Biology subjects

Janke, C.

Publications and source records attributed to Janke, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Surface-induced tau condensation generates a selective microenvironment around microtubules

Tau is a neuron-specific microtubule-associated protein that can self-associate into pathological insoluble aggregates or phase separate into condensates whose (patho)physiological role is debated. Recent studies suggest that intracellular surfaces can locally promote biomolecular condensation, even at low molecular concentrations. While microtubules in neurons provide an abundant tau-interaction surface, their role in tau phase separation remains unclear. Through a dialogue between experiments and theory, we demonstrate that tau forms multilayered condensates on microtubules at physiological concentrations via a prewetting-like transition. Concomitant tau-microtubule and tau-tau interactions explain the experimentally observed cooperative binding of the innermost tau layer directly adsorbed to the microtubule. The formation of this layer is dictated by the spacing of tubulin dimers within the microtubule lattice. Additional tau layers, driven by tau-tau interactions and independent of lattice spacing, are finite in thickness and unstable away from the microtubule surface. While the microtubule-adsorbed tau can selectively restrict proteins from the microtubule surface, the multilayered tau condensates can recruit tau interactors, such as RNA or soluble tubulin, highlighting the distinct roles of the condensate layers. Our results suggest that a prewetting-like transition constitutes a general physical mechanism for organizing liquid-like biomolecular layers of defined composition on charged intracellular surfaces.

biophysics↗

bioRxiv2026

bgnorm: A Generative Statistical Framework for Background Correction, Normalisation, and Quality Control in Multiplex Spatial Proteomics

Multiplex spatial proteomics enables highly multiplexed in situ profiling but remains limited by technical variation arising from autofluorescence, non-specific antibody binding, instrument noise, and staining variability, affecting downstream biological tasks like cell typing. We present bgnorm, a statistical framework that describes fluorescence measurements using a generative mixture model of background, non-specific binding, and biological signal components. As natural statistical consequences, the model yielded three new methods: a background-correction method through probabilistic deconvolution of protein intensities, quality control metrics, and a quantile normalisation approach to unify measurements across markers, samples, and sequential slices. Across multiple multiplex imaging technologies, bgnorm improves signal separation and downstream marker positivity classification compared with existing preprocessing approaches. In expert-annotated datasets comprising over 406,000 marker positivity annotations, bgnorm achieved the highest classification performance and enabled accurate use of a single global positivity threshold across markers and samples. The method is implemented in the bgnormR and bgnormpy packages.

bioinformatics↗

bioRxiv2026

Reduced tubulin polyglutamylation perturbs neuronal function and causes late-onset neurodegeneration

Microtubules are essential for neuronal functions and are involved in many neurodegenerative disorders. A posttranslational modification (PTM) of neuronal microtubules, polyglutamylation, was shown to control cargo transport, Tau protein binding to microtubules and to induce early-onset neurodegeneration when exceeding physiological levels. To determine the physiological importance of this PTM, we characterised mouse models with reduced polyglutamylation. We demonstrate that these mice develop late-onset atrophy of the olfactory bulb and frontal cortex, progressively reduced dendritic spine density, and deficits in olfaction, memory, and cognitive control. This indicates an important role of polyglutamylation in neuronal function. To test whether the observed defects are linked to neurodegeneration, we combined mice with reduced polyglutamylation with a transgenic model for tauopathy. Strikingly, reduced polyglutamylation accelerated the premature demise of mice transgenic for human Tau carrying the P301S mutation. Our work thus reveals key roles of microtubule polyglutamylation in neuronal function with implications for late-onset neurodegeneration linked to Tau pathology.

neuroscience↗

bioRxiv2026

Loss of Tubulin Tyrosination in Purkinje Neurons Does Not Cause Their Degeneration

Posttranslational modifications (PTMs) of tubulin have been suggested to form a tubulin code to coordinate microtubule functions. While at the molecular level PTMs were demonstrated to act in a target-specific manner, an outstanding question is the selectivity with which they control cellular and physiological functions. For instance, several tubulin PTMs have been linked to neurodegeneration, suggesting that whichever PTM is dysregulated could be sufficient to induce microtubule perturbation and cause neuronal death. Here we test whether perturbation of tyrosination can cause degeneration of Purkinje neurons similar to what happens when the PTM polyglutamylation is abnormally accumulated. Strikingly, this is not the case: deletion of tubulin tyrosine ligase in Purkinje cells, while resulting in loss of tyrosinated tubulin in these neurons, does not affect their survival nor function for more than one year. Our results thus reveal a highly specific role of polyglutamylation in neuronal homeostasis that is not replicated by other tubulin PTMs, suggesting stringent functional specialisation of those PTMs at the physiological level.

neuroscience↗

bioRxiv2026

Coupling of tubulin acetylation to microtubule stabilization through molecular mimicry

Microtubules support diverse cellular functions through regulation by microtubule-associated proteins and tubulin post-translational modification, yet how these two layers are mechanistically integrated remains unclear. -tubulin acetylation marks mechanically resilient microtubules, and its incorporation in defined microtubule sub-populations is not well understood. Here, we identify MTCL1 as a molecular link between microtubule stabilization and post-translational modification installation. We find that MTCL1 stabilizes microtubules and alters the luminal surface when copolymerized with tubulin, remodeling -tubulin and enhancing TAT-mediated tubulin acetylation through molecular mimicry. This effect depends on assembly history and is not observed in pre-assembled microtubules. Targeted deletion of MTCL1 in zebrafish impacts axonal organization, leading to motor defects and increased seizure susceptibility. These findings establish MTCL1 as a licensing factor that couples microtubule stabilization with acetylation to regulate neuronal function.

biophysics↗

bioRxiv2026

Cytosolic factors govern vimentin network architecture and mechanics

Vimentin intermediate filaments are key cytoskeletal components forming networks with architectures distinct from other intermediate filament types, enabling specialized functions. Although assembly of individual filaments from soluble subunits is well characterized, dissecting how vimentin networks are organized has been challenging, as existing in vitro systems do not mimic the structures observed in cells. Thus, how cells establish higher-order vimentin organization remains unclear. Here, we reconstitute cell-like vimentin networks in vitro, using purified vimentin and extracts from mammalian and non-mammalian cells. Systematic variation of parameters reveals that cytosolic biomolecules, rather than intrinsic filament properties or generic ionic components, are the primary determinants of network architecture and mechanical behaviour. Importantly, network architecture is not universal but varies strongly according to the cell type from which the extract is derived, indicating that vimentin assemblies are tailored in a cell type-specific manner. Thus, this extract-based reconstitution system enables mechanistic dissection of intermediate filament regulation under near-native biochemical conditions, bridging the current gap between purified systems and the cellular environment. Our findings show that vimentin architecture and mechanics emerge primarily from cytosolic biomolecular factors that organize filaments into cell type-specific networks. These results establish cytosolic regulation as a central mechanism specifying intermediate filament network architecture and function.

cell biology↗

bioRxiv2025

Organelle distribution and protein translation defects in radial glia cells with mutations in Eml1

Cerebral cortical development is a finely regulated process, depending on key neuronal progenitors, giving rise to post-mitotic neurons that migrate to superficial regions in the developing brain. Disruptions can give rise to severe cortical malformations that are associated with intellectual disability and intractable epilepsy. Mutations in EML1/Eml1, coding for a microtubule (MT)-associated protein, lead to subcortical heterotopia (SH) in both humans and mice. In developing mutant mouse brains, ectopic apical radial glia (aRG) progenitors were found outside the normal proliferative ventricular zone (VZ). The normal role of Eml1 in these cells remains to be precisely determined. Using proteomics and imaging approaches, we compared brain tissue from control and mutant mice and identified microtubule and organelle dysregulation. Eml1 influences the trafficking of organelles to different compartments of aRG, where they may be involved in local functions. Thus, we find that mitochondrial and polysome distribution are altered in mutant conditions. Indeed, Eml1 interacts with translational machinery, and an overall downregulation of translation is observed when analysing the translatome of Eml1 mutant aRG. Paradoxically, an upregulation of translation-related mRNAs is also observed. These new data uncover an unprecedented function of Eml1, as well as associating novel subcellular patho-mechanisms with severe heterotopia formation.

neuroscience↗

bioRxiv2025

TTLL6-mediated Polyglutamylation of PurA Maintains Colonic Crypt Integrity

The tubulin tyrosine ligase-like (TTLL) family comprises enzymes catalyzing posttranslational modifications of tubulin, including glutamylation and glycylation. We previously described a critical role for the monoglycylase TTLL3 in colon. Here, we identified TTLL6 as the predominant polyglutamylase in the colon, specifically expressed in epithelial cells of distal and transverse segments. TTLL6 expression decreases during CRC progression, correlating with poor patient prognosis. Deletion of Ttll6 in mice resulted in elongated colonic crypts, expansion of stem and transit-amplifying compartments, and increased numbers of differentiated epithelial cells. Moreover, Ttll6-deficient mice showed an elevated susceptibility to chemically induced colon carcinogenesis. Notably, we identified the nucleic acid-binding protein PurA as a novel TTLL6 substrate with both proteins mutually required for nuclear localization. Consistently, both nuclear polyglutamylation and PurA were present in the bottom compartment of control colons, but reduced in Ttll6-deficient colons. These findings reveal a TTLL6-PurA axis being critical in maintaining colonic homeostasis.

cell biology↗

bioRxiv2025

Tubulin isotypes polymerise into sectioned microtubules that locally regulate protein binding

Microtubules assemble from tubulin heterodimers composed of conserved - and {beta}- tubulins. In many species, including humans, tubulins are expressed from multiple genes. While the resulting tubulin isotypes show only subtle sequence differences, microtubules made of distinct isotypes can differ in their dynamic behaviour, as well as in their structure, for example in the number of their protofilaments. In cells, tubulin isotypes co-polymerize into mixed-isotype microtubules. How the mixing of tubulin isotypes affects microtubule functionality is unknown. Here we show that co-polymerization of recombinant tubulin dimers containing two different human {beta}-tubulin isotypes, 1{beta}3 and 1{beta}4, generates sectioned microtubules in which tubulin content and protofilament number differ from one section to the next. We demonstrate that two microtubule-associated proteins (MAPs), TPPP1 and optineurin (OPTN), bind differentially to these sections. Microtubules grown from natively mixed-isotype sources, either HeLa cells or mammalian brain tissue, also consist of sections that are differentially recognized by TPPP1 and OPTN, suggestive of isotype sorting. Our results demonstrate that co-polymerization of multiple tubulin isotypes can drive microtubules to assemble into distinct sections that locally regulate the interactions of MAPs with the microtubule lattice.

cell biology↗

bioRxiv2025

Cell-free Reconstitution Reveals Synergistic Stabilization of Microtubule Doublets by PACRG and FAP20

Ciliary microtubule doublets, consisting of an incomplete B-tubule on the surface of a complete A-microtubule, form the fundamental structural framework of motile and sensory cilia/flagella. Ciliary proteins PACRG and FAP20, which are essential for flagellar stability and function, localize to the junction between the A-and the B-tubule. However, their precise role in microtubule doublet assembly and dynamics remains unknown. Here, using a cell-free assay, TIRF-microscopy, and cryo-electron tomography, we demonstrate that in combination PACRG and FAP20 stabilize B-tubule dynamics and microtubule doublet architecture. We show that together these proteins localize to the B-tubules in distinct, high-density patches, which locally stabilize B-tubule by decreasing its depolymerization velocity and increasing its rescue frequency. Cryo-tomography of in vitro reconstructed microtubule doublets in presence of PACRG and FAP20 reveals reduced B-tubule curvature fluctuations, promoting a more rigid and aligned conformation. Altogether, our findings suggest that PACRG and FAP20 function synergistically to reinforce microtubule doublet stability, ensuring ciliary integrity and function.

cell biology↗

bioRxiv2025

Microtubule remodeling by the innate immune factor Trim69 compromises dynein-dependent migration of HIV virion cores towards the nucleus

Like many viruses, HIV relies on the microtubule (MT) cytoskeleton for successful infection. MT-associated proteins (MAPs) regulate MT functions and thus bear the potential to modulate viral infection. However, while several MAPs are known to exert pro-viral effects on HIV, little is known about antiviral ones. We previously described the Tripartite motif protein 69 (Trim69) as an innate immune factor that remodels MTs, leading to inhibition of a broad spectrum of viruses, including HIV. Through in silico modeling, TIRF microscopy and cell-based assays, we determine that Trim69 binding to MTs is determined by a basic surface in its SPRY domain that interacts with the C-terminal tails of tubulins. This surface is conserved among mammalian Trim69s and is critical for its functions. We demonstrate that by binding and remodeling MTs, Trim69 inhibits the docking and the migration of virion cores on MTs by promoting the stalling of the dynein/dynactin motor complexes. Altogether, these findings shed light on a novel mechanism of viral defense that involves an innate immune regulation of the MT cytoskeleton.

cell biology↗

bioRxiv2025

Mass spectrometry reveals novel features of tubulin polyglutamylation in the flagellum of Trypanosoma brucei

Tubulin polyglutamylation is a prominent feature of eukaryotic cilia and flagella. In the protist parasite Trypanosoma brucei, nine different tubulin tyrosine ligase-like (TTLL) enzymes are potentially involved in tubulin glutamylation. Given this enzymatic diversity, it is important to qualitatively and quantitatively characterize tubulin polyglutamylation, in order to understand the trypanosome tubulin code and pave the way for functional assignments of different TTLLs. Robust mass spectrometry (MS) based proteomics approaches for analysis of posttranslational modifications have been developed in recent years, but none to study polyglutamylation. We therefore optimized a nanoLC-MS/MS pipeline, from sample preparation to data analysis, using synthetic polyglutamylated peptides for quantification. Applied to the flagellum of T. brucei, our approach enabled the detection and quantification of C-terminal tubulin peptides with up to eleven supplementary glutamates on -, and five on {beta}-tubulin. In addition to the known E445 on - and E435 on {beta}-, a novel glutamylation site of {beta}-tubulin was discovered at residue E438. Furthermore, our data revealed an increase in enzymatic detyrosination with increasing length of the glutamate chains, especially for -tubulin. This indicates crosstalk between the modifications and different rates of detyrosination of the two tubulin types. Our results complement the existing knowledge of the tubulin code in T. brucei and introduce an efficient analytical pipeline for the characterization of polyglutamylated proteins.

biochemistry↗

bioRxiv2025

Prolyl isomerase FKBP12 reduces axon growth and negatively regulates microtubule polymerization by inhibiting CRMP2A

Prolyl isomerases are enzymes catalyzing conformational change of the peptide bond between proline and the preceding amino acid, regulating the function and stability of their substrates. We have previously identified CRMP2A - the longer isoform of a microtubule-associated protein Collapsin response mediator protein 2 - as a substrate of the phospho-specific prolyl isomerase Pin1. CRMP2A is negatively regulated and destabilized by CDK5 phosphorylation in the distal axons and growth cones. Pin1 specifically binds to phosphorylated CRMP2A and stabilizes it by inducing conformational changes. However, the conformational regulation of unphosphorylated CRMP2 remains unknown. Here, we show that the prolyl isomerase FKBP12 specifically binds to unphosphorylated CRMP2A and regulates its activity. Using in vitro microtubule polymerization assays we demonstrate that CRMP2A promotes microtubule growth and that this function is inhibited by FKBP12. Next, using GFP-EB3 microtubule plus-end tracking assay, we demonstrate that FKBP12 inhibits CRMP2A-mediated microtubule polymerization also in cells. Furthermore, we show that FKBP12 co-localizes with unphosphorylated CRMP2A in growth cones and that expression of FKBP12 reduces axon growth in microfluidic chambers, while FKBP12 knockdown enhances it. Together, we demonstrate that FKBP12 is a negative regulator of microtubule dynamics and axon growth. Moreover, we show that two prolyl isomerases can differentially (positively or negatively) regulate activity of a common substrate depending on its phosphorylation. This provides an additional layer of phosphorylation-dependent or -independent control of protein activity, microtubule dynamics, and neuronal growth. Given the broad substrate specificity of FKBP12 and Pin1, this regulatory mechanism likely contributes to the modulation of diverse proteins and cellular processes in the nervous system and beyond.

neuroscience↗

bioRxiv2024

Multivariate Trait Evolution: Models for the Evolution of the Quantitative Genetic G-Matrix on Phylogenies.

Genetic covariance matrices (G-matrices) are a key focus for research and predictions from quantitative genetic evolutionary models of multiple traits. There is a consensus among quantitative geneticists that the G-matrix can evolve through deep time. Yet, quantitative genetic models for the evolution of the G-matrix are conspicuously lacking. In contrast, the field of macroevolution has several stochastic models for univariate traits evolving on phylogenies. However, despite much research into multivariate phylogenetic comparative methods, analytical models of how multivariate trait matrices might evolve on phylogenies have not been considered. Here we show how three analytical models for the evolution of matrices and multivariate traits on phylogenies, based on Lie group theory, Riemannian geometry and stochastic differential (diffusion) equations, can be combined to unify quantitative genetics and macroevolutionary theory in a coherent mathematical framework. The models provide a basis for understanding how G-matrices might evolve on phylogenies, and we show how to fit models to data via simulation using Approximate Bayesian Computation. Such models can be used to generate and test hypotheses about the evolution of genetic variances and covariances, together with the evolution of the traits themselves, and how these might vary across a phylogeny. This unification of macroevolutionary theory and quantitative genetics is an important advance in the study of phenotypes, allowing for the construction of a synthetic quantitative theory of the evolution of species and multivariate traits over deep time. Lay SummaryWe unite Quantitative Genetics, the major mathematical theory of multivariate quantitative trait microevolution, with the mathematical theory of multivariate macroevolution. To do this, we allow the key component of quantitative genetic theory, the matrix of additive genetic variances and covariances (the G-matrix) to evolve along evolutionary trees. This is an advance because the G-matrix is assumed to be constant in quantitative genetics (for convenience), but it has been recognised that it evolves on macroevolutionary timescales (in deep time). Uniting Quantitative Genetics with macroevolutionary theory allows for a more complete mathematical description of Darwins theory of evolution, and allows for further testing of evolutionary hypotheses.

evolutionary biology↗

bioRxiv2024

Glutamylation imbalance leads to photoreceptor degeneration

The stereotypic structure of microtubules, assembled from conserved /{beta}-tubulin dimers is subject to a complex diversity of Post-translational Modifications (PTMs). PTMs are predicted to fine-tune microtubule properties and interactions with other proteins, thus allowing microtubules to perform specific functions. Cilia accumulate several types of tubulin PTMs, such as polyglutamylation, polyglycylation, detyrosination and acetylation, whose functions are not yet fully understood. Recently, mutations of AGBL5, coding for the deglutamylating enzyme CCP5, have been associated to retinitis pigmentosa, suggesting that perturbation of polyglutamylation leads to the degeneration of photoreceptor cells. However, the molecular mechanisms underlying this degeneration remain unknown. Here, using super-resolution Ultrastructure Expansion Microscopy in mouse and human photoreceptor cells, we found that most tubulin PTMs are accumulated at the level of the connecting cilium, a structure linking the outer and inner segments of photoreceptor cells. Using mouse models with increased glutamylation (Ccp5-/- and Ccp1-/-), or loss of tubulin acetylation (Atat1-/-), we demonstrated that aberrant glutamylation, but not loss of acetylation, resulted in perturbed molecular architecture of the outer segment, with the loss of the bulge region and destabilization of the distal axoneme. Concurrently, we observed a substantial impairment in tubulin glycylation and intraflagellar transport. Altogether our results indicate that glutamylation plays a crucial role in the maintenance of the molecular architecture of the outer segment and point to tubulin PTM imbalance as possible culprit in retinal degeneration.

cell biology↗

bioRxiv2024

Tau phosphorylation impedes functionality of protective tau envelopes

Tau, an axonal microtubule-associated protein, is a critical regulator of microtubule function and stability. Tau interaction with microtubules is regulated by tau phosphorylation. Tau hyperphosphorylation is implicated in microtubule destabilization related to neurodegenerative disorders. How tau phosphorylation leads to microtubule destabilization is however unknown. Recently, it was shown that tau molecules on microtubules cooperatively assemble into cohesive layers termed envelopes. Tau envelopes protect microtubules against degradation by microtubule-severing enzymes, suggesting a functional link between envelopes and microtubule stability. Here we show that tau phosphorylation has deleterious effects on the microtubule-protective function of tau envelopes. Using reconstitution and live-cell experiments, we found that tau phosphorylation destabilizes tau envelopes and decreases their integrity, leading to reduced microtubule protection against microtubule-severing enzymes. Our data suggest that a perturbation of microtubule homeostasis linked to tau hyperphosphorylation in neurodegeneration, could be explained by the disassembly and impaired functionality of the tau envelopes.

cell biology↗

bioRxiv2024

Polyglutamylation of microtubules drives neuronal remodeling

Developmental remodeling shapes neural circuits via activity-dependent pruning of synapses and axons. The cytoskeleton is critical for this process, as microtubule loss via enzymatic severing is an early step of pruning across many circuits and species. However, how microtubule-severing enzymes, such as spastin, are activated in specific neuronal compartments remains unknown. Here, we reveal that polyglutamylation, a posttranslational tubulin modification that is enriched in neurons, plays an instructive role in developmental remodeling by tagging microtubules for severing. Motor neuron-specific gene deletion of enzymes that add or remove tubulin polyglutamylation--TTLL glutamylases vs. CCP deglutamylases--accelerates or delays neuromuscular synapse remodeling in a neurotransmission-dependent manner. This mechanism is not specific to peripheral synapses but also operates in central circuits, e.g., the hippocampus. Thus, tubulin polyglutamylation acts as an activity-dependent rheostat of remodeling and shapes neuronal morphology and connectivity.

cell biology↗

bioRxiv2023

A new mechanism of posttranslational polyglutamylation regulates phase separation and signaling of the Wnt pathway protein Dishevelled.

Polyglutamylation is a reversible post-translational modification that is catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Disheveled 3 (DVL3), thereby changing the interactome of DVL3, as well as it increases its capacity to get phosphorylated, to undergo liquid-liquid phase separation (LLPS), and to act in the non-canonical Wnt pathway. Both carboxyterminal polyglutamylation and the resulting reduction in LLPS capacity of DVL3 were reverted by the deglutamylating enzyme CCP6, which demonstrates the causal relationship between TTLL11-mediated polyglutamylation and LLPS. We thus discovered a novel type of posttranslational modification, which significantly broadens the range of proteins that can be modified by polyglutamylation and provides the first evidence that polyglutamylation can act as a regulator of protein LLPS.

cell biology↗