Biology subjects

Tyc, J.

Publications and source records attributed to Tyc, J..

5 recordsLinked to original sources

bioRxiv2023

Identification of 30 transition fibre proteins reveals a complex and dynamic structure with essential roles in ciliogenesis

Transition fibres are appendages that surround the distal end of mature basal bodies (also called distal appendages) and are essential for ciliogenesis, but only a small number of proteins have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 additional transition fibre proteins (TFPs) in the flagellated eukaryote Trypanosoma brucei and mapped the arrangement of the molecular components. We discovered TFPs recruited to the basal body pre- and post-initiation of ciliogenesis with differential expression of TFPs at the assembling new flagellum compared to the existing old flagellum. Knockdown by RNAi of 17 TFPs revealed 6 were necessary for ciliogenesis and a further 3 were necessary for normal flagellum length. We identified 9 TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work demonstrates that transition fibres are complex and dynamic in their composition throughout the cell cycle which relates to their essential roles in ciliogenesis and length regulation.

cell biology↗

bioRxiv2023

Unlocking Trypanosome Biology: A Comprehensive Protein-Tagging Toolkit for Localization and Functional Analysis

African trypanosomes are medically important parasites that cause Sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible protein tagging in trypanosomes, which enabled the localisation of all proteins in the trypanosome cell. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an expanded set of >100 vectors that utilizes universal primer annealing sequences, enabling protein tagging with a range of fluorescent and biochemical tags using five different selection markers. We evaluated the suitability of various fluorescent proteins for live cell imaging and determined their brightness and stability under different fixation conditions. Finally, we determined the optimal fluorescent protein for a set of specific experimental conditions demonstrating the utility of this toolkit.

cell biology↗

bioRxiv2022

Massive accumulation of strontium and barium in diplonemid protists

Barium (Ba) and strontium (Sr) are often used as proxies for the reconstruction of past marine productivity and global climate. The ability to accumulate Ba2+ and Sr2+ in the form of crystals is rare among eukaryotes. Here we report that unicellular heterotrophs called diplonemids (Euglenozoa), one of the most abundant groups of marine planktonic protists, accumulate conspicuous amounts of these trace elements in the form of intracellular barite (BaSO4) and celestite (SrSO4) crystals, in concentrations greater than in other known Ba/Sr-accumulating organisms. Moreover, these flagellates can uptake Sr2+ exclusively or together with Ba2+ and form (Ba,Sr)SO4. One species, Namystinia karyoxenos, is naturally capable of intracellular accumulation of Ba2+ and Sr2+ 42,000 and 10,000 times relative to the surrounding medium. Altering the amounts of both elements in the medium resulted in corresponding changes in the quantity and composition of crystals. Planktonic copepods fed with diplonemids produce celestite-rich fecal pellets, which facilitate deposition of these minerals on the seafloor. We propose that diplonemids, which emerged during the Neoproterozoic era, qualify as impactful players of Ba2+/Sr2+ cycling in the ocean that possibly contributed to sedimentary rock formation over long geological periods.

microbiology↗

bioRxiv2019

A key regulatory protein for flagellum length control in stable flagella

Cilia and flagella are highly conserved microtubule-based organelles that have important roles in cell motility and sensing [1]. They can be highly dynamic and short lived such as primary cilia or Chlamydomonas [2] or very stable and long lived such as those in spermatozoa [3] photoreceptors [4] or the flagella of many protist cells [3,4]. Although there is a wide variation in length between cell types, there is generally a defined length for a given cell type [1]. Many unicellular flagellated and ciliated organisms have an additional challenge as they must maintain flagella/cilia at a defined length whilst also growing new flagella/cilia in the same cell. It is not currently understood how this is achieved. A grow-and-lock model was proposed for the maintenance of stable flagella where a molecular lock is applied to prevent flagellum length change after assembly [5]. The molecular mechanisms of how this lock operates are unknown, but could be important in cells where an existing flagellum must be maintained whilst a new flagellum assembles. Here we show that Cep164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. It is only localised on the transition fibres of basal bodies of fully assembled flagella and missing from assembling flagella. In fact, basal bodies only acquire Cep164C in the third cell cycle after they assemble in trypanosomes. Depletion leads to dysregulation of flagellum growth with both longer and shorter flagella; consistent with defects in a flagellum locking mechanism. By controlling delivery of components into the old assembled flagellum, maintenance of stable flagella can occur but limits further growth. This offers an important explanation for how many eukaryotic unicellular cells maintain their existing flagella whilst growing new ones before these cells divide. This work also reveals additional regulatory roles for Cep164 in eukaryotic organisms.

cell biology↗

bioRxiv2017

A molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei

In almost all eukaryotes mitochondria maintain their own genome. Despite the discovery more than 50 years ago still very little is known about how the genome is properly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure proper segregation of the mitochondrial genome via the basal bodies movement, during cell cycle. Using super-resolution microscopy we precisely localize each of the currently known unique TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum towards the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on biochemical analysis the TAC consists of several non-overlapping subcomplexes suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC has an impact on mitochondrial organelle positioning however is not required for proper organelle biogenesis or segregation.\n\nSignificance StatementMitochondrial genome replication and segregation are essential processes in most eukaryotic cells. While replication has been studied in some detail much less is known about the molecular machinery required distribute the replicated genomes. Using super-resolution microscopy in combination with molecular biology and biochemistry we show for the first time in which order the segregation machinery is assembled and that it is assembled de novo rather than in a semi conservative fashion in the single celled parasite Trypanosoma brucei. Furthermore, we demonstrate that the mitochondrial genome itself is not required for assembly to occur. It seems that the physical connection of the mitochondrial genome to cytoskeletal elements is a conserved feature in most eukaryotes, however the molecular components are highly diverse.\n\nAbbreviation

cell biology↗