Biology subjects

Aumeier, C.

Publications and source records attributed to Aumeier, C..

15 recordsLinked to original sources

bioRxiv2026

Chemical Interrogation and Reprogramming of ATAT1-Mediated Tubulin Acetylation

Acetylation of -tubulin K40 by -tubulin acetyltransferase 1 (ATAT1) using acetyl-coenzyme A (Ac-CoA) marks stable microtubule populations, yet chemical tools to directly measure ATAT1 ligand engagement, inhibit its activity, or visualize ATAT1-mediated modification on intact microtubules remain limited. Through the development of a quantitative binding assay, we uncovered that ATAT1 can bind unnatural cofactors but fails to efficiently use them in acyl-transfer reactions. Structure-guided mutation subsequently yielded ATAT1-L163A, which successfully installed clickable handles at the native -tubulin K40 site of synthetic tubulin peptides, -tubulin, and intact microtubules. Cu(I)-catalyzed azide-alkyne cycloaddition enabled visualization of modified microtubules by in-gel fluorescence and microscopy. Moreover, we report a p11-CoA bisubstrate inhibitor that suppressed both native acetylation and engineered acylation. Together, these tools provide chemically controlled access to ATAT1 activity and a site-verified, clickable K40 modification on intact microtubules.

biochemistry↗

bioRxiv2026

A biochemical probe for microtubule lattice integrity uncovers motor-caused lattice damage

Microtubules experience mechanical and enzymatic stresses that can compromise lattice integrity, yet where and how lattice damage forms remains poorly understood due to the lack of tools that directly visualize damage as it occurs. Existing approaches infer damage indirectly through repair events or rely on static ultrastructural snapshots, precluding dynamic analysis. Here, we introduce MT-DS (Microtubule Damage Sensor), a damage-selective fluorescent probe that directly labels microtubule lattice openings. MT-DS combines taxane-based microtubule binding with a multivalent protein scaffold to restrict intraluminal diffusion and selectively retain the probe at sites of lattice openings. Using MT-DS, we visualize intrinsic lattice defects in stabilized microtubules, uncover a strong enrichment of damage at annealing sites, and demonstrate that kinesin-1{Delta}6 actively generates de novo lattice damage during motility. By enabling direct, time-resolved detection of microtubule damage, MT-DS establishes lattice integrity as an experimentally accessible parameter and provides a chemical tool to investigate how mechanical stress reshapes the microtubule cytoskeleton.

biochemistry↗

bioRxiv2025

Microtubule deacetylation drives kinesin-1 mediated mitochondrial transport accelerating breast cancer cell migration

Mitochondrial trafficking is reprogrammed in metastatic breast cancer cells to sustain their migratory and invasive behavior. Mitochondria repositioning to sites of high energy demand is governed by a balance between opposing dynein and kinesin-1 (KIF5B) molecular motors whose regulation remains incompletely understood. Here, we identify the SYBU gene as a candidate prognostic marker downregulated in metastatic disease. SYBU encodes syntabulin, a mitochondria outer membrane protein that interacts with dynein to counterbalance KIF5B-dependent anterograde transport to the cell cortex. Loss of SYBU disrupts the balance, causing excessive KIF5B-driven mitochondria movement, microtubule damage and deacetylation. In turn, microtubule deacetylation reinforces KIF5B-mediated transport, creating a positive feedback loop that drives mitochondria distribution close to the cell periphery and enhances cancer cell migration. Pharmacological inhibition of the tubulin deacetylase HDAC6 restores mitochondrial positioning and reduces cell migration in SYBU-deficient cells. Our findings identify SYBU as a key regulator of mitochondrial trafficking and pave the way to personalized therapeutic approaches for metastatic breast tumors with low SYBU expression.

cell biology↗

bioRxiv2025

Microtubule lattice conformation and integrity regulate α-tubulin acetylation

Microtubule acetylation of lysine 40 of -tubulin is a hallmark of stable microtubules. This luminal modification is catalyzed by -tubulin acetyltransferase 1 (TAT1) and reversed by histone deacetylase 6 (HDAC6). However, acetylation regulation within the microtubule lumen and the influence of lattice architecture on enzymatic activity remain poorly understood. Here, we reconstitute microtubule acetylation in vitro using purified TAT1 and HDAC6 on microtubules assembled with defined lattice conformations. We show that TAT1 overwrites HDAC6 enzymatic activity, but its acetylation efficiency decreases upon microtubule damage. Importantly, TAT1 efficiently acetylates microtubules in expanded lattices and twisted tubulin states, while compacted lattices impede its activity. Our findings reveal that both microtubule integrity and lattice conformation are critical regulators for TAT1 enzymatic activity, suggesting that dynamic lattice transitions modulate the acetylation pattern of microtubules in cells.

cell biology↗

bioRxiv2025

Phase Separation and a Hydrodynamic Instability Localize Proteins at Growing Microtubule Ends

Regulating microtubule dynamics is essential for cellular function, and precise localization of regulatory proteins at microtubule ends is critical. End-binding protein EB3, a key regulator of microtubule growth, accumulates over an extended region at the growing end, forming a comet that gradually fragments into transient droplets along the microtubule shaft. Here, we combine in vitro reconstitution experiments with theoretical analysis to show that surface-mediated condensation of EB3 effectively localizes the protein at microtubule ends. Our results reveal that a Rayleigh-Plateau instability limits the condensates extent, producing a finite comet length and discrete droplets along the shaft. Remarkably, the comet size is independent of the GTP-cap size. This finding is supported by experiments on cells showing that modulation of microtubule growth velocity and hence GTP-cap size do not consistently alter EB3 comet length. Furthermore, our theory shows that rapid droplet evaporation requires a transition of EB3 to a non-phase-separating state. Overall, our work challenges the view that comet length directly reflects GTP-cap size and highlights a novel mechanism for regulating microtubule dynamics.

biophysics↗

bioRxiv2024

Actin dynamics sustains spatial gradients of membrane tension in adherent cells

Tension propagates in lipid bilayers over hundreds of microns within milliseconds, precluding the formation of tension gradients. Nevertheless, plasma membrane tension gradients have been evidenced in migrating cells and along axons. Here, using a fluorescent membrane tension probe, we show that membrane tension gradients exist in all adherent cells, whether they migrate or not. Non-adhering cells do not display tension gradients. We further show that branched actin increases tension, while membrane-to-cortex attachments facilitate its propagation. Tension is the lowest at the edge of adhesion sites and highest at protrusions, setting the boundaries of the tension gradients. By providing a quantitative and mechanistic basis behind the organization of membrane tension gradients, our work explains how they are actively sustained in adherent cells.

cell biology↗

bioRxiv2024

Modulation of Microtubule Dynamics by Monovalent Ions

The microtubule cytoskeleton is a dynamic network essential for many cellular processes, influenced by physicochemical factor such as temperature, pH, dimer concentration and ionic environment. In this study, we used in vitro reconstitution assays to examine the effects of four monovalent ions (Na+, K+, Cl-, and Ac-) on microtubule dynamics, uncovering distinct effects for each ion. Na+ was found to increase microtubule dynamicity by raising catastrophe frequency, polymerization and depolymerization speeds, ultimately reducing microtubule lifetime by 80 %. Conversely, Ac boosts microtubule nucleation and stabilizes microtubules by increasing rescue frequency and preventing breakages, resulting in longer microtubules with extended lifetimes. Cl- appeared to potentiate the effects of Na+, while K+ had minimal impact on microtubule dynamic parameters. These findings demonstrate that Na+ and Ac- have opposing effects on microtubule dynamics, with Na+ destabilizing and Ac- stabilizing the microtubule structure. This ionic impact is mainly through modulation of tubulin-tubulin interactions rather than affecting the hydrolysis rate. In conclusion, ion identity plays a crucial role in modulating microtubule dynamics. Understanding the ionic environment is essential for microtubule-related research, as it significantly influences microtubule behavior, stability, and interactions with other proteins. SIGNIFICANCE STATEMENTThe microtubule cytoskeleton is vital for cellular processes and influenced by temperature, pH, dimer concentration, and ionic environment. Understanding how these physicochemical factors regulate microtubule polymerization is crucial for elucidating microtubule dynamics and stability. Our in vitro reconstitution assays reveal that Na+ and Ac- ions have opposing effects on microtubule dynamics. Na+ increases dynamicity by raising catastrophe frequency and reducing lifetime by 80 %, while Ac - enhances nucleation and stability, resulting in longer microtubules. Cl- potentiates the effects of Na+, and K+ has minimal impact. Our findings highlight that ion identity crucially modulates microtubule dynamics, significantly influencing stability and interactions.

biochemistry↗

bioRxiv2022

Running Kinesin-1 shapes the microtubule acetylation gradient

The properties of single microtubules within the microtubule network can be modulated through posttranslational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could either enter through the microtubule ends or at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen depends on kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase TAT1 is independent of kinesin-1 and shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de-)acetylation and scales with the size of the cells. The control of shaft damage represents a novel mechanism to regulate PTM inside the microtubule by giving access to the lumen.

cell biology↗

bioRxiv2022

Tubulin engineering by semisynthesis reveals that polyglutamylation directs detyrosination

Microtubules, a critical component of the cytoskeleton, carry combinations of post-translational modifications (PTMs), which are critical for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of -tubulin as well as polyglutamylation. Dysregulation of these PTMs results in disease, including developmental defects and neurodegeneration. Despite their importance, the mechanisms governing the emergence of such PTM patterns are not well understood, mostly because tools to dissect the function and regulation of tubulin PTMs have been lacking. Here, we report a chemical method to produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. Using a sortase- and intein-mediated tandem transamidation strategy, we ligate synthetic -tubulin tails, which are site-specifically glutamylated to specific extents, to recombinant human tubulin heterodimers. Using microtubules reconstituted with such designer tubulins, we show that polyglutamylation of -tubulin promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/SVBP in a manner dependent on the length of polyglutamyl chains. Moreover, modulating polyglutamylation levels in cells results in corresponding changes in detyrosination. Together, using synthetic chemistry to produce tubulins carrying defined PTMs, we can directly link the detyrosination cycle to polyglutamylation, connecting two key regulatory systems that control tubulin function.

biochemistry↗

bioRxiv2022

The effect of motor-induced shaft dynamics on microtubule stability and length

Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depends on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in presence of kinesin-1 molecular motors. Although the average microtubule dynamics can be captured by an effective rescue rate, the dynamics of individual microtubules differs dramatically when rescue occurs only at exchange sites. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network. SIGNIFICANCEThe microtubule network is essential for vital cellular processes like the organization of intracellular transport and division. Although microtubule assembly occurs at its tips, it has recently been reported that tubulin is exchanged along the microtubule shaft. Tubulin exchange plays an essential role in regulating microtubule dynamics and can be induced by molecular motors. Here, we provide the first systematic study of the impact of shaft dynamics on the regulation of rescue events, where a microtubule switches from shrinking to growing. Our results illustrate how the usage of microtubules as tracks for intracellular transport regulates the microtubule network and thus offers a novel perspective on intracellular organization.

biophysics↗

bioRxiv2021

A liquid +TIP-network drives microtubule dynamics through tubulin condensation

Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here we show that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid-networks. When CLIP-170 and EB3 act together the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells these liquid networks can condense tubulin. In vitro in the presence of microtubules, EB3/CLIP-170 phase separation can co-condense tubulin all along the microtubule. At this condition microtubule growth speed increases up to two-fold and depolymerization events are strongly reduced, compared to conditions with phase separation deficient networks. Our data show that phase separated EB3/CLIP-170 networks impact microtubule growth dynamics beyond direct protein-microtubule interactions.

cell biology↗

bioRxiv2021

Motor usage imprints microtubule stability in the shaft

Tubulin dimers assemble into dynamic microtubules which are used by molecular motors as tracks for intracellular transport. Organization and dynamics of the microtubule network is commonly thought to be regulated at the polymer ends, where tubulin-dimers can be added or removed. Here we show that molecular motors running on microtubules cause exchange of dimers along the shaft. These sites of dimer exchange act as rescue sites where depolymerising microtubules stop shrinking and start re-growing. Consequently, the average length of microtubules increases depending on how frequently they are used as motor tracks. An increase of motor activity densifies the cellular microtubule network and enhances cell polarity. Running motors leave marks in the shaft serving as traces of microtubule usage to organize the polarity landscape of the cell.

cell biology↗

bioRxiv2020

Kinesin-1 motility traced by an activity-based precipitating dye

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. As tracks for cargo delivery, kinesin-1 uses a subset of microtubules within the dense microtubule network. It is still debated what defines the specific binding of kinesin-1 to a subset of microtubules. Therefore, the ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate for kinesin-1 that yields a precipitating dye. The activity of kinesin-1 thus leaves a fluorescent trail along its walking path and can be visualized without loss of signal due to diffusion. Kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time.

biochemistry↗

bioRxiv2018

Self-repair protects microtubules from their destruction by molecular motors

Microtubules are dynamic polymers that are used for intracellular transport and chromosome segregation during cell division. Their instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Microtubules function in coordination with kinesin and dynein molecular motors, which use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the forces produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that the mechanical work of molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. This effect was not observed when free tubulin dimers were present in the assay. Using fluorescently labelled tubulin dimers we found that dimer removal by motors was compensated for by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of kinesin and dynein motors and the renewal of the microtubule lattice.

cell biology↗

bioRxiv2018

Lattice defects induce microtubule self-renewal

The dynamic instability of microtubules is powered by the addition and removal of tubulin dimers at the ends of the microtubule. Apart from the end, the microtubule shaft is not considered to be dynamic. However recent evidence suggests that free dimers can be incorporated into the shaft of a microtubule damaged by mechanical stress. Here we explored whether dimer exchange was a core property of the microtubule lattice independently of any external constraint. We found that dimers can be removed from and incorporated into the lattice at sites along the microtubule shaft. Furthermore, we showed by experiment and by modeling that rapid dimer renewal requires structural defects in the lattice, which occur in fast growing microtubules. Hence long-lived microtubules have the capacity to self-renew despite their apparent stability and thereby can potentially regulate signaling pathways and structural rearrangements associated with tubulin-dimer exchange at sites along their entire length.

cell biology↗