Comparative analysis of two Caenorhabditis elegans kinesins KLP-6 and UNC-104 reveals a common and distinct activation mechanism in kinesin-3.

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

Autoinhibition and activation of kinesin-1 and their involvement in amyotrophic lateral sclerosis.

Kinesin-1, composed of kinesin heavy chain and kinesin light chain, is a founding member of kinesin superfamily and transports various neuronal cargos. Kinesin-1 is one of the most abundant ATPases in the cell and thus need to be tightly regulated to avoid wastage of energy. It has been well established that kinesin-1 is regulated by the autoinhibition mechanism. This review focuses on the recent researches that have contributed to the understanding of mechanisms for the autoinhibition of kinesin-1 and its unlocking. Recent electron microscopic studies have shown an unanticipated structure of autoinhibited kinesin-1. Biochemical reconstitution have revealed detailed molecular mechanisms how the autoinhibition is unlocked. Importantly, misregulation of kinesin-1 is emerging as one of the major causes of amyotrophic lateral sclerosis.

Modeling the motion of disease-associated KIF1A heterodimers.

KIF1A is a member of the kinesin-3 motor protein family that transports synaptic vesicle precursors in axons. Mutations in the Kif1a gene cause neuronal diseases. Most patients are heterozygous and have both mutated and intact KIF1A alleles, suggesting that heterodimers composed of wild-type KIF1A and mutant KIF1A are likely involved in pathogenesis. In this study, we propose mathematical models to describe the motility of KIF1A heterodimers composed of wild-type KIF1A and mutant KIF1A. Our models precisely describe run length, run time, and velocity of KIF1A heterodimers using a few parameters obtained from two homodimers. The first model is a simple hand-over-hand model in which stepping and detachment rates from a microtubule of each head are identical to those in the respective homodimers. Although the velocities of heterodimers expected from this model were in good agreement with the experimental results, this model underestimated the run lengths and run times of some heterodimeric motors. To address this discrepancy, we propose the tethered-head affinity model, in which we hypothesize a tethered head, in addition to a microtubule-binding head, contributes to microtubule binding in a vulnerable one-head-bound state. The run lengths and run times of the KIF1A heterodimers predicted by the tethered-head affinity model matched well with experimental results, suggesting a possibility that the tethered head affects the microtubule binding of KIF1A. Our models provide insights into how each head contributes to the processive movement of KIF1A and can be used to estimate motile parameters of KIF1A heterodimers.

Generation of recombinant and chickenized scFv versions of an anti-kinesin monoclonal antibody H2.

Kinesin-1, a motor protein composed of the kinesin heavy chain (KHC) and the kinesin light chain (KLC), is essential for proper cellular morphogenesis and function. A monoclonal antibody (mAb) called H2 recognizes the KHC in a broad range of species and is one of the most widely used mAbs in cytoskeletal motor research. Here, we present vectors that express recombinant H2 in mammalian cells. We show the recombinant H2 performs as well as the hybridoma-derived H2 in both western blotting and immunofluorescence assays. Additionally, the recombinant H2 can detect all three human KHC isotypes (KIF5A, KIF5B, and KIF5C) and amyotrophic lateral sclerosis-associated KIF5A aggregates in cells. In addition, we developed a chickenized version of the H2 mAb's single chain variable fragment, which can be used in immunofluorescence microscopy and expands the potential applications of H2. Overall, our results demonstrate that recombinant H2 is a useful tool for studying the functions of KHCs.

Insight into the regulation of axonal transport from the study of KIF1A-associated neurological disorder.

Neuronal function depends on axonal transport by kinesin superfamily proteins (KIFs). KIF1A is the molecular motor that transports synaptic vesicle precursors, synaptic vesicles, dense core vesicles and active zone precursors. KIF1A is regulated by an autoinhibitory mechanism; many studies, as well as the crystal structure of KIF1A paralogs, support a model whereby autoinhibited KIF1A is monomeric in solution, whereas activated KIF1A is dimeric on microtubules. KIF1A-associated neurological disorder (KAND) is a broad-spectrum neuropathy that is caused by mutations in KIF1A. More than 100 point mutations have been identified in KAND. In vitro assays show that most mutations are loss-of-function mutations that disrupt the motor activity of KIF1A, whereas some mutations disrupt its autoinhibition and abnormally hyperactivate KIF1A. Studies on disease model worms suggests that both loss-of-function and gain-of-function mutations cause KAND by affecting the axonal transport and localization of synaptic vesicles. In this Review, we discuss how the analysis of these mutations by molecular genetics, single-molecule assays and force measurements have helped to reveal the physiological significance of KIF1A function and regulation, and what physical parameters of KIF1A are fundamental to axonal transport.

Dynein intermediate chains DYCI-1 and WDR-60 have specific functions in Caenorhabditis elegans.

Dynein is a microtubule-dependent motor protein required for cell division, retrograde intracellular transport, and intraflagellar transport (IFT). Dynein 1 and dynein 2 serve as molecular motors in the cytoplasm and cilia, respectively. Each dynein consists of multiple subunits. Although the components of dynein 1 and dynein 2 are different and specific in most species, a previous study has suggested that dynein intermediate chain subunit DYCI-1 is shared by both dynein 1 and 2 in Caenorhabditis elegans (C. elegans). Here, we show that C. elegans has two dynein intermediate chains-DYCI-1 and WDR-60-and their functions are different. Mutational analysis showed that dyci-1 is essential for the retrograde axonal transport of synaptic vesicles. In the same mutant allele, IFT is not affected at all. Instead, wdr-60 is essential for IFT. Thus, we suggest that dynein 1 and dynein 2 have specific intermediate chains in C. elegans as in other organisms.

Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 -tubulin complex.

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

From meter-long structures that allow nerve cells to stretch across a body to miniscule 'hairs' required for lung cells to clear mucus, many life processes rely on cells sporting projections which have the right size for their role. Networks of hollow filaments known as microtubules shape these structures and ensure that they have the appropriate dimensions. Controlling the length of microtubules is therefore essential for organisms, yet how this process takes place is still not fully elucidated. Previous research has shown that microtubules continue to grow when their end is straight but stop when it is curved. A family of molecular motors known as kinesin-4 participate in this process, but the exact mechanisms at play remain unclear. To investigate, Tuguchi, Nakano, Imasaki et al. focused on the KLP-12 protein, a kinesin-4 equivalent which helps to controls the length of microtubules in the tiny worm Caenorhabditis elegans. They performed genetic manipulations and imaged the interactions between KLP-12 and the growing end of a microtubule using X-ray crystallography. This revealed that KLP-12 controls the length of neurons by inhibiting microtubule growth. It does so by modulating the curvature of the growing end of the filament to suppress its extension. A 'snapshot' of KLP-12 binding to a microtubule at the resolution of the atom revealed exactly how the protein helps to bend the end of the filament to prevent it from growing further. These results will help to understand how nerve cells are shaped. This may also provide insights into the molecular mechanisms for various neurodegenerative disorders caused by problems with the human equivalents of KLP-12, potentially leading to new therapies.

De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors.

KIF1A is a kinesin superfamily motor protein that transports synaptic vesicle precursors in axons. Cargo binding stimulates the dimerization of KIF1A molecules to induce processive movement along microtubules. Mutations in human Kif1a lead to a group of neurodegenerative diseases called KIF1A-associated neuronal disorder (KAND). KAND mutations are mostly de novo and autosomal dominant; however, it is unknown if the function of wild-type KIF1A motors is inhibited by heterodimerization with mutated KIF1A. Here, we have established Caenorhabditis elegans models for KAND using CRISPR-Cas9 technology and analyzed the effects of human KIF1A mutation on axonal transport. In our C. elegans models, both heterozygotes and homozygotes exhibited reduced axonal transport. Suppressor screening using the disease model identified a mutation that recovers the motor activity of mutated human KIF1A. In addition, we developed in vitro assays to analyze the motility of heterodimeric motors composed of wild-type and mutant KIF1A. We find that mutant KIF1A significantly impaired the motility of heterodimeric motors. Our data provide insight into the molecular mechanism underlying the dominant nature of de novo KAND mutations.

Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity.

Kinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor. We show that the KLC inhibits the kinesin-microtubule interaction independently from the proposed intramolecular interaction within KHC. Cargo-adaptor proteins that bind the KLC stimulated processive movement, but the landing rate of activated kinesin complexes remained low. Mitogen-activated protein 7 (MAP7) enhanced motility by increasing the landing rate and run length of the activated kinesin motors. Our results support a model whereby the motor activity of the kinesin is regulated by synergistic inhibition mechanisms and that cargo-adaptor binding to the KLC releases both mechanisms. However, a non-motor MAP is required for robust microtubule association of the activated motor. Thus, human kinesin is regulated by synergistic autoinhibition and activation mechanisms.

CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation.

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.

An ALS-associated KIF5A mutant forms oligomers and aggregates and induces neuronal toxicity.

KIF5A is a kinesin superfamily motor protein that transports various cargos in neurons. Mutations in Kif5a cause familial amyotrophic lateral sclerosis (ALS). These ALS mutations are in the intron of Kif5a and induce mis-splicing of KIF5A mRNA, leading to splicing out of exon 27, which in human KIF5A encodes the cargo-binding tail domain of KIF5A. Therefore, it has been suggested that ALS is caused by loss of function of KIF5A. However, the precise mechanisms regarding how mutations in KIF5A cause ALS remain unclear. Here, we show that an ALS-associated mutant of KIF5A, KIF5A(Δexon27), is predisposed to form oligomers and aggregates in cultured mouse cell lines. Interestingly, purified KIF5A(Δexon27) oligomers showed more active movement on microtubules than wild-type KIF5A in vitro. Purified KIF5A(∆exon27) was prone to form aggregates in vitro. Moreover, KIF5A(Δexon27)-expressing Caenorhabditis elegans neurons showed morphological defects. These data collectively suggest that ALS-associated mutations of KIF5A are toxic gain-of-function mutations rather than simple loss-of-function mutations.

Analyzing the Impact of Gene Mutations on Axonal Transport in Caenorhabditis Elegans.

The development and functions of neurons are supported by axonal transport. Axonal transport is a complex process whose regulation involves multiple molecules, such as microtubules, microtubule-associated proteins, kinases, molecular motors, and motor binding proteins. Gain of function and loss of function mutations of genes that encode these proteins often lead to human axonal neuropathy. Caenorhabditis elegans provides a powerful genetic system to study the consequences of gene mutations for axonal transport. Here, we discuss advantages and limitations of using C. elegans, propose standard criteria, and describe methods to analyze the impact of gene mutations on axonal transport in C. elegans. To obtain solid conclusions, it is necessary to image single neurons in vivo labeled by a specific promoter and to confirm that a mutation changes the localization of a cargo. The motility parameters of the transported cargo should then be analyzed in the mutant. This method enables the axonal transport of proteins and organelles, such as synaptic vesicle precursors and mitochondria, to be analyzed.

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.

Neural and behavioral control in Caenorhabditis elegans by a yellow-light-activatable caged compound.

Caenorhabditis elegans is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist N-vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illumination-induced uncaging enables optical control of the behavior of freely moving C. elegans without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.

Effects of dynein inhibitor on the number of motor proteins transporting synaptic cargos.

Synaptic cargo transport by kinesin and dynein in hippocampal neurons was investigated by noninvasively measuring the transport force based on nonequilibrium statistical mechanics. Although direct physical measurements such as force measurement using optical tweezers are difficult in an intracellular environment, the noninvasive estimations enabled enumerating force-producing units (FPUs) carrying a cargo comprising the motor proteins generating force. The number of FPUs served as a barometer for stable and long-distance transport by multiple motors, which was then used to quantify the extent of damage to axonal transport by dynarrestin, a dynein inhibitor. We found that dynarrestin decreased the FPU for retrograde transport more than for anterograde transport. This result indicates the applicability of the noninvasive force measurements. In the future, these measurements may be used to quantify damage to axonal transport resulting from neuronal diseases, including Alzheimer's, Parkinson's, and Huntington's diseases.

A Combinatorial MAP Code Dictates Polarized Microtubule Transport.

Many eukaryotic cells distribute their intracellular components asymmetrically through regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors and unveil MAP9 as a positive modulator of kinesin-3 motility. From these data, we propose a general "MAP code" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.

Going Too Far Is the Same as Falling Short†: Kinesin-3 Family Members in Hereditary Spastic Paraplegia.

Proper intracellular trafficking is essential for neuronal development and function, and when any aspect of this process is dysregulated, the resulting "transportopathy" causes neurological disorders. Hereditary spastic paraplegias (HSPs) are a family of such diseases attributed to over 80 spastic gait genes (SPG), specifically characterized by lower extremity spasticity and weakness. Multiple genes in the trafficking pathway such as those relating to microtubule structure and function and organelle biogenesis are representative disease loci. Microtubule motor proteins, or kinesins, are also causal in HSP, specifically mutations in Kinesin-I/KIF5A (SPG10) and two kinesin-3 family members; KIF1A (SPG30) and KIF1C (SPG58). KIF1A is a motor enriched in neurons, and involved in the anterograde transport of a variety of vesicles that contribute to pre- and post-synaptic assembly, autophagic processes, and neuron survival. KIF1C is ubiquitously expressed and, in addition to anterograde cargo transport, also functions in retrograde transport between the Golgi and the endoplasmic reticulum. Only a handful of KIF1C cargos have been identified; however, many have crucial roles such as neuronal differentiation, outgrowth, plasticity and survival. HSP-related kinesin-3 mutants are characterized mainly as loss-of-function resulting in deficits in motility, regulation, and cargo binding. Gain-of-function mutants are also seen, and are characterized by increased microtubule-on rates and hypermotility. Both sets of mutations ultimately result in misdelivery of critical cargos within the neuron. This likely leads to deleterious cell biological cascades that likely underlie or contribute to HSP clinical pathology and ultimately, symptomology. Due to the paucity of histopathological or cell biological data assessing perturbations in cargo localization, it has been difficult to positively link these mutations to the outcomes seen in HSPs. Ultimately, the goal of this review is to encourage future academic and clinical efforts to focus on "transportopathies" through a cargo-centric lens.

Vital roles of PCNA K165 modification during C. elegans gametogenesis and embryogenesis.

In eukaryotes, the DNA damage bypass pathway is promoted by ubiquitylation of PCNA at the conserved lysine 164. Using CRISPR-Cas9 system, we introduced amino acid substitution at K165 of C. elegans PCNA that corresponds to K164 in other characterised organisms and examined the contribution of this residue at a variety of stages during development. In the presence of UV-induced DNA lesions, PCNA-K165 is crucial for not only the early embryonic stages but also during larval development, implicating its functions for a broad time period during animal development. We also show that, without induction of DNA damage, concomitant inhibition of PCNA ubiquitylation and checkpoint activation causes abnormal gametogenesis events and severely impairs reproduction of worms. Our findings suggest a conserved function of PCNA ubiquitylation in tolerance of UV-induced damage and also propose that PCNA ubiquitylation contributes to gametogenesis during unperturbed C. elegans development.