bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.01.27.634986

A novel approach to tagging tubulin reveals microtubule assembly dynamics of the axoneme in Trypanosoma brucei

Abbuhl, D.·Pruzincova, M.·Stepanek, L.·Bouscasse, E.·Azevedo, R.·Matondo, M.·Varga, V.·BONNEFOY, S.·BASTIN, P.
View original source ↗Export metadata ↓

Abstract

The protozoan parasite Trypanosoma brucei is a mono-flagellated cell during the G1-phase of its cell cycle. In order to duplicate, it assembles a new flagellum alongside the mature one, in which further elongation is prevented. Our group proposed a model where the mature flagellum is locked after construction to full length (Bertiaux et al. 2018) and access of new building blocks for elongation is exclusive to the new flagellum. To test this hypothesis directly, we developed a tool for the inducible expression of tagged tubulin. Alpha-tubulin that was tagged with an intragenic Ty-1-epitope behaved indistinguishable from untagged tubulin. Its incorporation was monitored after inducible expression, to follow the assembly dynamics of microtubules in the cell body, the mitotic spindle and the flagellum. In this study we observed that integration of tubulin occurs at the distal flagellum tip at a linear rate and is indeed restricted to the new flagellum in bi-flagellated cells. This is direct evidence that trypanosomes avoid competition between the two flagella by allowing tubulin incorporation only in the new organelle. However, by tracing flagella over several cell cycles we could also show that mature flagella do not remain locked indefinitely. The restriction is lifted briefly after the bi-flagellated cell has divided and the daughter cell inheriting the old flagellum shows incorporation of newly synthesized building blocks again. It then has to lock again before the cell can assemble a new flagellum. Our findings suggest regular incorporation of tubulin at the tip of previously locked flagella. This evidence was supported with an orthogonal approach, with which we monitored the incorporation of HaloTag-tagged radial spoke protein 4/6. Since flagellum length in trypanosomes is stable, this indicates that the entire axoneme is subject to regular events of transient disassembly followed by assembly at its distal tip.

Source connections

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Abbuhl, D., Pruzincova, M., Stepanek, L., Bouscasse, E., Azevedo, R., Matondo, M., Varga, V., BONNEFOY, S., BASTIN, P.. 2025-01-27. A novel approach to tagging tubulin reveals microtubule assembly dynamics of the axoneme in Trypanosoma brucei. https://doi.org/10.1101/2025.01.27.634986

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A dynamic phosphoproteome reveals stage-specific regulation of cytokinesis in Leishmania

Leishmania parasites undergo extensive morphological and metabolic remodelling during their transition from motile insect promastigotes to non-motile intracellular amastigotes, yet how dynamic protein kinase signalling drives this process remains poorly understood. Here, we present a high-resolution temporal phosphoproteome of L. mexicana promastigote to axenic amastigote differentiation, profiling 16,495 phosphosites on 3,101 proteins across seven developmental time points and identifying 5,795 dynamically regulated sites. This temporal map reveals stage-specific signalling programs, including regulated sites in MPK2 and non-regulated sites in MRK1, protein kinases known to be essential for amastigote differentiation. Using analogue-sensitive (AS) chemical genetics, we show that acute inhibition of either kinase in axenic amastigotes disrupts the spatial coordination of cleavage furrow ingression with nuclear and kinetoplast segregation, revealing essential cytokinesis roles obscured in constitutive gene deletions. Despite generating similar cytokinesis phenotypes, genetic over-expression and cellular localisation analyses demonstrate that MPK2 and MRK1 act through spatially segregated signalling pathways. Time-resolved phosphoproteomics upon MPK2 inhibition places its activity upstream of cytoskeletal remodelling networks. Importantly, we show that the stage-specific requirement for MPK2 is defined by the parasite's morphology: while motile promastigotes tolerate MPK2 inhibition, genetic ablation of flagellar motility sensitises them to loss of MPK2 activity. Thus, flagellar motility acts as a mechanical buffer during promastigote division, explaining why non-motile amastigotes are vulnerable to MPK2 inhibition. Together, our findings provide a systems level framework for Leishmania signalling and demonstrate that cellular architecture and biomechanics dictate protein kinase dependency across developmental stages of the parasite.

cell biology↗
bioRxiv2026

Single-cell Transcriptome-Proteome-PTMome(x8)-Metabolome (scTPPM) co-profiling decodes multimodal regulation during mouse oocyte meiosis

Single-cell multi-omics technology is crucial for comprehensive characterization of cellular molecular features. Despite recent advances in single-cell multi-omics approaches, co-profiling of nucleic acids, proteins, metabolites, and post-translational modifications (PTMs) within the same cell individuals remains major challenges. Here, we developed a single-cell transcriptome, proteome, PTMome, and metabolome (scTPPM) profiling approach, enabling simultaneous analysis of transcriptomes, proteomes, eight types of PTMs (e.g., phosphorylation, methylation, ubiquitination, O_GlcNAc, acetylation, crotonylation, lactylation, and succinylation), and metabolomes in individual cells. We demonstrated the scTPPM's performance in tumor cell lines and oocyte samples, reaching total identification and quantification of 27,668 genes, 4,847 proteins, 1,508 PTM sites, and 680 metabolites in single mouse oocytes. We profiled previously-uncharacterized dynamic crosstalk among the entire metabolic pathways from metabolic enzyme expression and regulation to metabolite-mediated post-translational modifications during oocyte maturation. In summary, scTPPM provided an effective workflow for multi-omics co-profiling in single cells, enabling multi-layered insights into complex regulatory network.

cell biology↗
bioRxiv2026

Sequence context and partner availability specify divergent cell fates from a single transcription factor

Transcription factors drive the gene regulatory networks that control developmental transitions, yet which of their many targets a cell uses remains unresolved. Expressing Cebp, Cebp{beta} or Cebp{delta} identically, alone or with Oct4, Sox2, Klf4 and cMyc, gave four outcomes: adipocyte from fibroblasts and neural stem cells, trophectoderm from embryonic stem cells, and myeloid from B cells. Raising Cebp levels changed how many sites it reached, not which sequence it read: clustered motifs maintained somatic enhancers, while spillover onto low-copy sites created de novo enhancers carrying composites of Cebp and cell-type-restricted partners. In fibroblasts, Cebp also redistributed Oct4, Sox2 and Klf4 from early targets, blocking iPSC formation. Supplying the predicted partners rewrote the output: Spi1 redirected Cebp into parallel adipogenic and myeloid programs, via a transient PpargCD45 state using separate enhancers for each; TFAP2C abolished adipogenic conversion. Motif density and partner composites therefore assemble a factor's instructive output in the receiving cell.

cell biology↗