Minus Ends Research Articles

CAMSAP3 forms dimers via its α-helix domain that directly stabilize non-centrosomal microtubule minus ends.

Microtubules are vital components of the cytoskeleton. Their plus ends are dynamic and respond to changes in cell morphology, while the minus ends are stable and serve a crucial role in microtubule seeding and maintaining spatial organization. In mammalian cells, the calmodulin-regulated spectrin-associated proteins (CAMSAPs), play a key role in directly regulating the dynamics of non-centrosomal microtubules minus ends. However, the molecular mechanisms are not yet fully understood. Our study reveals that CAMSAP3 forms dimers through its C-terminal α-helix; this dimerization not only enhances the microtubule-binding affinity of the CKK domain but also enables the CKK domain to regulate the dynamics of microtubules. Furthermore, CAMSAP3 also specializes in decorating at the minus end of microtubules through the combined action of the microtubule-binding domain (MBD) and the C-terminal α-helix, thereby achieving dynamic regulation of the minus ends of microtubules. These findings are crucial for advancing our understanding and treatment of diseases associated with non-centrosomal microtubules.

Molecular Motors in Myelination and Their Misregulation in Disease.

Molecular motors are cellular components involved in the intracellular transport of organelles and materials to ensure cell homeostasis. This is particularly relevant in neurons, where the synaptic components synthesized in the soma need to travel overlong distances to their destination. They can walk on microtubules (kinesins and dyneins) or actin filaments (myosins), themajor components of cell cytoskeleton. While kinesins mostly perform the anterograde transport of intracellular components toward the plus ends of microtubules located distally in cell processes, cytoplasmic dyneins allow the retrograde flux of intracellular cargo toward the minus ends of microtubules located at the cell soma. Axon myelination represents a major aspect of neuronal maturation and is essential for neuronal function, as it speeds up the transmission of electrical signals. Increasing evidence supports a role for molecular motors in the homeostatic control of myelination. This role includes the trafficking of myelin components along the processes of myelinating cells and local regulation of pathways that ensure axon wrapping. Dysfunctional control of the intracellular transport machinery has thereforebeen linked to several brain pathologies, including demyelinating diseases. These disordersinclude a broad spectrum of conditions characterized by pathological demyelination of axons within the nervous system, ultimately leading to axonal degeneration and neuronal death, withmultiple sclerosis representing the most prevalent and studied condition. This review highlights the involvement of molecular motors in the homeostatic control of myelination. It also discusses studies that have yielded insights into the dysfunctional activity of molecular motors in the pathophysiology of multiple sclerosis.

A subpellicular microtubule dynein transport machinery regulates ookinete morphogenesis for mosquito transmission of Plasmodium yoelii

The cortical cytoskeleton of subpellicular microtubules (SPMTs) supports the Plasmodium ookinete morphogenesis during mosquito transmission of malaria. SPMTs are hypothesized to function as the cytoskeletal tracks in motor-driven cargo transport for apical organelle and structure assembly in ookinetes. However, the SPMT-based transport motor has not been identified in the Plasmodium. The cytoplasmic dynein is the motor moving towards the minus end of microtubules (MTs) and likely be responsible for cargo transport to the apical part in ookinetes. Here we screen 7 putative dynein heavy chain (DHC) proteins in the P. yoelii and identify DHC3 showing peripheral localization in ookinetes. DHC3 is localized at SPMTs throughout ookinete morphogenesis. We also identify five other dynein subunits localizing at SPMTs. DHC3 disruption impairs ookinete development, shape, and gliding, leading to failure in mosquito infection of Plasmodium. The DHC3-deficient ookinetes display defective formation or localization of apical organelles and structures. Rab11A and Rab11B interact with DHC3 at SPMTs in a DHC3-dependent manner, likely functioning as the receptors for the cargoes driven by SPMT-dynein. Disturbing Rab11A or Rab11B phenocopies DHC3 deficiency in ookinete morphogenesis. Our study reveals an SPMT-based dynein motor driving the transport of Rab11A- and Rab11B-labeled cargoes in the ookinete morphogenesis of Plasmodium.

Kinesin-5/Cut7 C-terminal tail phosphorylation is essential for microtubule sliding force and bipolar mitotic spindle assembly

Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms1,2,3,4,5,6,7,8,9,10,11: they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart.12,13,14,15,16,17 This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 where they show enhanced localization.3,5,7,27,29,32 The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-535,36,37 along with motility and spindle assembly in cells.27,38,39 The tail contains phosphorylation sites, particularly in the conserved BimC box.6,7,40,41,42,43,44 Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe,45,46,47,48 suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors.39,49,50,51,52 Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5.36 Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast.

Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons.

Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.

Modeling study of kinesin-13 MCAK microtubule depolymerase.

Mitotic centromere-associated kinesin (MCAK) motor protein is a typical member of the kinesin-13 family, which can depolymerize microtubules from both plus and minus ends. A critical issue for the MCAK motor is how it performs the depolymerase activity. To address the issue, the pathway of the MCAK motor moving on microtubules and depolymerizing the microtubules is presented here. On the basis of the pathway, the dynamics of both the wild-type and mutant MCAK motors is studied theoretically, which include the full-length MCAK, the full-length MCAK with mutations in the α4-helix of the motor domain, the mutant full-length MCAK with a neutralized neck, the monomeric MCAK and the mutant monomeric MCAK with a neutralized neck. The studies show that a single dimeric MCAK motor can depolymerize microtubules in a processive manner, with either one tubulin or two tubulins being removed per times. The theoretical results are in agreement with the available experimental data. Moreover, predicted results are provided.

The structure of the γ-TuRC at the microtubule minus end - not just one solution.

In cells, microtubules (MTs) assemble from α/β-tubulin subunits at nucleation sites containing the γ-tubulin ring complex (γ-TuRC). Within the γ-TuRC, exposed γ-tubulin molecules act as templates for MT assembly by interacting with α/β-tubulin. The vertebrate γ-TuRC is scaffolded by γ-tubulin-interacting proteins GCP2-6 arranged in a specific order. Interestingly, the γ-tubulin molecules in the γ-TuRC deviate from the cylindrical geometry of MTs, raising the question of how the γ-TuRC structure changes during MT nucleation. Recent studies on the structure of the vertebrate γ-TuRC attached to the end of MTs came to varying conclusions. In vitro assembly of MTs, facilitated by an α-tubulin mutant, resulted in a closed, cylindrical γ-TuRC showing canonical interactions between all γ-tubulin molecules and α/β-tubulin subunits. Conversely, native MTs formed in a frog extract were capped by a partially closed γ-TuRC, with some γ-tubulin molecules failing to align with α/β-tubulin. This review discusses these outcomes, along with the broader implications.

Stepping dynamics of dynein characterized by MINFLUX.

Cytoplasmic dynein is a dimeric motor that drives minus-end directed transport on microtubules (MTs). To couple ATP hydrolysis to a mechanical step, a dynein monomer must be released from the MT before undergoing a conformational change that generates a bias towards the minus end. However, the dynamics of dynein stepping have been poorly characterized by tracking flexible regions of the motor with limited resolution. Here, we developed a cysteine-light mutant of yeast dynein and site-specifically labeled its MT-binding domain in vitro. MINFLUX tracking at sub-millisecond resolution revealed that dynein hydrolyzes one ATP per step and takes multitudes of 8 nm steps at physiological ATP. Steps are preceded by the transient movement towards the plus end. We propose that these backward "dips" correspond to MT release and subsequent diffusion of the stepping monomer around its MT-bound partner before taking a minus-end-directed conformational change of its linker. Our results reveal the order of sub-millisecond events that result in a productive step of dynein.

Tether-scanning the kinesin motor domain reveals a core mechanical action

Natural kinesin motors are tethered to their cargoes via short C-terminal or N-terminal linkers, whose docking against the core motor domain generates directional force. It remains unclear whether linker docking is the only process contributing directional force or whether linker docking is coupled to and amplifies an underlying, more fundamental force-generating mechanical cycle of the kinesin motor domain. Here, we show that kinesin motor domains tethered via double-stranded DNAs (dsDNAs) attached to surface loops drive robust microtubule (MT) gliding. Tethering using dsDNA attached to surface loops disconnects the C-terminal neck-linker and the N-terminal cover strand so that their dock-undock cycle cannot exert force. The most effective attachment positions for the dsDNA tether are loop 2 or loop 10, which lie closest to the MT plus and minus ends, respectively. In three cases, we observed minus-end-directed motility. Our findings demonstrate an underlying, potentially ancient, force-generating core mechanical action of the kinesin motor domain, which drives, and is amplified by, linker docking.

The initiation and early development of apical-basal polarity in Toxoplasma gondii.

The human parasite Toxoplasma gondii has a distinctive body plan with a well-defined polarity. In the apical complex, the minus ends of the 22 cortical microtubules are anchored to the apical polar ring, a putative microtubule-organizing center. The basal complex caps and constricts the parasite posterior end, and is critical for cytokinesis. How this apical-basal polarity axis is initiated was unknown. Here we examined the development of the apical polar ring and the basal complex in nascent daughters using expansion microscopy. We found that different substructures in the apical polar ring have different sensitivity to stress. In addition, apical-basal differentiation is already established upon nucleation of the cortical microtubule array: arc forms of the apical polar ring and basal complex associate with opposite ends of the microtubules. As the construction of the daughter framework progresses towards the centrioles, the apical and the basal arcs co-develop in striking synchrony ahead of the microtubule array, and close into a ring-form before all the microtubules are nucleated. We also found that two apical polar ring components, APR2 and KinesinA, act synergistically. The removal of each protein individually has modest to no impact on the lytic cycle. However, the loss of both results in abnormalities in the microtubule array and highly reduced plaquing and invasion efficiency.

Tunable intracellular transport on converging microtubule morphologies

A common type of cytoskeletal morphology involves multiple microtubules converging with their minus ends at the microtubule organizing center (MTOC). The cargo-motor complex will experience ballistic transport when bound to microtubules or diffusive transport when unbound. This machinery allows for sequestering and subsequent dispersal of dynein-transported cargo. The general principles governing dynamics, efficiency, and tunability of such transport in the MTOC vicinity are not fully understood. To address this, we develop a one-dimensional model that includes advective transport toward an attractor (such as the MTOC) and diffusive transport that allows particles to reach absorbing boundaries (such as cellular membranes). We calculated the mean first passage time (MFPT) for cargo to reach the boundaries as a measure of the effectiveness of sequestering (large MFPT) and diffusive dispersal (low MFPT). We show that the MFPT experiences a dramatic growth, transitioning from a low to high MFPT regime (dispersal to sequestering) over a window of cargo on-/off-rates that is close to in vivo values. Furthermore, increasing either the on-rate (attachment) or off-rate (detachment) can result in optimal dispersal when the attractor is placed asymmetrically. Finally, we also describe a regime of rare events where the MFPT scales exponentially with motor velocity and the escape location becomes exponentially sensitive to the attractor positioning. Our results suggest that structures such as the MTOC allow for the sensitive control of the spatial and temporal features of transport and corresponding function under physiological conditions.

Stable GDP-tubulin islands rescue dynamic microtubules.

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.

Minimal Mechanisms of Microtubule Length Regulation in Living Cells.

The microtubule cytoskeleton is responsible for sustained, long-range intracellular transport of mRNAs, proteins, and organelles in neurons. Neuronal microtubules must be stable enough to ensure reliable transport, but they also undergo dynamic instability, as their plus and minus ends continuously switch between growth and shrinking. This process allows for continuous rebuilding of the cytoskeleton and for flexibility in injury settings. Motivated by in vivo experimental data on microtubule behavior in Drosophila neurons, we propose a mathematical model of dendritic microtubule dynamics, with a focus on understanding microtubule length, velocity, and state-duration distributions. We find that limitations on microtubule growth phases are needed for realistic dynamics, but the type of limiting mechanism leads to qualitatively different responses to plausible experimental perturbations. We therefore propose and investigate two minimally-complex length-limiting factors: limitation due to resource (tubulin) constraints and limitation due to catastrophe of large-length microtubules. We combine simulations of a detailed stochastic model with steady-state analysis of a mean-field ordinary differential equations model to map out qualitatively distinct parameter regimes. This provides a basis for predicting changes in microtubule dynamics, tubulin allocation, and the turnover rate of tubulin within microtubules in different experimental environments.

Modeling Studies of the Mechanism of Context-Dependent Bidirectional Movements of Kinesin-14 Motors.

Kinesin-14s, a subfamily of the large superfamily of kinesin motor proteins, function mainly in spindle assembly and maintenance during mitosis and meiosis. KlpA from Aspergillus nidulans and GiKIN14a from Giardia intestinalis are two types of kinesin-14s. Available experimental results puzzlingly showed that while KlpA moves preferentially toward the minus end in microtubule-gliding setups and inside parallel microtubule overlaps, it moves preferentially toward the plus end on single microtubules. More puzzlingly, the insertion of an extra polypeptide linker in the central region of the neck stalk switches the motility direction of KlpA on single microtubules to the minus end. Prior experimental results showed that GiKIN14a moves preferentially toward the minus end on single microtubules in either tailless or full-length forms. The tail not only greatly enhances the processivity but also accelerates the ATPase rate and velocity of GiKIN14a. The insertion of an extra polypeptide linker in the central region of the neck stalk reduces the ATPase rate of GiKIN14a. However, the underlying mechanism of these puzzling dynamical features for KlpA and GiKIN14a is unclear. Here, to understand this mechanism, the dynamics of KlpA and GiKIN14a were studied theoretically on the basis of the proposed model, incorporating potential changes between the kinesin head and microtubule, as well as the potential between the tail and microtubule. The theoretical results quantitatively explain the available experimental results and provide predicted results. It was found that the elasticity of the neck stalk determines the directionality of KlpA on single microtubules and affects the ATPase rate and velocity of GiKIN14a on single microtubules.

Structure of the native γ-tubulin ring complex capping spindle microtubules

Microtubule (MT) filaments, composed of α/β-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/β-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/β-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.

Structure of the γ-tubulin ring complex-capped microtubule.

Microtubules are composed of α-tubulin and β-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/β-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.3 MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryogenic electron microscopy reconstructions of γ-TuRC-capped microtubule minus ends reveal the extensive intra-domain and inter-domain motions of γ-TuRC subunits that accommodate luminal bridge components and establish lateral and longitudinal interactions between γ-tubulins and α-tubulins. Our structures suggest that γ-TuRC, an inefficient nucleation template owing to its splayed conformation, can transform into a compacted cap at the microtubule minus end and set the lattice architecture of cellular microtubules.

γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.

Opposing actions of JIP4 and RILPL1 provide antagonistic motor force to dynamically regulate membrane reformation during lysosomal tubulation/sorting driven by LRRK2.

Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by Leucine-Rich Repeat Kinase 2 [LRRK2]), wherein damaged lysosomes generate tubules sorted into mobile vesicles. LYTL is orchestrated by the Parkinson's disease-associated kinase LRRK2 that recruits the motor adaptor protein and RHD family member JIP4 to lysosomes via phosphorylated RAB proteins. To identify new players involved in LYTL, we performed unbiased proteomics on isolated lysosomes after LRRK2 kinase inhibition. Our results demonstrate that there is recruitment of RILPL1 to ruptured lysosomes via LRRK2 activity to promote phosphorylation of RAB proteins at the lysosomal surface. RILPL1, which is also a member of the RHD family, enhances the clustering of LRRK2-positive lysosomes in the perinuclear area and causes retraction of LYTL tubules, in contrast to JIP4 which promotes LYTL tubule extension. Mechanistically, RILPL1 binds to p150Glued, a dynactin subunit, facilitating the transport of lysosomes and tubules to the minus end of microtubules. Further characterization of the tubulation process revealed that LYTL tubules move along tyrosinated microtubules, with tubulin tyrosination proving essential for tubule elongation. In summary, our findings emphasize the dynamic regulation of LYTL tubules by two distinct RHD proteins and pRAB effectors, serving as opposing motor adaptor proteins: JIP4, promoting tubulation via kinesin, and RILPL1, facilitating tubule retraction through dynein/dynactin. We infer that the two opposing processes generate a metastable lysosomal membrane deformation that facilitates dynamic tubulation events.

The mechanism and energetics of the dynein priming stroke

Dyneins are an AAA+ motor responsible for motility and force generation toward the minus end of microtubules. Dynein motility is powered by nucleotide-dependent transitions of its linker domain, which transitions between straight (post-powerstroke) and bent (pre-powerstroke) conformations. To understand the dynamics and energetics of the linker, we performed all-atom molecular dynamics simulations of human dynein-2 primed for its power stroke. Simulations revealed that the linker can adopt either a bent conformation or a semi-bent conformation, separated by a 5.7 kT energy barrier. The linker cannot switch back to its straight conformation in the pre-powerstroke state due to a steric clash with the AAA+ ring. Simulations also showed that an isolated linker has a free energy minimum near the semi-bent conformation in the absence ofthe AAA+ ring, indicating that the linker stores energy as it bends and releases this energy during the powerstroke.

CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.