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 are predicted to produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC-derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single-molecule, cellular, and organism levels. Our results show that ΔExon27 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 ΔExon27 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.

Sequences in the stalk domain regulate auto-inhibition and ciliary tip localization of the immotile kinesin-4 KIF7.

The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by auto-inhibition, as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for auto-inhibition of KIF7, whereas the adjacent regulatory coiled coil (rCC) that contributes to auto-inhibition of the motile kinesin-4s KIF21A and KIF21B is not sufficient for KIF7 auto-inhibition. Disease-associated mutations in the inhCC relieve auto-inhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC, and could be increased by disease-associated mutations regardless of the auto-inhibition state of the protein. These findings suggest that loss of KIF7 auto-inhibition and/or altered cilium tip localization can contribute to the pathogenesis of human disease.

Intramolecular strain coordinates kinesin stepping behavior along microtubules.

Kinesin advances 8 nm along a microtubule per ATP hydrolyzed, but the mechanism responsible for coordinating the enzymatic cycles of kinesin's two identical motor domains remains unresolved. Here, we have tested whether such coordination is mediated by intramolecular tension generated by the "neck linkers," mechanical elements that span between the motor domains. When tension is reduced by extending the neck linkers with artificial peptides, the coupling between ATP hydrolysis and forward stepping is impaired and motor's velocity decreases as a consequence. However, speed recovers to nearly normal levels when external tension is applied by an optical trap. Remarkably, external load also induces bidirectional stepping of an immotile kinesin that lacks its mechanical element (neck linker) and fuel (ATP). Our results indicate that the kinesin motor domain senses and responds to strain in a manner that facilitates its plus-end-directed stepping and communication between its two motor domains.

Optical Control of Mitosis with a Photoswitchable Eg5 Inhibitor.

Eg5 is a kinesin motor protein that is responsible for bipolar spindle formation and plays a crucial role during mitosis. Loss of Eg5 function leads to the formation of monopolar spindles, followed by mitotic arrest, and subsequent cell death. Several cell-permeable small molecules have been reported to inhibit Eg5 and some have been evaluated as anticancer agents. We now describe the design, synthesis, and biological evaluation of photoswitchable variants with five different pharmacophores. Our lead compound Azo-EMD is a cell permeable azobenzene that inhibits Eg5 more potently in its light-induced cis form. This activity decreased the velocity of Eg5 in single-molecule assays, promoted formation of monopolar spindles, and led to mitotic arrest in a light dependent way.

Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains.

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

Structure and Function of Dynein's Non-Catalytic Subunits.

Dynein, an ancient microtubule-based motor protein, performs diverse cellular functions in nearly all eukaryotic cells, with the exception of land plants. It has evolved into three subfamilies-cytoplasmic dynein-1, cytoplasmic dynein-2, and axonemal dyneins-each differentiated by their cellular functions. These megadalton complexes consist of multiple subunits, with the heavy chain being the largest subunit that generates motion and force along microtubules by converting the chemical energy of ATP hydrolysis into mechanical work. Beyond this catalytic core, the functionality of dynein is significantly enhanced by numerous non-catalytic subunits. These subunits are integral to the complex, contributing to its stability, regulating its enzymatic activities, targeting it to specific cellular locations, and mediating its interactions with other cofactors. The diversity of non-catalytic subunits expands dynein's cellular roles, enabling it to perform critical tasks despite the conservation of its heavy chains. In this review, we discuss recent findings and insights regarding these non-catalytic subunits.

Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies.

Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and kinesin superfamilies play critical roles in transporting biological payloads (such as proteins, organelles, and vesicles) along microtubule pathways, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes to progeny cells. Understanding the underlying mechanisms and behaviors of motor proteins is critical to provide better strategies for the treatment of motor protein-related diseases. Here, we provide detailed protocols for the recombinant expression of the Kinesin-1 motor KIF5C using a baculovirus/insect cell system and provide updated protocols for performing single-molecule studies using total internal reflection fluorescence microscopy and optical tweezers to study the motility and force generation of the purified motor.

Cryo-EM unveils kinesin KIF1A's processivity mechanism and the impact of 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 2.7-3.5 Å 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, KIF1A's class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both α- and ß-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12's conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1A's superprocessive motility. Our findings advance the understanding of KIF1A's molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.

Measurements of the Force-Dependent Detachment Rates of Cytoplasmic Dynein from Microtubules.

Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules (MT). Despite its essential roles in eukaryotic cells, dynein's molecular mechanism, the regulatory functions of its subunits and accessory proteins, and the consequences of human disease mutations on dynein force generation remain largely unclear. Recent work combining mutagenesis, single-molecule fluorescence, and optical tweezers-based force measurement have provided valuable insights into how dynein's multiple AAA+ ATPase domains regulate dynein's attachment to MTs. Here, we describe detailed protocols for the measurements of the force-dependent dynein-MT detachment rates. We provide updated and optimized protocols for the expression and purification of a tail-truncated single-headed Saccharomyces cerevisiae dynein, for polarity-marked MT polymerization, and for the non-covalent attachment of MTs to cover glass surfaces for the measurement of dynein-MT detachment forces.

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.

Cryo-EM Unveils the Processivity Mechanism of Kinesin KIF1A and the Impact of 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 Å 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, KIF1A's class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both α- and ß-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12's conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1A's superprocessive motility. Our findings advance the understanding of KIF1A's molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.

Kinesin-73 is a processive motor that localizes to Rab5-containing organelles.

Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73. We find that constructs containing the motor and the conserved short stretches of putative coiled-coil-forming regions are predominantly monomeric in vitro, but that dimerization allows for fast, processive movement and high force production (7 piconewtons). In Drosophila cell lines, we present evidence that Khc-73 can dimerize in vivo. We also show that Khc-73 is recruited specifically to Rab5-containing endosomes through its "tail" domain. Our results suggest that the N-terminal half of Khc-73 can undergo a monomer-dimer transition to produce a fast processive motor and that its C-terminal half possesses a specific Rab5-vesicle binding domain.

Single-Molecule Studies on the Motion and Force Generation of the Kinesin-3 Motor KIF1A.

KIF1A is a neuron-specific member of the kinesin-3 family of microtubule (MT) plus-end-directed motor proteins. It powers the migration of nuclei in differentiating brain stem cells and the transport of synaptic precursors and dense core vesicles in axons. Its dysfunction causes severe neurodevelopmental and neurodegenerative diseases termed KIF1A-associated neurological disorders (KAND). KAND mutations span the entirety of the KIF1A protein sequence, of which the majority are located within the motor domain and are thus predicted to affect the motor's motility and force-generating properties. Unfortunately, the molecular etiologies of KAND remain poorly understood, in part because KIF1A's molecular mechanism remains unclear. Here, we describe detailed methods for how to express a tail-truncated dimeric KIF1A in E. coli cells and provide step-by-step protocols for performing single-molecule studies with total internal reflection fluorescence microscopy and optical tweezers assays, which, when combined with structure-function studies, help to decipher KIF1A's molecular mechanism.

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 in neurons from Dcx-/y or Dcx-/y;Dclk1-/- mice by reducing dynein's association with MTs and 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 regulating the composition of the dynein motor complex.

Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3.

OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3-null mutation, suggesting that autoinhibition is important for OSM-3's biological function.

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 facilitates long-distance transport, but little is known about its force-generating properties. Using optical tweezers, we demonstrate that KIF1A stalls at an opposing load of ~3 pN but more frequently detaches at lower forces. KIF1A rapidly reattaches to the microtubule to resume motion due to its class-specific K-loop, resulting in a unique clustering of force generation events. To test the importance of neck linker docking in KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders. Molecular dynamics simulations predict that V8M and Y89D mutations impair neck linker docking. Indeed, both mutations dramatically reduce the force generation of KIF1A but not the motor's ability to rapidly reattach to the microtubule. Although both mutations relieve autoinhibition of the full-length motor, the mutant motors display decreased velocities, run lengths, and landing rates and delayed cargo transport in cells. These results advance our understanding of how mutations in KIF1A can manifest in disease.

Genotype and defects in microtubule-based motility correlate with clinical severity in KIF1A-associated neurological disorder.

KIF1A-associated neurological disorder (KAND) encompasses a group of rare neurodegenerative conditions caused by variants in KIF1A,a gene that encodes an anterograde neuronal microtubule (MT) motor protein. Here we characterize the natural history of KAND in 117 individuals using a combination of caregiver or self-reported medical history, a standardized measure of adaptive behavior, clinical records, and neuropathology. We developed a heuristic severity score using a weighted sum of common symptoms to assess disease severity. Focusing on 100 individuals, we compared the average clinical severity score for each variant with in silico predictions of deleteriousness and location in the protein. We found increased severity is strongly associated with variants occurring in protein regions involved with ATP and MT binding: the P loop, switch I, and switch II. For a subset of variants, we generated recombinant proteins, which we used to assess transport in vivo by assessing neurite tip accumulation and to assess MT binding, motor velocity, and processivity using total internal reflection fluorescence microscopy. We find all modeled variants result in defects in protein transport, and we describe three classes of protein dysfunction: reduced MT binding, reduced velocity and processivity, and increased non-motile rigor MT binding. The rigor phenotype is consistently associated with the most severe clinical phenotype, while reduced MT binding is associated with milder clinical phenotypes. Our findings suggest the clinical phenotypic heterogeneity in KAND likely reflects and parallels diverse molecular phenotypes. We propose a different way to describe KAND subtypes to better capture the breadth of disease severity.

A highly conserved 310 helix within the kinesin motor domain is critical for kinesin function and human health.

KIF1A is a critical cargo transport motor within neurons. More than 100 known mutations result in KIF1A-associated neurological disorder (KAND), a degenerative condition for which there is no cure. A missense mutation, P305L, was identified in children diagnosed with KAND, but the molecular basis for the disease is unknown. We find that this conserved residue is part of an unusual 310 helix immediately adjacent to the family-specific K-loop, which facilitates a high microtubule-association rate. We find that the mutation negatively affects several biophysical parameters of the motor. However, the microtubule-association rate of the motor is most markedly affected, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that the 310 helix facilitates a specific K-loop conformation that is critical for its function. We find that the function of this proline is conserved in kinesin-1, revealing a fundamental principle of the kinesin motor mechanism.

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 dynein's 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 dynein's stepping cycle and underscores the complexity and crosstalk among the motor's multiple AAA+ domains.

Force production of human cytoplasmic dynein is limited by its processivity.

Cytoplasmic dynein is a highly complex motor protein that generates forces toward the minus end of microtubules. Using optical tweezers, we demonstrate that the low processivity (ability to take multiple steps before dissociating) of human dynein limits its force generation due to premature microtubule dissociation. Using a high trap stiffness whereby the motor achieves greater force per step, we reveal that the motor's true maximal force ("stall force") is ~2 pN. Furthermore, an average force versus trap stiffness plot yields a hyperbolic curve that plateaus at the stall force. We derive an analytical equation that accurately describes this curve, predicting both stall force and zero-load processivity. This theoretical model describes the behavior of a kinesin motor under low-processivity conditions. Our work clarifies the true stall force and processivity of human dynein and provides a new paradigm for understanding and analyzing molecular motor force generation for weakly processive motors.