Biology subjects

Blisnick, T.

Publications and source records attributed to Blisnick, T..

9 recordsLinked to original sources

bioRxiv2026

The IFT-A complex plays a major role in the assembly of anterograde intraflagellar transport trains

Cilia are assembled by intraflagellar transport (IFT), which relies on two protein complexes: IFT-A and IFT-B. It is generally assumed that IFT-B and IFT-A are critical for anterograde and retrograde transport, respectively. However, full deletion of IFT-A genes in several organisms suggests a possible contribution to anterograde transport. In many species, cilia collapse when IFT is altered, hindering functional studies. Here, we investigated the role of IFT-A in the protist Trypanosoma brucei, where IFT is not required for cilium maintenance. Following the inducible knockdown of IFT88 (an IFT-B member) or IFT140 (an IFT-A member), we monitored the fate of several IFT proteins in preassembled cilia using live imaging and evaluated the consequences on train formation by volumetric electron microscopy. Surprisingly, both IFT88 and IFT140 turned out to be essential for anterograde train assembly. Their depletion initially led to the formation of shorter trains and subsequently to an inhibition of train injection. We propose a model to reconcile the diverging phenotypes reported in the literature.

cell biology↗

bioRxiv2026

Investigating the axoneme CCDC40 protein reveals new insights in trypanosome morphogenesis and division

Cilia and flagella contribute to cell morphogenesis in multiple organisms. In the parasite Trypanosoma brucei, the flagellum is attached along the length of the cell body and acts as a guide for cell division, while its motility function is required for the completion of cytokinesis. To tease apart the contributions of flagellum length and motility to trypanosome morphogenesis, we investigated the coiled-coil containing domain 40 (CCDC40 or FAP172) protein. Iterative Ultrastructure Expansion Microscopy (iU-ExM) revealed that CCDC40 is associated to the 96-nm repeats of the trypanosome axoneme. CCDC40 depletion by RNAi leads to loss of components from the dynein regulatory complex, inner dynein arms and radial spokes, resulting in disconnected microtubule doublets and disorganised axoneme structure, abrogating motility and resulting in flagella and cell bodies 2-3 times shorter than normal. We show for the first time that this short flagellum phenotype is associated to slower tubulin incorporation and premature acquisition of the maturation marker FLAM8 but not of the locking protein CEP164C. Surprisingly, these short and immotile trypanosomes grow and divide normally. We discuss the significance of these observations for trypanosome morphogenesis and division.

cell biology↗

bioRxiv2026

Life without heterotrimeric kinesins: trypanosomatids use a combination of homodimeric kinesin-2 motors to drive intraflagellar transport

Heterotrimeric kinesin 2 is the canonical motor protein for anterograde intraflagellar transport (IFT), driving movement of protein complexes towards the tip of cilia and flagella. Here, we show that all members of the Euglenozoa group lack genes for heterotrimeric kinesins and instead possess a variable number of genes for two homodimeric kinesins termed KIN2A and KIN2B. When expressed in vitro, both Trypanosoma brucei kinesins form homodimers and move processively along brain microtubules, KIN2A being faster than KIN2B. Studies in T. brucei and Leishmania mexicana show anterograde and retrograde IFT of both kinesins, with KIN2A travelling throughout the whole length of the flagellum, while KIN2B is concentrated at its base. In the proximal portion of the flagellum, most KIN2B molecules travel without IFT proteins, except for a few particles that are associated with IFT proteins and reach the tip. Surprisingly, the absence of KIN2A has mild effects on IFT and flagellum assembly, whereas KIN2B is essential for both. Investigation of trypanosome flagella deprived of KIN2B revealed that IFT proteins do not access these flagella but that KIN2A can still circulate. We propose a division-of-labour model where KIN2B would be responsible for the import of IFT proteins while KIN2A would ensure most of the anterograde transport.

cell biology↗

bioRxiv2025

Wnt and Nodal asymmetries stratify mouse laterality phenotypes in the absence of node flow

Left-right symmetry breaking in mice is considered to occur via node leftward fluid flow. The molecular cascade within the node has been reconstructed at high resolution. However, its importance for organogenesis remains poorly understood. Here we show in mutants for the motile cilium component CCDC40 that 70% of mice develop normal situs at birth despite abrogation of node flow. The discrete morphospace output, including situs inversus totalis and heterotaxy with left isomerism, supports a novel model of symmetry-breaking, in which node flow only biases asymmetry orientation, while another mechanism, potentially self-amplifying, generates it. Longitudinal, quantitative and paired transcriptomic analyses uncover the molecular signature of laterality clusters, highlighting WNT in addition to NODAL pathway asymmetry. We identify asymmetry of cardiopulmonary progenitors, the disruption of which is associated with combined heart and lung defects in mutants. Our work refines the role of the node in the establishment of asymmetry.

developmental biology↗

bioRxiv2024

The ANKS3/BICC1 protein complex is a master post-transcriptional regulator of NPHP1 ciliopathy gene transcripts

Ciliopathies are a class of multi-systemic genetic diseases characterized by ciliary dysfunction. Here, we report a novel ANKS3 variant in patients with a renal ciliopathy known as nephronophthisis (NPH) associated with hepatic defects. ANKS3 is an ankyrin and sterile alpha motif domain-containing protein that interacts with many NPH proteins as well as with BICC1, an RNA-binding protein involved in renal cystic diseases. The pathogenic effect of the ANKS3 mutation was validated in the zebrafish mutant and knock-in rat model, the latter showing urine concentration defect and tubular dilatations similar to NPH patients. In addition, cilia morphology and function as well as epithelialization of kidney tubular cells was affected by loss or mutation of ANKS3. Finally, our results evidenced that these classically renal ciliopathy-associated phenotypes were linked to the negative regulation of BICC1 by ANKS3 which binds to transcripts of the major NPH gene NPHP1 and mediates their decay through the AGO2-RISC complex and recruitment into P-bodies. Altogether, our findings suggest that the ANKS3/BICC1 complex is a key post-transcriptional regulator of NPHP1 transcript stability, providing another level of regulation of cilium biogenesis and kidney homeostasis, as well as an unusual mechanism leading to NPH-related ciliopathies.

pathology↗

bioRxiv2020

Dynamic High-Content Imaging Reveals Surface Exposure Of Virulent Leishmania Amastigotes In Infected Macrophages Undergoing Pyroptosis

Leishmania spp are obligate intracellular parasites that infect vertebrate phagocytes, notably macrophages. We previously reported that Leishmania amazonensis (L. am) subvert the host cell pro-inflammatory response by dampening the macrophage NLRP3 inflammasome. No information is available on how Leishmania infection affects inflammatory cell death termed pyroptosis, known to limit microbial infection. Here, we provide first evidence that L. amazonensis-infected macrophages can undergo pyroptosis when subjected to pro-inflammatory stimuli. We analyzed the dynamics of the pyroptotic process and the fate of intracellular amastigotes at the single cell level using spinning disk confocal microscopy and high-content, real-time imaging. Bone marrow-derived macrophages (BMDMs) were infected with L. am amastigotes isolated from footpad lesions and sequentially treated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) for canonical NLRP3 inflammasome priming and activation. Real-time monitoring was performed for 240 min post ATP addition. Longitudinal analyses revealed distinct phases of the pyroptotic process, including rapid decay of the parasitophorous vacuole (PV) as monitored by the pH-sensitive lysotracker fluid phase marker, progressive decrease in macrophage viability as monitored by accumulation of the nuclear dye YO-PRO-1, followed by translocation of the luminal PV membrane to the cell surface observed for 40% of macrophages, resulting in the extracellular exposure of amastigotes that remained anchored to the PV membrane. Scanning and transmission electron microscopy analyses revealed a highly polarized orientation of parasites with exclusive exposure of the anterior pole toward the extracellular milieu, and an attachment site forming a potential biological junction between the parasite posterior pole and the PV membrane. We showed that the exposed parasites are resistant to the cytolytic host cell activities linked to pyroptosis and retain their full infectious potential in reinfection experiments using naive macrophages. Together these data uncover a novel Leishmania immune subversion strategy that may allow stealthy parasite dissemination via the uptake of pyroptotic host debris by uninfected phagocytes.

immunology↗

bioRxiv2018

IFT25 is required for the construction of the trypanosome flagellum

Intraflagellar transport (IFT), the movement of protein complexes responsible for the assembly of cilia and flagella, is remarkably well conserved from protists to humans. However, two IFT components (IFT25 and IFT27) are missing from multiple unrelated eukaryotic species. In mouse, IFT25 and IFT27 are not required for assembly of several cilia with the noticeable exception of the flagellum of spermatozoa. Here we show that the Trypanosoma brucei IFT25 protein is a proper component of the IFT-B complex and displays typical IFT trafficking. Using bimolecular fluorescence complementation assays, we reveal that IFT25 and IFT27 interact within the flagellum in live cells during the IFT transport process. IFT25-depleted cells construct tiny disorganised flagella that accumulate IFT-B proteins (with the exception of IFT27, the binding partner of IFT25) but not IFT-A proteins. This phenotype is comparable to the one following depletion of IFT27 and shows that IFT25/IFT27 constitute a specific module requested for proper IFT and flagellum construction in trypanosomes. We discuss the possible reasons why IFT25/IFT27 would be required for only some types of cilia.

cell biology↗

bioRxiv2018

Bidirectional intraflagellar transport is restricted to only two microtubule doublets in the trypanosome flagellum

Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. Here we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei. Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3-4 and 7-8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occur on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3-4 seconds while remaining on the same side of the axoneme.

cell biology↗

bioRxiv2018

Control of flagellum length by a grow-and-lock model

Several models have been proposed to explain how eukaryotic cells control the length of their cilia and flagella. Here, we investigated this process in the protist Trypanosoma brucei, an excellent system for cells with stable cilia like photoreceptors or spermatozoa. We show that the total amount of intraflagellar transport material (IFT, the machinery responsible for flagellum construction) increases during flagellum elongation, consistent with constant delivery of precursors and the previously reported linear growth. Reducing the IFT frequency by RNAi knockdown of the IFT kinesin motors slows down the elongation rate and results in the assembly of shorter flagella. These keep on elongating after cell division but fail to reach the normal length. This failure is neither due to an absence of precursors nor to a morphogenetic control by the cell body. We propose that the flagellum is locked after cell division, preventing further elongation or shortening. This is supported by the fact that subsequent increase in the IFT rate does not lead to further elongation. The distal tip FLAM8 protein was identified as a marker for the locking event. It is initiated prior cell division, leading to an arrest of elongation in the daughter cell. Here, we propose a new model termed grow-and-lock where the flagellum elongates until a locking event takes place in a timely defined manner hence fixing length. Alteration in the growth rate and/or in the timing of the locking event would lead to the formation of flagella of different lengths.

cell biology↗