Biology subjects

Steinmetz, M. O.

Publications and source records attributed to Steinmetz, M. O..

16 recordsLinked to original sources

bioRxiv2026

Structural basis of microtubule destabilization by GTP hydrolysis

Microtubules are cytoskeletal filaments that dynamically grow and shrink to support vital biological functions. Their behavior is regulated by GTP hydrolysis in tubulin incorporated into the lattice, but the underlying chemical transitions and their impact on microtubule stability remain unclear. Using cryo-electron microscopy, we resolved microtubule structures at 1.9-2.2 [A] in four different nucleotide states, revealing the roles of amino acids, ions, and hundreds of water molecules in the hydrolysis process. We found that compaction of the GTP-bound lattice into a more stable pre-hydrolysis state activates a catalytic water molecule for {gamma}-phosphate cleavage. Hydrolysis intermediates are stabilized by strengthened lattice contacts, but in the final GDP state, release of reaction products disrupts these contacts and destabilizes the lattice. Our results explain how chemical changes in tubulins nucleotide site drive lattice transitions and offer an atomistic framework for understanding microtubule dynamic instability.

biochemistry↗

bioRxiv2026

Metabolites Shift Equilibria of Biomolecular Condensates

Biomolecular condensates compartmentalize biochemistry in living cells. While in vitro models of condensates involve only a few components, the cytoplasm is a complex mixture with thousands of components, including many small molecules. While many macromolecular drivers of phase separation have been revealed, the contributions from small molecules have received little attention. To quantify the impact of solutes on biomolecular condensates, we introduce susceptibility, a dimensionless descriptor of condensate response to solute perturbations. We measured how three model condensates, assembled by distinct cohesive mechanisms, respond to diverse solutes including amino acids and nucleotides. Generically, solutes shift condensate phase equilibria, with susceptibilities spanning over four orders of magnitude. These values reflect underlying molecular interactions, consistent with theoretical descriptions including Flory-Huggins and polyphasic linkages. As one example of the predictive power of susceptibility, we exploit enzymatic activity to induce condensation and modulate material properties. Our work establishes susceptibility as an indicator of the sensitivity of biomolecular condensates to solutes, with implications for cell physiology and therapeutic design. SIGNIFICANCE STATEMENTCells compartmentalize biochemistry using biomolecular condensates formed through phase separation. Although cells contain thousands of small molecules, little is known about their influence on condensation. In such complex mixtures, mapping full phase diagrams is infeasible. Alternatively, we introduce susceptibility to characterize system response around a working composition. Using three distinct model condensates and over a dozen solutes, we observe susceptibilities varying over four orders of magnitude. We provide a general thermodynamic framework that clarifies the driving forces behind these responses, rationalizing their magnitude and their dependence on location in the phase diagram. Our work provides a framework for understanding and harnessing solutes to regulate biomolecular condensation, with implications for cell physiology and therapeutic design.

biophysics↗

bioRxiv2025

Effective Tubulin Degradation by Rationally Designed Proteolysis Targeting Chimeras

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that induce the degradation of proteins of interest (POIs) via the ubiquitin-proteasome pathway by recruiting E3 ligases to form a ternary complex with the POI. In this study, we rationally designed and synthesized PROTACs targeting the {beta}-tubulin heterodimer, the building block of microtubules (MTs) that are essential for numerous cellular functions and represent important therapeutic targets in cancer and neurodegenerative diseases. Maytansinol, a known MT-destabilising agent, was selected as the POI ligand, functionalised and conjugated to linkers bearing cereblon or Von Hippel-Lindau ligands as E3 ligase recruiters. Four compounds were synthesized and characterized through structural, biophysical and cell biology studies to evaluate their ability to form degradation-prone tubulin-PROTAC-E3 ligase ternary complexes. We confirmed that the PROTACs effectively bind tubulin and recruit E3 ligases. Remarkably, two PROTACs exhibited cellular degradation activity, representing an important advancement in chemically inducing tubulin-E3-ligase interactions. This work integrates rational design, biophysical and structural validation, and cell-based studies to establish a robust framework for developing tubulin-targeting PROTACs, offering significant implications for basic research and therapeutic developments.

biochemistry↗

bioRxiv2024

Phase separation of a microtubule plus-end tracking protein into a fluid fractal network

Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based cellular processes, and it has been recently proposed that they function as liquid condensates. However, the formation process and internal organization of +TIP condensates are poorly understood. Here, we have investigated the phase separation of the CLIP-170 family member Bik1, a key +TIP implicated in budding yeast cell division. We found that Bik1 is a rod-shaped dimer whose conformation is dominated by its central coiled-coil domain. Liquid condensation is accompanied by Bik1 conformational rearrangements, leading to a 2-3-fold rise in interactions between the proteins folded and disordered domains. In contrast to classical liquids, the supramolecular structure of the Bik1 condensate is heterogeneous, with a fractal structure of protein-rich and protein-free domains. This observation provides structural evidence in support of recent models of biomolecular condensates based on percolation. More broadly, our results provide insights into the structure, dynamic rearrangement, and organization of a complex, multidomain protein in its dilute and condensed phases. Our experimental framework can be extended to other biomolecular condensates, including more intricate +TIP networks.

biochemistry↗

bioRxiv2022

VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules

The detyrosination/tyrosination cycle of -tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzyme complexes involved in microtubule detyrosination. However, little is known about their mode of action. Here, we show in reconstituted systems and in cells that VASH1-SVBP and VASH2-SVBP drive global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of human VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We further show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH domain is critical for microtubule-detachment and diffusion of VASH-SVBP enzymes on the lattice. Together, our results suggest a mechanism by which these enzymes could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions. SUMMARYVASH1-SVBP and VASH2-SVBP produce global and local detyrosination patterns of microtubule lattices, respectively. These activities rely on the interplay between the N- and C-terminal disordered regions of the enzymes, which determine their differential molecular mechanism of action. GRAPHICAL ABSTRACTSchematic representation of divergent molecular mechanisms of action of VASH-SVBP detyrosination complexes.

cell biology↗

bioRxiv2021

Microtubule plus-end regulation by centriolar cap proteins

Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, grow very slowly and are very stable. The complex of centriolar proteins CP110 and CEP97 forms a cap that stabilizes the distal centriole end and prevents its over-elongation. Here, we used in vitro reconstitution assays to show that whereas CEP97 does not interact with microtubules directly, CP110 specifically binds microtubule plus ends, potently blocks their growth and induces microtubule pausing. Cryo-electron tomography indicated that CP110 binds to the luminal side of microtubule plus ends and reduces protofilament peeling. Furthermore, CP110 directly interacts with another centriole biogenesis factor, CPAP/SAS- 4, which tracks growing microtubule plus ends, slows down their growth and prevents catastrophes. CP110 and CPAP synergize in inhibiting plus-end growth, and this synergy depends on their direct binding. Together, our data reveal a molecular mechanism controlling centriolar microtubule plus- end dynamics and centriole biogenesis.

cell biology↗

bioRxiv2021

High interaction valency ensures cohesion and persistence of a microtubule +TIP body at the plus-end of a single specialized microtubule in yeast

Microtubule plus-end tracking proteins (+TIPs) control microtubule specialization and are as such essential notably during eukaryotic cell division. Here, we investigated interactions and functions of the budding yeast Kar9 network consisting of the core +TIPs components Kar9 (functional homologue of APC, MACF, and SLAIN), Bim1 (orthologue of EB1), and Bik1 (orthologue of CLIP-170). Our data indicate that a redundant, multivalent web of interactions links the three +TIPs together to form a "Kar9 body" at the tip of a single cytoplasmic microtubule. They further suggest that this body is a liquid-like condensate, allowing it to persist on both growing and shrinking microtubule tips, and functions as a mechanical coupling device between microtubules and actin cables during mitosis. Our study underlines the power of dissecting the web of low-affinity interactions driving liquid-liquid phase separation of proteins in order to demonstrate the importance and establish the functional roles of condensation processes in vivo.

cell biology↗

bioRxiv2021

Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol

Paclitaxel (Taxol(R)) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drugs mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 [A] resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the {beta}M loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, binding of the taxane core to the taxane site displaces the S9-S10 loop of {beta}-tubulin resulting in microtubule expansion. Our results provide detailed new insights into the mechanism of microtubule-stabilization by taxanes.

biophysics↗

bioRxiv2021

Structural heterogeneity of the microtubule lattice

Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions including motility, compartmentalization, and division. They are considered as pseudo-helical polymers whose constituent {beta}-tubulin heterodimers share lateral homotypic interactions, except at one unique region called the seam. Here, we used a segmented sub-tomogram averaging strategy to reassess this paradigm and analyze the organization of the {beta}-tubulin heterodimers in microtubules assembled from purified porcine brain tubulin in the presence of GTP and GMPCPP, and in Xenopus egg cytoplasmic extracts. We find that in all conditions, microtubules incorporate variable protofilament and/or tubulin subunit helical-start numbers, as well as variable numbers of seams. Strikingly, the seam number and location vary along individual microtubules, generating holes of one to a few subunits in size within their lattices. Together, our results reveal that the formation of mixed and discontinuous microtubule lattices is an intrinsic property of tubulin that requires the formation of unique lateral interactions without longitudinal ones. They further suggest that microtubule assembly is tightly regulated in a cytoplasmic environment.

cell biology↗

bioRxiv2021

In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M

Photoswitchable reagents to modulate microtubule stability and dynamics are an exciting tool approach towards micron- and millisecond-scale control over endogenous cytoskeleton-dependent processes. When these reagents are globally administered yet locally photoactivated in 2D cell culture, they can exert precise biological control that would have great potential for in vivo translation across a variety of research fields and for all eukaryotes. However, photopharmacologys reliance on the azobenzene photoswitch scaffold has been accompanied by a failure to translate this temporally- and cellularly-resolved control to 3D models or to in vivo applications in multi-organ animals, which we attribute substantially to the metabolic liabilities of azobenzenes. Here, we optimised the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based instead on the styrylbenzothiazole (SBT) photoswitch scaffold, that are non-responsive to the major fluorescent protein imaging channels and so enable multiplexed imaging studies. We applied these reagents to 3D systems (organoids, tissue explants) and classic model organisms (zebrafish, clawed frog) with one- and two-protein imaging experiments. We successfully used systemic treatment plus spatiotemporally-localised illuminations in vivo to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in these systems with cellular precision and second-level resolution. These nanomolar, in vivo-capable photoswitchable reagents can prove a game-changer for high-precision cytoskeleton research in cargo transport, cell motility, cell division and development. More broadly, their straightforward design can also inspire the development of similarly capable optical reagents for a range of protein targets, so bringing general in vivo photopharmacology one step closer to productive realisation.

cell biology↗

bioRxiv2020

The mechanism of selective kinesin inhibition by kinesin binding protein.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, crescent-shaped right-handed -solenoid that sequesters the tubulin-binding surface of the kinesin motor domain, structurally distorting the motor domain and sterically blocking MT attachment. KBP uses its -solenoid concave face and edge loops to bind the kinesin motor domain and selective mutation of this extended binding surface disrupts KBP inhibition of kinesin transport in cells. The KBP-interacting surface of the motor domain contains motifs exclusively conserved in KBP-interacting kinesins, providing a basis for kinesin selectivity.

biochemistry↗

bioRxiv2020

The inner scaffold protects from centriole fracture

Centrioles are characterized by a nine-fold arrangement of long-lived microtubule triplets that are held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy (U-ExM) for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the centriolar microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein with a predicted structurally homology with the ciliary inner junction protein FAP20. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in both human and Chlamydomonas cells. Altogether, this work highlights that POC16/WDR90 is a crucial evolutionary conserved molecular player participating in centriole architecture integrity.

cell biology↗

bioRxiv2020

Pharmaceutical-grade Rigosertib is a Microtubule-destabilizing Agent

We recently used CRISPRi/a-based chemical-genetic screens and targeted cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. In a recent manuscript, Reddy and co-workers suggest that this microtubule-destabilizing activity of rigosertib is mediated not by rigosertib itself but by a contaminating degradation product of rigosertib, ON01500, present in formulations obtained from commercial vendors (Baker et al., 2019). Here, we demonstrate that treatment of cells with pharmaceutical-grade rigosertib (>99.9% purity) results in qualitatively indistinguishable phenotypes as treatment with commercially obtained rigosertib across multiple assays. The two compounds have indistinguishable chemical-genetic interactions with genes involved in modulating the microtubule network (KIF2C and TACC3), both destabilize microtubules in cells and in vitro, and both show substantially reduced toxicity in cell lines expressing a rationally-designed mutant of tubulin (L240F TUBB mutant), in which the rigosertib binding site in tubulin is mutated. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents, which is disputed by Reddy and co-workers, was recently confirmed by an independent research group (Patterson et al., 2019). We conclude that rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

cancer biology↗

bioRxiv2019

Photoswitchable microtubule inhibitors enabling robust, GFP-orthogonal optical control over the tubulin cytoskeleton

Here we present GFP-orthogonal optically controlled reagents for reliable and repetitive in cellulo modulation of microtubule dynamics and its dependent processes. Optically controlled reagents (\"photopharmaceuticals\") have developed into powerful tools for high-spatiotemporal-precision control of endogenous biology, with numerous applications in neuroscience, embryology, and cytoskeleton research. However, the restricted chemical domain of photopharmaceutical scaffolds has constrained their properties and range of applications. Styrylbenzothiazoles are an as-yet unexplored scaffold for photopharmaceuticals, which we now rationally design to feature potent photocontrol, switching microtubule cytoskeleton function off and on according to illumination conditions. We show more broadly that this scaffold is exceptionally chemically and biochemically robust as well as substituent-tolerant, and offers particular advantages for intracellular biology through a range of desirable photopharmaceutical and drug-like properties not accessible to the current classes of photoswitches. We expect that these reagents will find powerful applications enabling robust, high precision, optically controlled cell biological experimentation in cytoskeleton research and beyond.

cell biology↗

bioRxiv2019

Structural determinants of microtubule minus end preference in CAMSAP CKK domains

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which has lost minus-end specificity. Near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit subtly different footprints on microtubules. Whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. NMR experiments show that HsCKK is remarkably rigid, supporting this remodelling ability. Thus, in contrast to many MAPs, CKK domains can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.

biophysics↗

bioRxiv2019

Mechanisms of motor-independent membrane remodeling driven by dynamic microtubules

Microtubule-dependent organization of membranous organelles, such as the endoplasmic reticulum, occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. How highly transient protein-protein interactions occurring at growing microtubule tips can induce load-bearing processive motion is currently unclear. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, End-Binding (EB) proteins and a membrane-targeted protein that interacts with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation by growing microtubule ends, motor-independent membrane sliding along microtubule shafts and pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Force spectroscopy revealed that attachments to growing and shrinking microtubule ends can sustain forces of [~]0.5 and [~]5 pN, respectively. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear sufficient load to induce membrane deformation and motility.

cell biology↗