Biology subjects

Cajanek, L.

Publications and source records attributed to Cajanek, L..

8 recordsLinked to original sources

bioRxiv2026

An Analog-Sensitive Tau Tubulin Kinase 2 Enables Acute Chemical-Genetic Control of Primary Cilia

Kinases regulate a broad spectrum of cellular processes, including the formation of primary cilia, microtubule-based organelles that protrude from the cell surface and play important roles in development and disease. TTBK2 is a key ciliary kinase that controls primary cilium assembly and maintenance. However, highly selective small-molecule inhibitors of TTBK2 are still lacking, representing a major bottleneck for investigating the temporal requirements and dynamics of TTBK2-dependent processes in cilia. Here, we report the first analog-sensitive variant of TTBK2, enabling rapid, reversible, and highly specific control of TTBK2 kinase activity in ciliogenesis-relevant hTERT-RPE1 cells. Using this system, we define the kinetics of CEP164 phosphorylation, cilium shortening and reassembly, and IFT88 retention at the ciliary base following acute TTBK2 inhibition and subsequent inhibitor washout. Together, our findings provide new insight into TTBK2-dependent pathways and establish analog-sensitive TTBK2 as a powerful tool for investigating the dynamics of TTBK2-regulated processes with high temporal resolution.

cell biology↗

bioRxiv2026

Development of Potent and Cell Active 5-Azaindole-Based Tau Tubulin Kinase Inhibitors

We have developed and characterized a potent and cell active tau tubulin kinase 1 and 2 (TTBK1 and TTBK2) inhibitor, 13. Compound 13 demonstrates in-cell, kinome-wide selectivity, and potently inhibits both TTBK1 and TTBK2. As part of our medicinal chemistry campaign, we also identified a structurally similar negative control, compound 5, which lacks in-cell affinity for TTBK1 and TTBK2. Based on their substrates, which include TDP-43, tau, and tubulin, TTBK1 and TTBK2 inhibition has been pursued as a therapeutic approach for Alzheimers disease, frontotemporal lobe dementia, and amyotrophic lateral sclerosis. TTBK2 is also an effector of ciliogenesis, acting in concert with CEP164, CP110, and CEP83 to initiate the biogenesis of primary cilia. The development of selective chemical tools for these kinases facilitates investigation into TTBK1/2-mediated pathways and potential disease-altering ramifications linked to their pharmacological perturbation.

cell biology↗

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↗

bioRxiv2023

Tau-Tubulin Kinase 2 restrains microtubule-depolymerizer KIF2A to support primary cilia growth.

The initiation of assembly of primary cilia, organelles with crucial functions in development and disease, is under the control of Tau-Tubulin Kinase 2 (TTBK2). Recent work has implicated TTBK2 also in the regulation of primary cilia maintenance and function. However, the mechanisms underlying individual functions of TTBK2 in primary cilia are not fully understood. Here, to dissect the role of TTBK2 in primary cilia maintenance in human cells, we examined disease related TTBK2 truncations. We demonstrate that these truncated protein moieties show selective activity towards TTBK2 substrates. This creates a semi-permissive condition where partial TTBK2 activity suffices to support the initiation of ciliogenesis but fails to sustain primary cilia length. Subsequently, we show that the defects in primary cilia growth are linked to aberrant turnover of kinesin KIF2A at basal body. Furthermore, we demonstrate that TTBK2 regulates KIF2A by phosphorylation, which in turn restrains its levels at the ciliary base to promote primary cilia elongation and maintenance. Taken together, our data highlight the regulation of KIF2A by TTBK2 as an important mechanism governing primary cilia in human cells.

cell biology↗

bioRxiv2023

Tau tubulin kinase 1 and 2 regulate ciliogenesis and human pluripotent stem cells-derived neural rosettes

Primary cilia are key regulators of embryo development and tissue homeostasis. However, their mechanisms and functions, particularly in the context of human cells, are still unclear. Here, we analyzed the consequences of primary cilia modulation for human pluripotent stem cells (hPSCs) proliferation and differentiation. We show that neither activation of the cilia-associated Hedgehog signaling pathway nor ablation of primary cilia by CRISPR gene editing to knockout Tau Tubulin Kinase 2 (TTBK2), a crucial ciliogenesis regulator, affects the selfrenewal of hPSCs. In addition, we demonstrate that TTBK1, a closely related kinase without previous links to ciliogenesis, is upregulated during hPSCs-derived neural rosette differentiation to regulate primary cilia formation together with TTBK2. Finally, we show that TTBK1/2 and primary cilia are implicated in the regulation of the size of hPSCs-derived neural rosettes.

cell biology↗

bioRxiv2020

Primary cilia formation does not rely on WNT/β-catenin signaling

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-{beta}. However gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/{beta}-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/{beta}-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast second model proposes a positive role of WNT/{beta}-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/{beta}-catenin pathway, {beta}-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/{beta}-catenin pathway in cell lines (RPE-1, NIH3T3, HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6 or AXIN1/2 we show that neither activation nor deactivation of the WNT/{beta}-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/{beta}-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/{beta}-catenin pathway in some systems seems to moderately suppress ciliogenesis.

cell biology↗

bioRxiv2019

Phosphorylation of multiple proteins involved in ciliogenesis by Tau Tubulin kinase 2

Primary cilia (PC) are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling final step of cilia initiation. The function of TTBK2 in ciliogenesisis is critically dependent on its kinase activity, however, precise mechanism of TTBK2 action is so far incompletely understood, due to very limited information about its relevant substrates. In this study we identify CEP83, CEP89, CCDC92, Rabin8 and DVL3 as substrates of TTBK2 kinase activity. Further, we characterise a set of phosphosites of the newly identified substrates and CEP164, induced by TTBK2 in vitro and in vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we address functional relevance of selected phosphorylations of CEP164 and provide evidence that the examined TTBK2-induced phosphorylations of CEP164 are relevant for the process of cilia formation. In summary, our work provides important insight into substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.

cell biology↗

bioRxiv2019

Comparative phosphorylation map of Dishevelled3 (DVL3)

In the presented study we analyze phosphorylation of human Dishevelled 3 (DVL3) induced by its previously reported (CK1{varepsilon}, NEK2, PLK1, CK2, RIPK4, PKC{delta}) and the newly identified (TTBK2, Aurora A) kinases. DVL3 contains 131 Ser/Thr whose phosphorylation generates complex barcodes underlying diverse DVL3 functions in Wnt pathways and other processes. We use quantitative mass spectrometry and via several complementary pipelines calculate site occupancies and quantify phosphorylation of >80 phosphorylated residues. In order to visualize the complex phosphorylation patterns, we design a novel visualization diagram, phosphoplot. Finally, we compare the individual sample and data processing approaches, identify their strengths and weaknesses. Subsequently, we verified a set of anti-phospho-DVL antibodies and were able to successfully confirm induction for several of the phosphorylation sites. From the biological point of view, our data represent an important reference point and a toolbox for further analysis of DVL functions and phosphorylation events that control them.\n\nOne sentence summaryThe study provides a comprehensive comparison of the phosphorylation of DVL3 induced by eight Ser/Thr kinases and identifies phosphorylation signatures associated with individual kinases.

cell biology↗