Biology subjects

Cross, R. A.

Publications and source records attributed to Cross, R. A..

13 recordsLinked to original sources

bioRxiv2026

Mutating the interprotofilament interface allows microtubules to assemble in GDP

Microtubule dynamic instability, driven by GTP turnover, allows microtubules in cells to reorganise themselves adaptively. GTP promotes the assembly of tubulin into microtubules, but exactly how it does so is controversial. In some models, GTP mainly supports the assembly of tubulin into single protofilaments, as in prokaryotic tubulins. In others, GTP mainly supports the formation of lateral bonds between protofilaments. To investigate, we mutated the interprotofilament interface in human 1b{beta}3 and 1b{beta}4b tubulins, whose sequences diverge markedly in this region. We find that transplanting the 1b{beta}3 M-loop or its binding pocket into 1b{beta}4b tubulin creates tubulins that assemble in 1 mM GDP. We accordingly propose that GTP- and GDP-tubulins are captured equivalently at the tips of microtubules, but then differentially retained, based on their differing abilities to form stable interprotofilament bonds. This biased retention mechanism allows mosaic lattices to be built and dynamic instability to be tuned. One sentence summaryMicrotubules grow not by biased capture of GTP tubulins, but by biased retention of tubulins that form stable interprotofilament bonds.

cell biology↗

bioRxiv2025

ATPγS unbiases kinesin

Kinesin-1 microtubule motors are ATP-fuelled, twin-headed cargo transporters that step processively along microtubules, with a load-dependent directional bias. Here we show using single molecule optical trapping that 1 mM ATP{gamma}S, a slowly-hydrolysed analogue, substantially defeats the biasing mechanism, whereas 1 {micro}M ATP{gamma}S supports it. Our data argue that kinesin re-registers itself between steps into an Await-Isomerisation (AI) state that engenders both on-axis steps and off-axis missteps, and that missteps can be rescued. In the AI state, ATP or ATP{gamma}S is bound but neck-linker docking and nucleotide hydrolysis are inhibited. Load-dependent exit from the AI state establishes hydrolytic competence via catalytic site closure and coupled neck-linker docking, which guides the tethered head to its next on-axis site. By overpopulating the AI state, ATP{gamma}S reveals its pivotal role in the biasing mechanism, whose control logic maximises steered diffusion-to-capture of the leading kinesin-1 head under load, at the expense of futile nucleotide turnover.

biophysics↗

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↗

bioRxiv2024

The eduWOSM: an advanced optical microscope for education

To improve access to advanced optical microscopy in educational and resource-limited settings we have developed the eduWOSM (educational Warwick Open Source Microscope), an open hardware platform for transmitted-light and epifluorescence imaging in up to 4 colours, including single molecule imaging. EduWOSMs are robust, bright, compact, portable and ultra-stable. They are controlled entirely by open source hardware and software, with an option for remote control from a webpage. Here we describe the core eduWOSM technology and benchmark its performance using 3 example projects, single fluorophore tracking of tubulin heterodimers within gliding microtubules, 4D (deconvolution) imaging/tracking of chromosome motions in dividing human cells, and automated single particle tracking in vitro and in live cells with classification into subdiffusive, diffusive and superdiffusive motion.

cell biology↗

bioRxiv2023

Taxol acts differently on different tubulin isotypes

Taxol is a critically important cancer drug that stabilises microtubules. We report that taxol acts differently on different metazoan tubulin isotypes. 50 nM taxol blocks catastrophe of human or zebrafish 1{beta}4 but has no effect on human 1{beta}3 microtubules. 500 nM taxol blocks catastrophe in both 1{beta}3 and 1{beta}4 microtubules but introduces kinks only into 1{beta}4 microtubules. Taxol washout relaxes the kinks, suggesting taxol expands 1{beta}4 but not 1{beta}3 lattices. Kinesin-driven microtubule gliding detects this conformational shift - 1{beta}4 microtubules glide at ~450 nm/sec in 400 nM taxol, but at ~750 nm/sec in 10 M taxol, whereas 1{beta}3 microtubules glide at ~450 nm/sec, even in 10 M taxol. Thus, taxol readily stabilises 1{beta}4 GDP-tubulin lattices and shifts them to a fastgliding conformation, but stabilises 1{beta}3 lattices much less readily and without shifting their conformation. These isotype-specific actions of taxol may drive the switch to {beta}3 tubulin commonly seen in taxol-resistant tumours.

cell biology↗

bioRxiv2021

Torque force in the kinesin stroke drives coupled yaw axis and orbital rotations of kinesin coated gold nanorods

Kinesin motor domains generate impulses of force and movement that have both translational and rotational components, raising the question of how the rotational component contributes to motor functions. We used a new assay in which kinesin-coated gold nanorods (kinesin-GNRs) move on suspended microtubules, for two plus-end-directed and weakly-processive kinesins: single-headed KIF1A, dimeric ZEN-4. Polarization of the light scattered by two types of kinesin-GNRs periodically oscillated as they orbited the microtubule along a left-handed helical trajectory. Our analyses revealed that each kinesin-GNR unidirectionally rotates about its yaw axis as it translocates, and that the period of this yaw-axis rotation corresponds to two periods of its left-handed helical orbit around the microtubule axis. Theoretical analyses suggest that the yaw-axis rotation enhances biased lateral displacement of the kinesin team. Our study reveals biaxial rotation as a new mode of motility in kinesin teams that may help the team to sidestep roadblocks.

biophysics↗

bioRxiv2021

Force generation of KIF1C is impaired by pathogenic mutations

Intracellular transport is essential for neuronal function and survival. The fastest neuronal transporter is the kinesin-3 KIF1C. Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients. However, neither the force generation of the KIF1C motor protein, nor the biophysical and functional properties of pathogenic mutant proteins have been studied thus far. Here we show that full length KIF1C is a processive motor that can generate forces up to 5.7 pN. We find that KIF1C single molecule processivity relies on its ability to slip and re-engage under load and that its slightly reduced stall force compared to kinesin-1 relates to its enhanced probability to backslip. Two pathogenic mutations P176L and R169W that cause hereditary spastic paraplegia in humans maintain fast, processive single molecule motility in vitro, but with decreased run length and slightly increased unloaded velocity compared to the wildtype motor. Under load in an optical trap, force generation by these mutants is severely reduced. In cells, the same mutants are impaired in producing sufficient force to efficiently relocate organelles. Our results establish a baseline for the single molecule mechanics of Kif1C and explain how pathogenic mutations at the microtubule-binding interface of KIF1C impair the cellular function of these long-distance transporters and result in neuronal disease.

cell biology↗

bioRxiv2020

Activation of cytoplasmic dynein through microtubule crossbridging

Cytoplasmic dynein is the main microtubule-minus-end-directed transporter of cellular cargo in animal cells [1, 2]. Cytoplasmic dynein also functions in the organisation and positioning of mitotic spindles [3, 4] and the formation of ordered microtubule arrays in neurons and muscle [5, 6]. Activation of the motor for cargo transport is thought to require formation of a complex with dynactin and a cargo adapter [7-10]. Here we show that recombinant human dynein can crossbridge neighbouring microtubules and can be activated by this crossbridging to slide and polarity-sort microtubule bundles. While single molecules of human dynein are predominantly static or diffusive on single microtubules, they walk processively for 1.5 m on average along the microtubule bundles they form. Speed and force output of dynein are doubled on bundles compared to single microtubules, indicating that the crossbridging dynein steps equivalently on two microtubules. Our data are consistent with a model of autoactivation through the physical separation of dynein motor domains when crossbridging two microtubules. This enables cytoplasmic dynein to function effectively as a microtubule organiser and transporter without needing to first form a complex with dynactin and a cargo adapter.

biophysics↗

bioRxiv2019

Proposed mechanism of kinesin backstepping

Kinesin-1 is an ATP-driven molecular motor that transports cellular cargo along microtubules. At low loads, kinesin-1 almost always steps forwards, towards microtubule plus ends, but at higher loads, it can also step backwards. Backsteps are usually 8 nm, but can be larger. These larger backwards events of 16 nm, 24 nm or more are thought to be slips rather than steps, because they are too fast to consist of multiple, tightly-coupled 8 nm steps. Here we propose that not just these larger backsteps, but all kinesin-1 backsteps, are slips. We show firstly that kinesin waits before forward steps for less time than before backsteps and detachments; secondly that kinesin waits for the same amount of time before backsteps and detachments and thirdly that by varying the microtubule type we can change the ratio of backsteps to detachments, without affecting forward stepping. Our findings indicate that backsteps and detachments originate from the same state and that this state arises later in the mechanochemical cycle than the state that gives rise to forward steps. To explain our data, we propose that in each cycle of ATP turnover, forward kinesin steps can only occur before Pi release, whilst backslips and detachments can only occur after Pi release. In the scheme we propose, Pi release gates access to a weak binding K.ADP-K.ADP state that can slip back along the microtubule, re-engage, release ADP and try again to take an ATP-driven forward step. We predict that this rescued detachment pathway is key to maintaining kinesin processivity under load. Significance statementKinesin-1 molecular motors are ATP-driven walking machines that typically step forward, towards microtubule plus ends. But they can also step backwards, especially at high load. Backsteps are currently thought to occur by directional reversal of forwards walking. To the contrary, we propose here that kinesin backsteps are not steps, but slips. We show that backwards translocations originate from a different and later state in the kinesin mechanism than the state that generates forward steps. To explain this, we propose that following ATP binding, kinesin molecules that fail to step forward within a load-dependent time window convert to a state that can slip back, rebind to the microtubule, and try again to step forward.

biophysics↗

bioRxiv2018

Stabilising and destabilising kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends

In fission yeast, the length of interphase microtubule (iMT) arrays are adapted to cell length so as to maintain cell polarity and to help centre the nucleus and cell division ring. Here we show that length regulation of iMTs is dictated by spatially-regulated competition between MT-stabilising Tea2/Tip1/Mal3 (Kinesin-7) and MT-destabilising Klp5/Klp6/Mcp1 (Kinesin-8) complexes at iMT plus tips. During MT growth, the Tea2/Tip1/Mal3 complex remains bound to the plus tips of iMT bundles and restricts access to the plus tips by Klp5/Klp6/Mcp1, which accumulates behind it. At cell ends, Klp5/Klp6/Mcp1 invades the space occupied by the Tea2/Tip1/Tea1 kinesin complex triggering its displacement from iMT plus tips and MT catastrophe. These data show that in vivo, whilst the \"antenna model\" for iMT length- and age-dependent catastrophase accumulation has validity, length control is an emergent property reflecting spatially-regulated competition between multiple complexes at the MT plus tip.

cell biology↗

bioRxiv2018

TOG domain MT polymerases accelerate MT plus end growth via electrostatically-steered diffusion-to-capture and electrostatic templating of GTP-tubulin

TOG domain microtubule polymerases track microtubule plus ends, bind GTP-tubulin and catalyse microtubule growth, by mechanisms that are not yet understood. In this work, we use computational analysis and simulation to probe the detailed mechanism of tubulin capture and exchange by TOG domains. TOG domains display a ridge of 5 surface loops that form the core of the TOG-tubulin interface. Using computational mutagenesis, we confirm that this row of loops, which is positively charged, plays a dominant role in setting the overall electrostatic field on the TOG domain. Brownian dynamics simulations establish that diffusion-to-capture of TOGs by tubulin is very strongly electrostatically steered. Under a range of conditions and in all trajectories examined, TOGs are initially captured and oriented by tubulin at high radius so that their basic loops faced inwards towards the tubulin. Thereafter, the loops continue to face inwards towards the tubulin and to scan its surface until stereospecific docking the crystallographic binding site occurs. We find that the acidic C-terminal tails of tubulin are not required for electrostatic steering, but instead serve to widen the acceptance angles for electrostatically steered diffusion-to-capture. All-atom normal mode analysis indicates that TOGs are remarkably stiff, enabling them to drive free GTP-tubulin into a partially-curved state by conformational selection. Electrostatic free energy calculations show that the complex that each TOG makes with its cognate tubulin is stable. Our work argues that TOGs accelerate microtubule plus end growth by two complementary electrostatic mechanisms, first by electrostatically steered diffusion-to-capture, and second by electrostatic stabilisation of a partially bent conformation of GTP-tubulin that exchanges rapidly into the tip-lattice. To explain this rapid exchange, we propose a model in which simultaneous binding of the GTP-tubulin to the TOG and the microtubule tip-lattice can occur and is required to de-stabilise the crystallographic complex and release and recycle the TOG.\n\nAuthor SummaryTOG domain microtubule polymerases are protein machines that accelerate the growth of microtubule plus ends by capturing tubulin building blocks from solution and feeding them into the growing microtubule tip. Exactly how TOGs manage to do this remains unclear. Several lines of evidence suggest that electrostatic interactions play a key role, but the detailed role of electrostatics in the polymerase mechanism of TOGs is so far little explored. Here using linked computational approaches we analyse the electrostatic fields of TOGs from the TOG polymerase superfamily and simulate their tubulin binding trajectories. We find that each TOG domain has a shaped electrostatic field that is precisely matched to its tubulin binding partner, such that each TOG is electrostatically orientated at high radius and thereafter electrostatically guided to its capture site. Our work shows that electrostatic steering dramatically accelerates the diffusion-to-capture of tubulin by TOGs. The resulting TOG-tubulin complexes are electrostatically stabilized and we suggest that release of the TOG from this complex requires that tubulin first be incorporated into the growing microtubule, thereby being driven into a TOG-incompatible conformation.

biophysics↗

bioRxiv2017

Single molecule mechanics reveal Kif15 as an active molecular ratchet with acute strain sensitivity

Human Kif15 is a tetrameric kinesin-12 that contributes critically to bipolar spindle assembly in eukaryotes. Here we examine its single molecule mechanics. Under hindering loads, Kif15 steps predominantly towards microtubule plus ends, with its forestep:backstep ratio decreasing exponentially with load and stall occurring at ~6pN. Between steps, Kif15 binds stably, usually via a single head domain. By complete contrast, under assisting loads, Kif15 detaches rapidly, even in AMPPNP. Furthermore, Kif15 can autoinhibit, via an interaction requiring its C-terminus. Autoinhibited Kif15 binds microtubules nucleotide-independently, resists both hindering and assisting loads, and is further stabilized by Tpx2, which interacts with the Kif15 C-terminus. Our data reveal the mechanics of Kif15 to be extraordinarily sensitive to loading direction. When unloaded, it walks rapidly; when pulled forwards it slips and when pulled backwards it grips. We discuss the implications of this unique mechanical behaviour for the roles of Kif15 in spindle function.

cell biology↗

bioRxiv2017

Structural-mechanical remodelling of GDP-microtubules by kinesin

Kinesin-1 is a nanoscale molecular motor that walks towards the fast growing (plus) ends of microtubules (MTs), hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organisation of eukaryotic cells and shows great promise as a tool for nano-engineering1,2. Recent work hints that kinesin may also play a role in modulating the stability of its MT track, both in vitro3-5 and in vivo6, but results are conflicting7-9 and mechanisms are unclear. Here we report a new dimension to the kinesin-MT interaction, whereby strong-state (ATP-bound and apo) kinesin-1 motor domains inhibit the shrinkage of GDP-MTs by up to 2 orders of magnitude and expand their lattice spacing by ~1.6%. Our data reveal an unexpected new mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, allowing motile kinesins to influence the structure, stability and mechanics of their MT track.

biophysics↗