Biology subjects

Fort, C.

Publications and source records attributed to Fort, C..

8 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

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

Axonemal dynein contributions to flagellar beat types and waveforms

Eukaryotic flagella, or motile cilia, are iconic molecular machines whose beating drives cell propulsion and fluid transport across diverse organisms. Beat type and waveform are tailored to function, differing between species and cell types, and individual flagella can switch between beat types. Aberrant beating causes ciliopathies and infertility in humans1 and prevents unicellular parasite transmission2. Eight distinct dynein motor protein complexes bind to axonemal doublet microtubules (DMTs) within flagella and drive beating, yet despite extensive structural analysis3-5, how this machinery achieves different beat types is unknown. Here, using the flagellate unicellular parasite Leishmania, we show a division of labour where specific dyneins drive specific beat types. Using cryo-EM, we determined the structure of the 96-nm repeat unit of the DMT and identified its dynein composition. We used CRISPR-Cas9 to systematically delete all 96-nm repeat proteins, comprehensively mapping necessity for swimming, and determined the contribution of each dynein to incidence and waveform of the preferred beat types. Outer dynein arms (ODAs) were required for symmetric tip-to-base beats, specific single-headed inner dynein arms (IDAs) were important for asymmetric base-to-tip beats (IDAd), and double-headed IDAf important for both. This systematic analysis indicates that the prevailing dogma that ODAs drive and IDAs shape the beat6-9 is either incomplete or not universal, and establishes new hypotheses for how different species, cell types and individual flagella achieve their necessary beat types.

biophysics↗

bioRxiv2024

Intraflagellar Transport Selectivity Occurs with the Proximal Portion of the Trypanosome Flagellum

Intraflagellar transport (IFT) trains move bidirectionally along the doublet microtubules (DMTs) of the axoneme within the flagellum. In Trypanosoma brucei, IFT trains predominantly associate with four of the nine DMTs. Using high-resolution microscopy, we reveal how this selective association is put in place. IFT proteins form a ring surrounding the 9 DMTs on top of the transition fibres. Volume electron microscopy revealed densities along all DMTs in the proximal portion of the flagellum, exhibiting thinner, shorter profiles with branches absent in mature IFT trains. As the axoneme extends within the flagellar pocket, IFT trains are detected but are often positioned outside DMTs 3-4/7-8. After the axoneme exits the flagellar pocket, IFT trains localise exclusively to DMTs 3-4 and 7-8. Super-resolution and expansion microscopy demonstrated that IFT proteins follow the same distribution as the IFT-like densities. This suggests they represent IFT trains undergoing assembly and/or disassembly and reveals their unexpected ability to shift from one DMT to another. SummaryIn Trypanosoma brucei, intraflagellar transport (IFT) trains selectively associate with specific axonemal microtubules. Using advanced microscopy, this study reveals how this restriction occurs at the proximal portion of the flagellum during the assembly and/or disassembly of IFT trains.

cell biology↗

bioRxiv2024

Proteins with proximal-distal asymmetries in axoneme localisation control flagellum beat frequency

The 9+2 microtubule-based axoneme within motile flagella is well known for its symmetry. However, examples of asymmetric structures and proteins asymmetrically positioned within the 9+2 axoneme architecture have been identified in multiple different organisms, particularly involving the inner or outer dynein arms, with a range of functions. Here, mapped, genome-wide, conserved proximal-distal asymmetries in the uniflagellate trypanosomatid eukaryotic parasites. Building on the genome-wide localisation screen in Trypanosoma brucei we identified conserved proteins with an analogous asymmetric localisation in the related parasite Leishmania mexicana. Using deletion mutants, we map which are necessary for normal cell swimming, flagellum beat parameters and axoneme ultrastructure, and using combinatorial endogenous fluorescent tagging and deletion, map co-dependencies for assembly into their normal asymmetric localisation. This revealed 15 proteins, 8 known and 7 novels, with a conserved proximal or distal axoneme-specific localisation. Most were outer dynein arm associated, and showed that there are at least two distinct classes of proximal-distal asymmetry - one dependent on the docking complex, and one independent. Many were necessary for normal frequency of the tip-to-base symmetric flagellar waveform, and our comprehensive mapping reveals unexpected contribution of proximal-specific axoneme components to frequency of distal waveform initiation.

cell biology↗

bioRxiv2021

Concentration of intraflagellar transport proteins at the ciliary base is required for proper train injection

Construction of cilia and flagella relies on Intraflagellar Transport (IFT). Although IFT proteins can be found in multiple locations in the cell, transport has only been reported along the axoneme. Here, we reveal that IFT concentration at the base of the flagellum of Trypanosoma brucei is required for proper assembly of IFT trains. Using live cell imaging at high resolution and direct optical nanoscopy with axially localized detection (DONALD) of fixed trypanosomes, we demonstrate that IFT proteins are localised around the 9 doublet microtubules of the proximal portion of the transition zone, just on top of the transition fibres. Super-resolution microscopy and photobleaching studies reveal that knockdown of the RP2 transition fibre protein results in reduced IFT protein concentration and turnover at the base of the flagellum. This in turn is accompanied by a 4- to 8-fold drop in the frequency of IFT train injection. We propose that the flagellum base provides a unique environment to assemble IFT trains.

cell biology↗

bioRxiv2020

Flagella Ca2+ elevations regulate pausing of retrograde intraflagellar transport trains in adherent Chlamydomonas flagella

The movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using IFT to move adhesive glycoproteins (FMG-1B) in the flagella membrane. Although Ca2+ signalling contributes directly to the gliding process, uncertainty remains over the mechanisms through which Ca2+ acts to influence the movement of IFT trains. Here we show that flagella Ca2+ elevations regulate IFT primarily by initiating the movement of paused retrograde IFT trains. Flagella Ca2+ elevations exhibit complex spatial and temporal properties, including high frequency repetitive Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. We show that flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane. The results suggest that flagella Ca2+ elevations directly disrupt the interaction between retrograde IFT particles and flagella membrane glycoproteins to modulate gliding motility and the adhesion of the flagellum to a surface.

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↗