Early endosomes sort transmembrane cargo either for lysosomal degradation or retrieval to the plasma membrane or the Golgi complex. Endosomal retrieval in eukaryotes is governed by the anciently homologous retromer or retriever complexes. Each comprises a core tri-protein subcomplex, membrane-deformation proteins and interacting partner complexes, together retrieving a variety of known cargo proteins. Trichomonas vaginalis, a sexually transmitted human parasite, uses the endomembrane system for pathogenesis. It has massively and selectively expanded its endomembrane protein complement, the evolutionary path of which has been largely unexplored. Our molecular evolutionary study of retromer, retriever and associated machinery in parabasalids and its free-living sister lineage of Anaeramoeba demonstrates specific expansion of the retromer machinery, contrasting with the retriever components. We also observed partial loss of the Commander complex and sorting nexins in Parabasalia but complete retention in Anaeramoeba. Notably, we identified putative parabasalid sorting nexin analogs. Finally, we report the first retriever protein localization in a non-metazoan group along with retromer protein localization in T. vaginalis.

• Klíčová slova
• Endomembrane, Evolution, Parabasalids, Phylogenomics, Retriever, Retromer,
• MeSH
• endozomy * metabolismus   MeSH
• fylogeneze MeSH
• Golgiho aparát metabolismus   MeSH
• lidé MeSH
• molekulární evoluce MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• transport proteinů MeSH
• Trichomonas vaginalis metabolismus   genetika   MeSH
• třídící nexiny metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH
• třídící nexiny MeSH

Mitosis is a crucial stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe demonstrates catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from, for example, mutations in the acetyl-CoA/biotin carboxylase (cut6), fatty acid synthase (fas2, also known as lsd1) or transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It has been previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride supplementation. In this study, we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity.

• Klíčová slova
• Closed mitosis, Cut, Lipid metabolism, Mitotic catastrophe, Nitrogen availability, TOR,
• MeSH
• dusík * metabolismus   MeSH
• metabolismus lipidů * MeSH
• mitóza * MeSH
• mutace genetika   MeSH
• Schizosaccharomyces pombe - proteiny * metabolismus   genetika   MeSH
• Schizosaccharomyces * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dusík * MeSH
• Schizosaccharomyces pombe - proteiny * MeSH

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

• Klíčová slova
• Trypanosoma, Cilia, Ciliogenesis, Distal appendages, Flagella, Transition fibres,
• MeSH
• bazální tělíska metabolismus   MeSH
• časové faktory MeSH
• cilie genetika   metabolismus   MeSH
• flagella genetika   metabolismus   MeSH
• konzervovaná sekvence MeSH
• protozoální proteiny * genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• transport proteinů MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny * MeSH

CSL proteins [named after the homologs CBF1 (RBP-Jκ in mice), Suppressor of Hairless and LAG-1] are conserved transcription factors found in animals and fungi. In the fission yeast Schizosaccharomyces pombe, they regulate various cellular processes, including cell cycle progression, lipid metabolism and cell adhesion. CSL proteins bind to DNA through their N-terminal Rel-like domain and central β-trefoil domain. Here, we investigated the importance of DNA binding for CSL protein functions in fission yeast. We created CSL protein mutants with disrupted DNA binding and found that the vast majority of CSL protein functions depend on intact DNA binding. Specifically, DNA binding is crucial for the regulation of cell adhesion, lipid metabolism, cell cycle progression, long non-coding RNA expression and genome integrity maintenance. Interestingly, perturbed lipid metabolism leads to chromatin structure changes, potentially linking lipid metabolism to the diverse phenotypes associated with CSL protein functions. Our study highlights the critical role of DNA binding for CSL protein functions in fission yeast.

• Klíčová slova
• CSL proteins, Cell adhesion, Chromatin structure, DNA binding, Fission yeast, Genome integrity, Lipid metabolism,
• MeSH
• buněčný cyklus genetika   MeSH
• DNA fungální metabolismus   genetika   MeSH
• DNA vazebné proteiny metabolismus   genetika   MeSH
• metabolismus lipidů genetika   MeSH
• proteiny buněčného cyklu * MeSH
• regulace genové exprese u hub MeSH
• Schizosaccharomyces pombe - proteiny * metabolismus   genetika   MeSH
• Schizosaccharomyces * metabolismus   genetika   MeSH
• transkripční faktory * MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• DNA vazebné proteiny MeSH
• DSC1 protein, S pombe MeSH Prohlížeč
• proteiny buněčného cyklu * MeSH
• Schizosaccharomyces pombe - proteiny * MeSH
• transkripční faktory * MeSH

The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.

• Klíčová slova
• Actin, Lipid, Micropattern, Microtubule, Reconstitution assays,
• MeSH
• aktiny * fyziologie   MeSH
• mikrofilamenta chemie   MeSH
• mikrotubuly * fyziologie   MeSH
• polarita buněk MeSH
• pseudopodia MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny * MeSH

The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input.

• Klíčová slova
• Tetrahymena, Contractile vacuole complex, Organelle biogenesis, Organelle dynamics, Osmotic regulation,
• MeSH
• endozomy MeSH
• mitóza MeSH
• proteiny metabolismus   MeSH
• Tetrahymena thermophila * MeSH
• vakuoly * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny MeSH

Mitotic fidelity is crucial for the faithful distribution of genetic information into the daughter cells. Many fungal species, including the fission yeast Schizosaccharomyces pombe, undergo a closed form of mitosis, during which the nuclear envelope does not break down. In S. pombe, numerous processes have been identified that contribute to successful completion of mitosis. Notably, perturbations of lipid metabolism can lead to catastrophic mitosis and the 'cut' phenotype. It has been suggested that these mitotic defects are caused by insufficient membrane phospholipid supply during the anaphase nuclear expansion. However, it is not clear whether additional factors are involved. In this study, we characterized in detail mitosis in an S. pombe mutant lacking the Cbf11 transcription factor, which regulates lipid metabolism genes. We show that in cbf11Δ cells mitotic defects have already appeared prior to anaphase, before the nuclear expansion begins. Moreover, we identify altered cohesin dynamics and centromeric chromatin structure as additional factors affecting mitotic fidelity in cells with disrupted lipid homeostasis, providing new insights into this fundamental biological process.

• Klíčová slova
• CSL transcription factor, Chromatin structure, Closed mitosis, Cohesin, Lipid metabolism, Mitotic defects,
• MeSH
• koheziny MeSH
• metabolismus lipidů genetika   MeSH
• mitóza genetika   MeSH
• Schizosaccharomyces pombe - proteiny * metabolismus   MeSH
• Schizosaccharomyces * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Schizosaccharomyces pombe - proteiny * MeSH

Eisosomes are large hemitubular structures that underlie the invaginated microdomains in the plasma membrane of various ascomycetous fungi, lichens and unicellular algae. In fungi, they are organized by BAR-domain containing proteins of the Pil1 family. Two such proteins, Pil1 and Lsp1, participate in eisosome formation in the yeast Saccharomyces cerevisiae. Under normal laboratory conditions, deletion of the PIL1 gene results in the inability of cells to assemble wild-type-like eisosomes. We found that under certain stress conditions, Lsp1 partially substitutes for the Pil1 function and mediates assembly of eisosomes, specifically following a decrease in the activity of serine palmitoyltransferase, for example, in response to hyperosmotic stress. Besides Lsp1, the assembly of eisosomes lacking Pil1 also requires Seg1 and Nce102 proteins. Using next-generation sequencing, we found that the seg1Δnce102Δpil1Δ strain, which is unable to form eisosomes, overexpresses genes coding for proteins of oxidative phosphorylation and tricarboxylic acid cycle. By contrast, genes involved in DNA repair, ribosome biogenesis and cell cycle are downregulated. Our results identify Lsp1 as a stress-responsive eisosome organizer and indicate several novel functional connections between the eisosome and essential cellular processes.

• Klíčová slova
• Eisosome, Lsp1, Membrane compartment of Can1, Pil1, Sphingolipid, Stress,
• MeSH
• buněčná membrána metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• LSP1 protein, S cerevisiae MeSH Prohlížeč
• membránové proteiny MeSH
• NCE102 protein, S cerevisiae MeSH Prohlížeč
• PIL1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny * MeSH

SART3 is a multifunctional protein that acts in several steps of gene expression, including assembly and recycling of the spliceosomal U4/U6 small nuclear ribonucleoprotein particle (snRNP). In this work, we provide evidence that SART3 associates via its N-terminal HAT domain with the 12S U2 snRNP. Further analysis showed that SART3 associates with the post-splicing complex containing U2 and U5 snRNP components. In addition, we observed an interaction between SART3 and the RNA helicase DHX15, which disassembles post-splicing complexes. Based on our data, we propose a model that SART3 associates via its N-terminal HAT domain with the post-splicing complex, where it interacts with U6 snRNA to protect it and to initiate U6 snRNA recycling before a next round of splicing.

• Klíčová slova
• Recycling, Splicing, U2 snRNP, U6 snRNA,
• MeSH
• malý jaderný ribonukleoprotein U2 genetika   metabolismus   MeSH
• malý jaderný ribonukleoprotein U4-U6 genetika   metabolismus   MeSH
• malý jaderný ribonukleoprotein U5 genetika   metabolismus   MeSH
• ribonukleoproteiny malé jaderné genetika   metabolismus   MeSH
• RNA malá jaderná genetika   metabolismus   MeSH
• sestřih RNA * genetika   MeSH
• spliceozomy * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U2 MeSH
• malý jaderný ribonukleoprotein U4-U6 MeSH
• malý jaderný ribonukleoprotein U5 MeSH
• ribonukleoproteiny malé jaderné MeSH
• RNA malá jaderná MeSH

Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found in a widely-used human EGFP-coilin construct that has a dominant-negative effect on Cajal body formation. We show that this coilin-K496E variant fails to rescue Cajal bodies in cells lacking endogenous coilin, whereas the wild-type construct restores Cajal bodies in mouse and human coilin-knockout cells. In cells containing endogenous coilin, both the wild-type and K496E variant proteins accumulate in Cajal bodies. However, high-level overexpression of coilin-K496E causes Cajal body disintegration. Thus, a mutation in the C-terminal region of human coilin can disrupt Cajal body assembly. Caution should be used when interpreting data from coilin plasmids that are derived from this variant (currently deposited at Addgene).

• Klíčová slova
• Cajal bodies, Coilin, Mutation,
• MeSH
• bodová mutace * genetika   MeSH
• Cajalova tělíska * genetika   MeSH
• HeLa buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mutace genetika   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• jaderné proteiny MeSH