Biology subjects

Verhey, K.

Publications and source records attributed to Verhey, K..

8 recordsLinked to original sources

bioRxiv2026

CLASP2 promotes repair of kinesin-1 damage to the microtubule lattice

Microtubules are cytoskeletal polymers that play essential roles in eukaryotic cells, including structural support, cell division, and intracellular transport. During intracellular transport, kinesin motor proteins move cargo along microtubule tracks via their processive stepping. Recent studies have shown that the kinesin-1 KIF5C can damage the microtubule lattice while stepping. Microtubule damage can be repaired through incorporation of new tubulin subunits, however, excessive lattice damage results in microtubule breakage and disassembly. To identify cellular factors involved in microtubule repair, we performed an siRNA screen targeting microtubule-associated proteins (MAPs) known to regulate microtubule dynamics and stability. Based on the results, we investigated whether the end binding protein EB1 and cytoplasmic linker-associated protein 2 (CLASP2) contribute to repair of microtubule damage. To test this, we used a microtubule destruction assay in which damage was induced in microtubules gliding over surfaces coated with wild-type or mutant KIF5C proteins. Our findings suggest that CLASP2 directly facilitates microtubule repair, whereas EB1 does not. We further examined CLASP function using a microtubule repair assay and found that CLASP2 promotes repair by enhancing tubulin incorporation and reducing microtubule breakage. Together, these findings demonstrate that CLASP proteins play an important role in repairing and protecting against lattice damage caused by kinesin-1 motor activity. Our results further suggest that MAPs can directly regulate microtubule lattice integrity under mechanical stress generated by motor protein-driven intracellular transport.

Cell Biology↗

bioRxiv2026

Taxol exploits molecular switches within tubulin to stabilize microtubules.

Microtubules are dynamic filaments of tubulin heterodimers that comprise an essential part of the eukaryotic cytoskeleton1. The nucleotide state of tubulin controls microtubule dynamics: stable GTP-microtubules favor polymerization, whereas unstable GDP-microtubules drive depolymerization2. Anticancer compounds such as Taxol (paclitaxel) target microtubule dynamicity by preventing microtubule depolymerization3,4. Despite decades of work, the molecular basis of microtubule dynamics remains poorly defined. Using cryo-EM, we determined [~]2.2 [A] structures of human microtubules in GTP-like (GMPCPP) and GDP states. Comparison of these two states revealed switch-like structural changes as tubulins transition from the pre-hydrolysis (GMPCPP) to the post-hydrolysis (GDP) state. Additional structure determination of Taxol-bound microtubules at [~]2.2 [A] showed that Taxol binding converts the microtubule lattice into a pre-hydrolysis state by reversing the structural switches flipped during GTP hydrolysis. Focusing our analysis on the microtubule seam shows that the pre-hydrolysis conformation of GMPCPP or Taxol-GDP exhibits favorable lateral interactions at the seam, with lattice deformations clearly visible at the GDP seam. Together, our data show the existence of structural switches in tubulin that are coupled to the nucleotide state and are exploited by Taxol to stabilize microtubules into a pre-hydrolysis-like state. (191 words)

molecular biology↗

bioRxiv2025

Micron-scale protein transport along microtubules by kinesin-driven shepherding

How microscopic interactions give rise to cellular-scale order in the crowded environment of cells remains an open problem. Far-from-equilibrium mixtures of active and passive molecules self-organize, but the principles remain unclear. Microtubules are cytoskeletal polymers composed of 13 protofilaments bound by mixtures of actively moving motors and passively diffusing microtubule-associated proteins (MAPs), serving as a model system for self-organization on a multi-lane lattice. Here, we combine computational modeling, analytic theory, and in vitro reconstitution to demonstrate that motors can "shepherd" diffusive MAPs by rectifying Brownian motion into net directional drift without motor-MAP binding. Our model reveals that biased movement increases when fewer protofilaments are accessible or when lateral diffusion between protofilaments is limited, identifying local dimensionality and diffusion as key parameters governing spatial patterning. Notably, shepherding depends not on individual motor processivity but on the total number of motors bound to microtubules. We experimentally recapitulate micron-scale shepherding with a kinesin-1 motor (K401) and a diffusive MAP (PRC1), which show no detectable binding. These findings reveal shepherding as an emergent mechanism by which ensembles of motors generate micron-scale spatial patterning and transport of MAPs without direct binding interactions.

biophysics↗

bioRxiv2025

Multi-kinesin clusters impart mechanical stress that reveals mechanisms of microtubule breakage in cells

Microtubules are cytoskeletal filaments that provide structural support for numerous cellular processes. Despite their high rigidity, microtubules can be dramatically bent in cells and it is unknown how much force a microtubule can withstand before breaking. We find that liquid-liquid phase separation of the kinesin-3 motor KIF1C results in multi-kinesin clusters that entangle neighboring microtubules and impose a high level of mechanical stress that results in microtubule breakage and disassembly. Combining computational simulations and experiments, we show that microtubule fragmentation is enhanced by having a highly processive kinesin motor domain, a stiff clustering mechanism, and sufficient drag force on the microtubules. We estimate a rupture force for microtubules in cells of 70-120 pN, which is lower than previous estimates based on in vitro studies with taxol-stabilized microtubules. These results indicate that the presence of multiple kinesins on a cargo has the potential to cause microtubule breakage. We propose that mechanisms exist to protect microtubule integrity by releasing either the motor-cargo or motor-microtubule interaction, thereby preventing the accumulation of mechanical stress upon the engagement of multi-motor clusters with microtubules.

cell biology↗

bioRxiv2023

Kif6 regulates cilia motility and polarity in brain ependymal cells

Ependymal cells, lining brain ventricular walls, display tufts of cilia that beat in concert promoting laminar Cerebrospinal fluid (CSF) flow within brain ventricles. The ciliary axonemes of multiciliated ependymal cells display a 9+2 microtubule array common to motile cilia. Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how Kinesin motors contribute to cilia motility. Here, we define the function of Kinesin family member 6 (Kif6) using a mutation that lacks a highly conserved C-terminal tail domain (Kif6p.G555fs) and which displays progressive hydrocephalus in mice. An analogous mutation was isolated in a proband displaying macrocephaly, hypotonia, and seizures implicating an evolutionarily conserved function for Kif6 in neurodevelopment. We find that loss of Kif6 function caused decreased ependymal cilia motility and subsequently decreased fluid flow on the surface of brain ventricular walls. Kif6 protein was localized at ependymal cilia and displayed processive motor movement (676 nm/s) on microtubules in vitro. Loss of the Kif6 C-terminal tail domain did not affect the initial ciliogenesis in vivo, but did result in defects in cilia orientation, the formation of robust apical actin networks, and stabilization of basal bodies at the apical surface. This suggests a novel role for the Kif6 motor in maintenance of ciliary homeostasis of ependymal cells. Summary statementWe found that Kif6 is localized to the axonemes of ependymal cells. In vitro analysis shows that Kif6 moves on microtubules and that its loss mice decrease cilia motility and cilia-driven flow, resulting in hydrocephalus.

developmental biology↗

bioRxiv2022

Dual and Opposing Roles for the Kinesin-2 Motor, KIF17, in Hedgehog-dependent Cerebellar Development

While the kinesin-2 motors KIF3A and KIF3B have essential roles in ciliogenesis and Hedgehog (HH) signal transduction, potential role(s) for another kinesin-2 motor, KIF17, in HH signaling have yet to be explored. Here, we investigated the contribution of KIF17 to HH-dependent cerebellar development, where Kif17 is expressed in both HH-producing Purkinje cells and HH-responding cerebellar granule neuron progenitors (CGNPs). Germline Kif17 deletion in mice results in cerebellar hypoplasia due to reduced CGNP proliferation, a consequence of decreased HH pathway activity mediated through decreased Sonic HH (SHH) protein. Notably, Purkinje cell-specific Kif17 deletion phenocopies Kif17 germline mutants. Surprisingly, CGNP-specific Kif17 deletion results in the opposite phenotype- increased CGNP proliferation and HH target gene expression due to altered GLI transcription factor processing. Together these data identify KIF17 as a key regulator of HH-dependent cerebellar development, with dual and opposing roles in HH-producing Purkinje cells and HH-responding CGNPS. TeaserDuring cerebellar development, the KIF17 microtubule motor performs opposing roles in HH-producing and HH-responding cells.

developmental biology↗

bioRxiv2021

Zn2+ acts as a brake signal for axonal transport by directly inhibiting motor protein progression

Accurate delivery of cargo over long distances through axonal transport requires precise spatiotemporal regulation and relies on microtubule function. Here we discover that Zn2+ influx via depolarization inhibits axonal transport. Zn2+-mediated inhibition is nonselective for cargo. Elevated Zn2+ (IC50 >> 5-10 nM) reduces both lysosomal and mitochondrial motility in primary rat hippocampal neurons and HeLa cells. We further reveal that Zn2+ directly binds to microtubules, inhibiting movement of motor proteins (kinesin and dynein) and promoting detachment of neuronal-specific MAPs (Tau, DCX, and MAP2C). We finally provide a detailed model of microtubule interactions with Tau, DCX, dynein, kinesin, and predict microtubule Zn2+ binding sites. Our results reveal that Zn2+ acts to inhibit the microtubule binding of tau, DCX, and MAP2C and can directly block the progression of motor proteins on microtubules. Intraneuronal Zn2+, therefore, is a critical signal for regulating axonal transport and microtubulebased processes.

cell biology↗

bioRxiv2020

Pathogenic Mutations in the Kinesin-3 Motor KIF1A Diminish Force Generation and Movement Through Allosteric Mechanisms

The kinesin-3 motor KIF1A functions in neurons where its fast and superprocessive motility is thought to be critical for long-distance transport. However, little is known about the force-generating properties of kinesin-3 motors. Using optical tweezers, we demonstrate that KIF1A and its C. elegans homolog UNC-104 undergo force-dependent detachments at ~3 pN and then rapidly reattach to the microtubule to resume motion, resulting in a sawtooth pattern of clustered force generation events that is unique among the kinesin superfamily. Whereas UNC-104 motors stall before detaching, KIF1A motors do not. To examine the mechanism of KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders, V8M and Y89D, based on their location in structural elements required for force generation in kinesin-1. Molecular dynamics simulations predict that the V8M and Y89D mutations impair docking of the N-terminal ({beta}9) or C-terminal ({beta}10) portions of the neck linker, respectively, to the KIF1A motor domain. Indeed, both mutations dramatically impair force generation of KIF1A but not the motors ability to rapidly reattach to the microtubule track. Homodimeric and heterodimeric mutant motors also display decreased velocities, run lengths, and landing rates and homodimeric Y89D motors exhibit a higher frequency of non-productive, diffusive events along the microtubule. In cells, cargo transport by the mutant motors is delayed. Our work demonstrates the importance of the neck linker in the force generation of kinesin-3 motors and advances our understanding of how mutations in the kinesin motor domain can manifest in disease.

biophysics↗