Biology subjects

Lansky, Z.

Publications and source records attributed to Lansky, Z..

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

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

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↗

bioRxiv2025

Taxane-Induced Conformational Changes in the Microtubule Lattice Activate GEF-H1-Dependent RhoA Signaling

Taxanes are widely used chemotherapeutic agents that perturb cell division. They also exert effects during interphase, but the underlying mechanisms are poorly understood. Here, we show that taxanes activate RhoA signaling and induce actin remodeling by displacing the RhoA activator GEF-H1 from microtubules. This taxane-induced release of GEF-H1 occurs rapidly, is independent of tubulin post-translational modifications, and can be recapitulated using purified proteins. In vitro reconstitution assays combined with analyses of microtubule structure revealed that microtubule binding by GEF-H1 is inhibited by microtubule-stabilizing agents that expand the microtubule lattice, such as taxanes and GMPCPP, but not by others, including GTP{gamma}S and discodermolide, which stabilize a compacted microtubule lattice. Our findings demonstrate that alterations in microtubule lattice conformation can activate key signaling pathways, offering new insights into the mode of action of taxanes and the possible origins of their side effects.

cell biology↗

bioRxiv2025

An atlas of microtubule lattice parameters regulated through ligand binding to the microtubule stabilizing sites.

Microtubules (MTs) are dynamic cytoskeletal polymers whose lattice architecture regulates force generation, nucleotide hydrolysis, and recognition by motor proteins and microtubule-associated proteins (MAPs). Microtubule-stabilizing agents (MSAs), including taxanes and laulimalide/peloruside-site ligands, suppress depolymerization by binding to defined lattice sites, yet stabilization is not structurally neutral. How ligand chemistry reshapes lattice organization and function remains unresolved. Here, we address three mechanistic questions. First, do distinct ligand classes induce defined lattice states? Using X-ray fiber diffraction, we show that MSAs selectively stabilize two preferred longitudinal conformations, a compact state ([~]4.06 nm monomer rise) and an expanded state ([~]4.17 nm), while modulating lateral organization reflected in shifts in mean MT radius. These axial spacings cluster around discrete values across chemotypes, indicating stabilization of pre-existing conformational minima rather than continuous distortion. Second, are these states interconvertible upon changes in ligand occupancy? Time-resolved diffraction reveals that longitudinal transitions occur within seconds of ligand addition even at substoichiometric occupancy; whereas, lateral equilibration proceeds slower, consistent with redistribution within heterogeneous protofilament organizations. Third, do such structural states alter nucleotide hydrolysis and motor/MAP behavior? Expanded lattices are associated with reduced apparent GTP hydrolysis rates under steady-state assembly conditions and altered kinesin motility, whereas compact lattices preferentially promote tau binding and distinct motor interaction profiles. Together, these findings establish longitudinal lattice conformation as a regulatory parameter and position microtubule-stabilizing agents as chemical tools that bias a dynamic structural landscape with predictable catalytic and transport consequences. Significance statementMicrotubules generate force and support intracellular transport through lattice geometries selectively recognized by motor proteins and microtubule-associated proteins. Microtubule-stabilizing drugs such as taxanes and epothilones are widely used in chemotherapy but can cause neurotoxicity, likely because stabilization alters lattice architecture rather than simply preventing depolymerization. Here, we show that stabilizing ligands bias microtubules between two preferred longitudinal conformations, compact and expanded, that can switch within seconds of binding, while lateral organization adjusts slower. These structural states differentially regulate GTP hydrolysis and recognition by tau and kinesin, linking lattice geometry to catalytic and transport functions. By establishing lattice conformation as a tunable regulatory parameter, this work provides a framework for interpreting drug effects and designing structure-selective stabilizers with improved therapeutic profiles.

biophysics↗

bioRxiv2025

Dissecting structural and functional determinants of microtubule stabilization through guided chemical modulation

Paclitaxel (PTX) is a widely used chemotherapeutic, but its efficacy is limited by peripheral neuropathy, likely due to its structural impact on neuronal microtubules (MTs). To decouple MT stabilization from adverse structural changes, we designed, synthetized and characterized several PTX analogues. Our compound 1b retains PTXs stabilizing activity in vitro and in cells, and the 1b-bound MTs preserve a native-like structure. These facts allowed us to investigate the influence of specific MT structural features on motor proteins behavior and interaction with MT-associated protein Tau. PTX-induced lattice expansion disrupted dynein-mediated retrograde transport and altered kinesin-1 motility. Additionally, PTX reduced Taus initial non-cooperative binding and envelopes growth rate while increasing dwell time and suppressing dynamic binding. In contrast, 1b preserved more physiological Tau dynamics. These findings reveal that MT stabilization and structural modulation can be separated and highlight the functional importance of MT heterogeneity in maintaining neuronal transport and MAP interactions.

molecular 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↗

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

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↗

bioRxiv2025

Anillin directly cross-links microtubules with filamentous actin

Complex morphogenetic processes such as cell division require a tight coordination of the activities of microtubules and actin filaments. There is evidence that anillin, conventionally known as an actin-binding and bundling protein, regulates microtubule-actin crosstalk during cell division. However, it is unknown whether anillin binds directly to microtubules and whether it is sufficient to establish crosslinking between microtubules and actin filaments. Here we address both questions by developing an in-vitro system to observe anillin-mediated interactions with actin filaments and dynamic microtubules using total internal reflection fluorescence microscopy. We find that anillin can interact directly with microtubules and promote microtubule bundling. We confirm that anillin binds and bundles actin filaments and find that it has a strong preference for actin bundles over individual filaments. Moreover, we show that anillin can directly crosslink microtubules and actin filaments, can cause sliding of actin filaments on the microtubule lattice, and can transport actin filaments by the growing microtubule tip. Our findings indicate that anillin can potentially serve as a direct regulator of microtubule-actin crosstalk, e.g. during cell division.

biophysics↗

bioRxiv2024

Optineurin-facilitated axonal mitochondria delivery promotes neuroprotection and axon regeneration

Optineurin (OPTN) mutations are linked to amyotrophic lateral sclerosis (ALS) and normal tension glaucoma (NTG), but a relevant animal model is lacking, and the molecular mechanisms underlying neurodegeneration are unknown. We found that OPTN C-terminus truncation (OPTN{Delta}C) causes late-onset neurodegeneration of retinal ganglion cells (RGCs), optic nerve (ON), and spinal cord motor neurons, preceded by a striking decrease of axonal mitochondria. Surprisingly, we discover that OPTN directly interacts with both microtubules and the mitochondrial transport complex TRAK1/KIF5B, stabilizing them for proper anterograde axonal mitochondrial transport, in a C- terminus dependent manner. Encouragingly, overexpressing OPTN/TRAK1/KIF5B reverses not only OPTN truncation-induced, but also ocular hypertension-induced neurodegeneration, and promotes striking ON regeneration. Therefore, in addition to generating new animal models for NTG and ALS, our results establish OPTN as a novel facilitator of the microtubule-dependent mitochondrial transport necessary for adequate axonal mitochondria delivery, and its loss as the likely molecular mechanism of neurodegeneration.

neuroscience↗

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↗

bioRxiv2023

A novel, RAS-independent role for NF1 in microtubular dynamics and damage repair dictates sensitivity to T-DM1 in HER2-positive breast cancer

Antibody-Drug Conjugates (ADC) have revolutionized the treatment of several tumors, and extensive research is being devoted to the identification of predictive biomarkers. These are particularly sought after in fields, like breast cancer, in which multiple ADCs with identical target but different payloads have been approved. NF1 is a tumor suppressor widely mutated across several cancers, best characterized as an inhibitor of RAS signaling. Additional functions have been proposed but not deeply investigated, due to its large size and complex domain structure. Whether somatic NF1 mutations can be used to guide clinical decisions is not known. Here, combining patient data, in vitro/in vivo models and protein biochemistry, we show that NF1 loss sensitizes cancer cells to T-DM1, the first approved ADC in breast cancer, through a novel, RAS-independent function on microtubular dynamics and repair. NF1 exhibits all biochemical properties of a bona fide Microtubule-Associated Protein (MAP) and specifically enhances intratubular repair, a recently discovered phenomenon whose regulation remains poorly characterized. NF1 loss results in mitotic defects and low-grade aneuploidy in cell lines and patients. Increased sensitivity to T-DM1 upon NF1 loss is confirmed in breast cancer patients analysed across institutions in Europe and USA. Our results define NF1 as a key regulatory factor for microtubular repair and the first ADC payload-associated predictive biomarker identified to date.

cancer biology↗

bioRxiv2023

MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement

Microtubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.

cell biology↗

bioRxiv2022

The Colon Mucosal Sialylglycome Is Redox-Regulated by the Golgi Enzyme QSOX1

Mucus shields the intestinal epithelium from pathogens and provides a supportive environment for commensal bacteria. Mucus is composed of enormous, heavily glycosylated proteins called mucins, which become disulfide crosslinked in a multi-step biosynthetic pathway culminating in the Golgi apparatus and secretory granules of goblet cells. We observed that knockout mice lacking the Golgi-localized disulfide catalyst QSOX1 produced poorly protective colon mucus, were hypersensitive to induced colitis, and had an altered microbiome. The initial hypothesis arising from these observations was that QSOX1 catalyzes disulfide crosslinking of mucins. Contrary to this hypothesis, the disulfide-mediated polymerization of mucins and related glycoproteins proceeded normally without QSOX1. Instead, we found that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases and thereby promotes effective sialylation of the colon glycome. Our findings reveal that enzymatic control of Golgi redox state impacts glycan elaboration in goblet cells, and that this pathway is crucial for maintaining mucosal function.

cell biology↗

bioRxiv2021

Microtubule Lattice Spacing Governs Cohesive Envelope Formation of Tau Family Proteins

Tau is an intrinsically-disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive "envelopes" around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here, we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau-envelope disassembly. Investigating other members of the tau-MAP family, we find MAP2 similarly forms envelopes governed by lattice-spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct segments. We conclude that the interdependent allostery between lattice-spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

biophysics↗

bioRxiv2021

Fast leaps between millisecond confinements govern Ase1 diffusion along microtubules

Diffusion is the most fundamental mode of protein translocation within cells. Confined diffusion of proteins along the electrostatic potential constituted by the surface of microtubules, although modeled meticulously in molecular dynamics simulations, has not been experimentally observed in real-time. Here, we used interferometric scattering microscopy to directly visualize the movement of the microtubule-associated protein Ase1 along the microtubule surface at nanometer and microsecond resolution. We resolved millisecond confinements of Ase1 and fast leaps between these positions of dwelling preferentially occurring along the microtubule protofilaments, revealing Ase1s mode of diffusive translocation along the microtubules periodic surface. The derived interaction potential closely matches the tubulin-dimer periodicity and the distribution of the electrostatic potential on the microtubule lattice. We anticipate that mapping the interaction landscapes for different proteins on microtubules, finding plausible energetic barriers of different positioning and heights, will provide valuable insights into regulating the dynamics of essential cytoskeletal processes, such as intracellular cargo trafficking, cell division, and morphogenesis, all of which rely on diffusive translocation of proteins along microtubules.

biophysics↗