Biology subjects

Stepanek, L.

Publications and source records attributed to Stepanek, L..

4 recordsLinked to original sources

bioRxiv2025

Kinesin-3 KIF14 Regulates Intraflagellar Transport Dynamics in Primary Cilia

The primary cilium is an antenna-like organelle that plays a crucial role in development and homeostasis. Its growth and functions depend on the transport of cargo from the cilia base to the tip by intraflagellar transport (IFT) proteins and kinesin-2 motors. Primary cilia exhibit great variation in morphology and function across cell types and organisms. This diversity is thought to be conferred by the modulation of IFT by additional factors. However, examples of such non-canonical regulators, such as kinesin motors distinct from kinesin-2, are sparse. Thus, the involvement of non-canonical ciliary kinesins in intraciliary transport is unclear. Here, we show that a poorly characterized member of the kinesin-3 family, KIF14, plays a prominent role in primary cilia trafficking in human cells. Using live cell imaging and expansion microscopy, we demonstrate that KIF14 depletion leads to impaired IFT. Furthermore, using TIRF microscopy we show that the motility of KIF14 and its effects on cilia trafficking rely on the motor domain of KIF14, which drives the processive runs along the ciliary axoneme in cells and in vitro, in co-operation with the C-terminal CC1 domain. Finally, we demonstrate that C-terminal truncations of KIF14, including patient mutation Q1380x, disrupt IFT by causing traffic jam-like accumulations of ciliary components in the distal part of the primary cilia, leading to bulged tips of cilia. In summary, our data exemplify the role of a non-canonical ciliary kinesin in the regulation of ciliary trafficking and suggest a new paradigm for how kinesin-related trafficking defects may contribute to the pathology of disorder with a ciliary contribution.

cell biology↗

bioRxiv2025

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

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.

cell biology↗

bioRxiv2024

Tubulin tyrosination/detyrosination governs the spatial segregation of intraflagellar transport trains on axonemal microtubule doublets

Assembly and function of cilia rely on the continuous transport of ciliary components between the cell body and the ciliary tip. This is performed by specialized molecular machines, known as Intraflagellar Transport (IFT) trains. Anterograde IFT trains are powered by kinesin-2 motors and move along the B-tubules (enriched in detyrosinated tubulin) of the ciliary microtubule doublets. Conversely, retrograde IFT trains are moved by dynein-1b motors along the A-tubules (enriched in tyrosinated tubulin) back to the cell body. The segregation of oppositely directed trains on A-tubules or B-tubules is thought to prevent traffic jams in the cilium, but the mechanism by which opposite polarity trains are sorted onto either tubule, and whether tubulin tyrosination/detyrosination plays a role in that process, is unknown. Here, we show that CRISPR-mediated knock-out of VashL, the enzyme that detyrosinates microtubules, causes recurrent stoppages of IFT trains and reduces the rate of ciliary growth in Chlamydomonas reinhardtii. To test whether the observed stoppages, potentially caused by collisions between oppositely directed IFT trains, are ascribable to direct interactions between IFT trains and tubulin tyrosination/detyrosination, we developed methods to reconstitute the motility of native IFT trains from cilia on de-membranated axonemes and ex vivo on synthetically polymerized microtubules. We show that anterograde trains have higher affinity for detyrosinated microtubules (analogous to B-tubules), while retrograde trains for tyrosinated microtubules (analogous to A-tubules). We conclude that tubulin tyrosination/detyrosination is required for the spatial segregation of oppositely directed trains and for their smooth uninterrupted motion. Our results provide a model for how the tubulin code governs molecular transport in cilia.

cell biology↗

bioRxiv2021

Intraflagellar transport trains can turn around without the ciliary tip complex

Cilia and flagella are microtubule doublet based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavourable to diffusive transport for their assembly and maintenance. Instead, a highly dynamic system of intraflagellar transport (IFT) trains moves rapidly up and down the cilium. However, the mechanism of how these trains turn around upon reaching the ciliary tip has remained elusive. It has been hypothesized that there exists a dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate this conversion. In this work, we use physical and chemical methods to manipulate IFT in the cilia of the unicellular green alga Chlamydomonas reinhardtii to show that no such stationary tip-machinery is required for IFT turnaround. Instead, we demonstrate that the conversion from anterograde to retrograde IFT trains is a calcium independent intrinsic ability of the IFT system.

cell biology↗