (FPC-IF), both of which appear to play crucial roles in linking and organizing cellular components during cell division. By uncovering these novel aspects of FPC biogenesis, our study significantly advances the understanding of cytoskeletal organization in trypanosomes and opens new avenues for exploring the functional significance of these structures.

microbiology↗

bioRxiv2024

Striated fibre assemblins localise in the feeding groove of the 'typical excavate' Paratrimastix pyriformis

The spatial arrangement of microtubules in the cytoskeleton in the cells of protists has been used for decades for taxonomy and phylogenetic inference at various levels. In contrast, the protein composition of non-microtubular structures is mostly unknown. Exceptions are system I fibers in algae, which are built of striated fiber assemblins (SFAs). Interestingly, SFAs are also components of a range of other, dissimilar structures, playing a role in the cortex of ciliates, cell division in apicomplexans, and adhesion of the parasite Giardia to the intestine. In a broad bioinformatic survey, we show the existence of three ancestral eukaryotic paralogs of SFA, and note that they are present in all "typical excavates": small heterotrophic flagellates bearing a ventral feeding groove. In one representative, Paratrimastix pyriformis, we detected two SFA paralogs using specific antibodies and expansion microscopy. We show that they co-localize selectively with several microtubules and structures attached to the basal body of the posterior flagellum, namely the right microtubular root, B-fiber, C-fiber, and composite fiber. We demonstrate that one of the paralogs self-assembles in vitro into striated filaments which, under negative staining and cryo-electron microscopy, resemble system I fibers as seen in previous studies. Given the facts that all three SFA paralogs appear to be ancestral to most eukaryotic lineages, as is probably the morphology of "typical excavates" with a ventral groove, we speculate that these proteins played roles in the support and development of the feeding apparatus of the last eukaryotic common ancestor. SIGNIFICANCE STATEMENTWe identified three paralogues of intermediate filament-like proteins from the family of striated fiber assemblins in the Last Eukaryotic Common Ancestor (LECA). Using expansion microscopy, we demonstrated that two of these proteins form a complex cytoskeleton that supports the feeding groove of Paratrimastix pyriformis. It is widely accepted that this flagellated protist retains the morphological and feeding characteristics of ancestral eukaryotes. Therefore, our results suggest that the striated fiber assemblin proteins, which form diverse structures in various extant eukaryotes, were initially components of the feeding apparatus in LECA.

microbiology↗

bioRxiv2024

Imaging Giardia intestinalis cellular organisation using expansion microscopy revealed atypical centrin localisation.

Advanced imaging of microorganisms, including protists, is challenging due to their small size. Specimen expansion prior to imaging is thus beneficial to increase resolution and cellular details. Here, we present a sample preparation workflow for improved observations of the single-celled eukaryotic pathogen Giardia intestinalis (Excavata, Metamonada). The binucleated trophozoites colonize the small intestine of humans and animals and cause a diarrhoeal disease. Their remarkable morphology includes two nuclei and a pronounced microtubular cytoskeleton enabling cell motility, attachment and proliferation. By use of expansion and confocal microscopy, we resolved in a great detail subcellular structures and organelles of the parasite cell. The acquired spatial resolution of 43 nm enabled novel observations of centrin localisation at Giardia basal bodies. Interestingly, non-luminal centrin localization between the Giardia basal bodies was observed, which is an atypical eukaryotic arrangement. Our protocol includes antibody staining and can be used for the localisation of epitope-tagged proteins, as well as for differential organelle labelling by amino reactive esters. This fast and simple protocol is suitable for routine use without a superresolution microscopy equipment.

microbiology↗

bioRxiv2023

Characterisation of the SUF FeS cluster machinery in the amitochondriate eukaryote Monocercomonoides exilis

Monocercomonoides exilis is the first eukaryotic organism described as a complete amitochondriate, yet it shares common features with heterotrophic anaerobic/microaerophilic protists, some of which bear divergent mitochondrion-related organelles or MROs. It has been postulated that the retention of these organelles stems from their involvement in the assembly of essential cytosolic and nuclear FeS proteins, whose maturation requires the evolutionarily conserved mitochondrial ISC and cytosolic CIA machineries. The amitochondriate M. exilis lacks genes encoding the ISC machinery yet contains a bacteria-derived SUF system (MeSuf), composed of the cysteine desulphurase SufS fused to SufD and SufU, as well as the FeS scaffolding components MeSufB and MeSufC. Here, we show that expression of the M. exilis SUF genes, either individually or in tandem, can restore the maturation of the FeS protein IscR in the Escherichia coli double mutants of {Delta}sufS {Delta}iscS and {Delta}sufB {Delta}iscUA. In vivo and in vitro studies indicate that purified MeSufB, MeSufC and MeSufDSU proteins interact suggesting that they act as a complex in the protist. MeSufBC can undergo conformational changes in the presence of ATP and assemble FeS clusters under anaerobic conditions in presence and absence of ATP in vitro. Altogether, these results indicate that the dynamically interacting MeSufDSUBC proteins may function as an FeS cluster assembly complex in M. exilis thereby being capable of replacing the organelle-enclosed ISC system of canonical eukaryotes.

biochemistry↗