Biology subjects

Gennerich, A.

Publications and source records attributed to Gennerich, A..

13 recordsLinked to original sources

bioRxiv2026

The P3018S disease variant reveals how dynein's trailing motor sets ensemble velocity.

Mutations in the cytoplasmic dynein heavy chain (DYNC1H1) underlie a range of neurodevelopmental disorders yet how individual variants perturb dynein function remains poorly understood. We characterize the disease-associated P3018S mutation, located in the AAA4 module and within the Lis1-interacting region of the motor. Dynein-dynactin-BicD2 (DDB) complexes containing P3018S dynein move at half the wild-type velocity and generate reduced stall forces but retain the ability to assume the inhibitory phi conformation that Lis1 suppresses, respond to Lis1, and assemble into higher-order force-generating states. Several Schizosaccharomyces species, which lack Lis1, naturally encode a serine at this position, suggesting evolutionary relevance for dynein activation. Using mixed wild-type-mutant assemblies, we find that the dynein occupying the trailing position on dynactin dictates ensemble velocity, and that the leading dyneins LIC enhances trailing-motor velocity by [~]130%, revealing a mechanism for leading-to-trailing motor stimulation within multi-dynein assemblies.

biophysics↗

bioRxiv2025

Pathogenic KIF1A R350 Variants Disrupt A Conserved Kinesin-Tubulin Salt Bridge

Pathogenic variants in the motor domain of the kinesin-3 motor protein KIF1A cause a range of neurodevelopmental and neurodegenerative conditions collectively termed KIF1A-associated neurological disorder (KAND). Among these, mutations at residue R350 are linked to hereditary spastic paraplegia and altered motor function. Yet, the structural basis for their pathogeny remains unclear. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of KIF1A R350G and R350W bound to microtubules in both the apo and AMP-PNP-bound states. We identify a previously unrecognized salt bridge between KIF1A residue R350 and -tubulin E415 that is disrupted in both mutants. This loss of electrostatic interaction correlates with increased velocity and reduced processivity, as demonstrated by single-molecule assays. Our results reveal a conserved electrostatic interaction at the motor-microtubule interface that regulates KIF1As motility behavior.

biochemistry↗

bioRxiv2025

Enhanced axonal transport in large vertebrates: KIF5A adaptations in giraffes and pythons

Efficient axonal transport is essential for neuronal function, particularly in species with exceptionally long axons. The Kinesin-1 motor protein KIF5A plays a key role in this process, but whether and how it adapts to the transport demands of large vertebrates remains unclear. Here, we show that KIF5A from giraffes (GcKIF5A) and pythons (PbKIF5A), moves 25% faster than its mouse counterpart (MmKIF5A) on neuronal microtubules in vitro and within axons of cultured mouse hippocampal neurons. This enhanced velocity, driven by three unique amino acid substitutions (R114Q, S155A and Y309F), facilitates long-distance transport in these species and represents a case of convergent evolution. Our structural analysis reveals that accelerated ADP release underlies the increased speed of GcKIF5A. Despite exhibiting a reduced force generation, GcKIF5A maintains efficient cargo transport under load. Furthermore, GcKIF5A exerts less drag in mixed-motor environments, an adaptation particularly beneficial for multiple motor-driven long-distance axonal transport. These findings reveal that KIF5A has evolved specific adaptations to facilitate efficient axonal transport in large vertebrates, highlighting the evolutionary plasticity of kinesin motors.

biophysics↗

bioRxiv2025

Distinct Clinical Phenotypes in KIF1A-Associated Neurological Disorders Result from Different Amino Acid Substitutions at the Same Residue in KIF1A

KIF1A is a neuron-specific kinesin motor responsible for intracellular transport along axons. Pathogenic KIF1A mutations cause KIF1A-associated neurological disorders (KAND), a spectrum of severe neurodevelopmental and neurodegenerative conditions. While individual KIF1A mutations have been studied, how different substitutions at the same residue affect motor function and disease progression remains unclear. Here, we systematically examine the molecular and clinical consequences of mutations at three key motor domain residues--R216, R254, and R307--using single-molecule motility assays and genotype-phenotype associations. Our findings reveal that mutations at R216 and R254 produce residue-specific effects, with some substitutions partially retaining function, whereas R307 mutations universally abolish motility, likely due to its critical role in microtubule binding. Mutant motor properties correlate with developmental outcomes, with R307 mutations linked to the most sever impairments. These results demonstrate that even single-residue substitutions can lead to distinct molecular and clinical phenotypes, highlighting the finely tuned mechanochemical properties of KIF1A. By establishing residue-specific genotype-phenotype relationships, this work provides fundamental insights into KAND pathogenesis and informs targeted therapeutic strategies.

biophysics↗

bioRxiv2025

A Two-Heads-Bound State Drives KIF1A Superprocessivity

KIF1A, a neuron-specific Kinesin-3 motor, is indispensable for long-distance axonal transport and nuclear migration, processes vital for neuronal function. Using MINFLUX tracking, we reveal that KIF1A predominantly adopts a two-heads-bound state, even under ATP-limiting conditions, challenging prior models proposing a one-head-bound rate-limiting step. This two-heads-bound conformation, stabilized by interactions between the positively charged K-loop and negatively charged tubulin tails, enhances microtubule affinity and minimizes detachment. The shorter neck linker facilitates inter-head tension, keeping the heads out of phase and enabling highly coordinated stepping. In contrast, Kinesin-1 (KIF5B) transitions to a one-head-bound state under similar conditions, limiting its processivity. Perturbing KIF1As mechanochemical cycle by prolonging its one-head-bound state significantly reduces processivity, underscoring the critical role of the two-heads-bound state in motility. These findings establish a mechanistic framework for understanding KIF1As adaptations for neuronal transport and dysfunction in neurological diseases.

biophysics↗

bioRxiv2025

The Power of Three: Dynactin associates with three dyneins under load for greater force production

Cytoplasmic dynein is an essential microtubule motor protein that powers organelle transport and mitotic spindle assembly. Its activity depends on dynein-dynactin-cargo adaptor complexes, such as dynein-dynactin-BicD2 (DDB), which typically function with two dynein motors. We show that mechanical tension recruits a third dynein motor via an auxiliary BicD adaptor binding the light intermediate chain of the third dynein, stabilizing multi-dynein assemblies and enhancing force generation. Lis1 prevents dynein from transitioning into a force-limiting phi-like conformation, allowing single-dynein DDB to sustain forces up to [~]4.5 pN, whereas force generation often ends at [~]2.5 pN without Lis1. Complexes with two or three dyneins generate [~]7 pN and [~]9 pN, respectively, consistent with a staggered motor arrangement that enhances collective output. Under load, DDB primarily takes [~]8 nm steps, challenging existing dynein coordination models. These findings reveal adaptive mechanisms that enable robust intracellular transport under varying mechanical demands.

biophysics↗

bioRxiv2023

Kinesin-14 HSET and KlpA are non-processive microtubule motors with load-dependent power strokes

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs minus ends at the poles. We investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA, revealing that both motors function as non-processive motors under load, producing single power strokes per MT encounter. Each homodimeric motor generates forces of [~]0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Importantly, cooperative activity among multiple motors leads to increased MT-sliding velocities. Our findings deepen our understanding of the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.

biophysics↗

bioRxiv2023

High-Resolution structures of microtubule-bound KIF1A and its pathogenic variant P305L

Mutations in the microtubule-associated motor protein KIF1A lead to severe neurological conditions known as KIF1A-associated neurological disorders (KAND). Despite insights into its molecular mechanism, high-resolution structures of KIF1A-microtubule complexes remain undefined. Here, we present 2.7-3.4 [A] resolution structures of dimeric microtubule-bound KIF1A, including the pathogenic P305L mutant, across various nucleotide states. Our structures reveal that KIF1A binds microtubules in one- and two-heads-bound configurations, with both heads exhibiting distinct conformations with tight inter-head connection. Notably, KIF1As class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both - and {beta}-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12s conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1As superprocessive motility. Our findings advance the understanding of KIF1As molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.

biochemistry↗

bioRxiv2022

Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking

Mutations in the microtubule (MT)-binding protein doublecortin (DCX) or in the MT- based molecular motor dynein result in lissencephaly. However, a functional link between DCX and dynein has not been defined. Here, we demonstrate that DCX negatively regulates dynein-mediated retrograde transport by reducing dyneins association with MTs and by disrupting the composition of the dynein motor complex. Previous work showed an increased binding of the adaptor protein C-Jun-amino-terminal kinase-interacting protein 3 (JIP3) to dynein in the absence of DCX. Using purified components, we demonstrate that JIP3 forms an active motor complex with dynein and its cofactor dynactin with two dyneins per complex. DCX competes with the binding of the second dynein, resulting in a velocity reduction of the complex. We conclude that DCX negatively regulates dynein-mediated retrograde transport through two critical interactions by regulating dynein binding to MTs and by regulating the association of JIP3 to the dynein motor complex.

cell biology↗

bioRxiv2022

ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain of function

Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and produce motor proteins with an altered C-terminal tail (referred to as {Delta}Exon27). However, the underlying pathogenic mechanism is still unknown. In this study, we performed a comprehensive analysis of {Delta}Exon27 at the single-molecule, cellular, and organism levels. Our results show that {Delta}Exon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of {Delta}Exon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis and premature death. Our results suggest gain of function as an underlying disease mechanism in KIF5A-associated ALS.

neuroscience↗

bioRxiv2020

A Highly Conserved 310-Helix Within the Kinesin Motor Domain is Critical for Kinesin Function and Human Health.

KIF1A, a kinesin-3 family member, plays critical roles as a long-distance cargo-transporter within neurons. Over 100 known KIF1A mutations in humans result in KIF1A Associated Neurological Disease (KAND), developmental and degenerative neurological conditions for which there is no cure. A de novo missense mutation, P305L, was recently identified in several children diagnosed with KAND, but the underlying molecular basis for the disease phenotype is unknown. Interestingly, this residue is highly conserved in kinesin-family proteins, and together with adjacent conserved residues also implicated in KAND, forms an unusual 310-helical element immediately C-terminal to loop-12 (L12, also known as the K-loop in KIF1A) in the conserved kinesin motor core. In KIF1A, the disordered K-loop contains a highly charged insertion of lysines that is thought to endow the motor with a high microtubule-association rate. Here, we characterize the molecular defects of the P305L mutation in KIF1A using genetic, biochemical, and single-molecule approaches. We find the mutation negatively impacts the velocity, run-length, and force generation of the motor. However, a much more dramatic effect is observed on the microtubule-association rate of the motor, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that an elusive K-loop conformation, mediated by formation of a highly conserved adjacent 310-helix that is modulated via P305, is critically important for the kinesin-microtubule interaction. Importantly, we find that the function of this proline is conserved in the canonical kinesin, KIF5, revealing a fundamental principle of the kinesin motor mechanism.

biophysics↗

bioRxiv2020

The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dyneins mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dyneins stepping cycle and underscores the complexity and crosstalk among the motors multiple AAA+ domains.

biophysics↗

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↗