Kinesin Motor Research Articles

F-box protein FBXB-65 regulates anterograde transport of the kinesin-3 motor UNC-104 through a PTM near its cargo-binding PH domain.

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse.

First person – Vidur Sabharwal

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Vidur Sabharwal is first author on ‘ F-box protein FBXB-65 regulates anterograde transport of the kinesin-3 motor UNC-104 through a PTM near its cargo-binding PH domain’, published in JCS. Vidur conducted the research described in this article while a PhD student in Sandhya P. Koushika's lab at the Tata Institute of Fundamental Research, Mumbai, India. He is now a postdoc in the lab of Abdel El Manira at Karolinska Institutet, Stockholm, Sweden, where he works to understand and describe how complex observations in biological systems emerge from simple underlying mechanisms.

Matrix mechanics regulates muscle regeneration by modulating kinesin-1 activity

Sarcopenia, a prevalent muscle disease characterized by muscle mass and strength reduction, is associated with impaired skeletal muscle regeneration. However, the influence of the biomechanical properties of sarcopenic skeletal muscle on the efficiency of the myogenic program remains unclear. Herein, we established a mouse model of sarcopenia and observed a reduction in stiffness within the sarcopenic skeletal muscle in vivo. To investigate whether the biomechanical properties of skeletal muscle directly impact the myogenic program, we established an in vitro system to explore the intrinsic mechanism involving matrix stiffness control of myogenic differentiation. Our findings identify the microtubule motor protein, kinesin-1, as a mechano-transduction hub that senses and responds to matrix stiffness, crucial for myogenic differentiation and muscle regeneration. Specifically, kinesin-1 activity is positively regulated by stiff matrices, facilitating its role in transporting mitochondria and enhancing translocation of the glucose transporter GLUT4 to the cell surface for glucose uptake. Conversely, the softer matrices significantly suppress kinesin-1 activity, leading to the accumulation of mitochondria around nuclei and hindering glucose uptake by inhibiting GLUT4 membrane translocation, consequently impairing myogenic differentiation. The insights gained from the in-vitro system highlight the mechano-transduction significance of kinesin-1 motor proteins in myogenic differentiation. Furthermore, our study confirms that enhancing kinesin-1 activity in the sarcopenic mouse model restores satellite cell expansion, myogenic differentiation, and muscle regeneration. Taken together, our findings provide a potential target for improving muscle regeneration in sarcopenia.

Abstract 5946: ZM-412, a potent and selective KIF18A inhibitor with robust efficacy as monotherapy in CIN-positive tumors and in combination with MMAE-conjugated ADC in CIN-negative tumors

Abstract Chromosomal instability (CIN), a hallmark of human cancer, is defined as the perpetual missegregation of whole chromosomes during mitosis. KIF18A has emerged as a potential therapeutic target of synthetic lethality to induce CIN tumor cell death because it is more required for the proliferation of CIN tumor cells than normal diploidy cells. Here, we identified a potent and selective KIF18A inhibitor ZM-412. In vitro, ZM-412 inhibited the kinesin motor enzymatic activity with an IC50 less than 30 nM, and arrested OVCAR3 cells in the G2/M phase with an EC50 around 2.0 nM. ZM-412 also selectively inhibited the proliferation of a panel of CIN-positive tumor cell lines with IC50 values less than 50 nM, whereas it didn’t affect the proliferation of CIN-negative cells such as PBMC and near-diploidy HCT116 as indicated by IC50 values over than 5 μM. A CIN-positive HCT116CIN model was established by cytochalasin D induction. Compared to parent HCT116 cells, it was sensitive to ZM-412 treatment, suggesting the high selectivity of ZM-412 to CIN tumors as monotherapy. Both KIF18A inhibitors and monomethyl auristatin E (MMAE) regulate microtubule dynamics, which provides a potential combination strategy. ZM-412 showed a significantly synergic effect with free MMAE or MMAE-conjugated ADC on the inhibition of proliferation in multiple kinds of cancer cell lines, including CIN-positive/negative tumor cell lines. In vivo, ZM-412 potently inhibited tumor growth in a dose-dependent manner in OVCAR3 and HT29 xenograft models. Moreover, ZM-412 showed a remarkable synergic effect on tumor growth inhibition with MMAE-conjugated LIV-1 ADC in OVCAR3 xenograft model. In conclusion, we discovered ZM-412 as a novel potent and selective KIF18A inhibitor, which has potential for the treatment of CIN cancers as monotherapy and for combination therapies for extended cancer types with MMAE-ADC. Citation Format: Feng Zhou, Lei Zhou, Ruina Wang, Liting Xue, Xiaokang Qin, Zhengtao Li, Jian Yu, Renhong Tang. ZM-412, a potent and selective KIF18A inhibitor with robust efficacy as monotherapy in CIN-positive tumors and in combination with MMAE-conjugated ADC in CIN-negative tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5946.

Abstract 5723: Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay

Abstract Kinesin superfamily proteins (KIFs) are a large family of molecular motor proteins, that share a highly conserved motor domain. KIFs play an important role in cell division and transport of vesicles and organelles within cells. Mitotic kinesins hydrolyze ATP through its ATPase activity to move along the spindle microtubules and carry out several functions during mitosis to facilitate precise chromosome segregation. Altered expressions of several kinesins are shown in various cancers, fueling the aggressive nature of cancers leading to genomic instability. KIF11/Eg5 overexpression is shown in various cancers including hepatic carcinoma, lung, prostate, colorectal, gastric, and pancreatic cancers. Small molecule inhibitors of KIF11/Eg5 and CENP-E are shown to result in mitotic arrest leading to apoptosis in tumor cell lines. Despite the poor clinical outcome of several Eg5 and CENP-E inhibitors entered in clinical trials, targeting kinesins remains an attractive anti-cancer therapeutic approach. Recently, an inhibitor of KIF18A has entered clinical trial for treatment of solid tumors with high chromosomal instability. Here we show development of a microplate based biochemical screening assays for a panel of kinesin motor domain proteins. We summarize kinetic characterization of kinesin proteins and study the selectivity of various previously reported KIF18A, Eg5, CENP-E inhibitors across a kinesin selectivity panel. Citation Format: Brenna P. Lee, Sung Won Oh, Kurumi Y. Horiuchi, Safnas F. AbdulSalam. Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5723.

Kinesin-7 CENP-E in tumorigenesis: Chromosome instability, spindle assembly checkpoint, and applications.

Kinesin motors are a large family of molecular motors that walk along microtubules to fulfill many roles in intracellular transport, microtubule organization, and chromosome alignment. Kinesin-7 CENP-E (Centromere protein E) is a chromosome scaffold-associated protein that is located in the corona layer of centromeres, which participates in kinetochore-microtubule attachment, chromosome alignment, and spindle assembly checkpoint. Over the past 3decades, CENP-E has attracted great interest as a promising new mitotic target for cancer therapy and drug development. In this review, we describe expression patterns of CENP-E in multiple tumors and highlight the functions of CENP-E in cancer cell proliferation. We summarize recent advances in structural domains, roles, and functions of CENP-E in cell division. Notably, we describe the dual functions of CENP-E in inhibiting and promoting tumorigenesis. We summarize the mechanisms by which CENP-E affects tumorigenesis through chromosome instability and spindle assembly checkpoints. Finally, we overview and summarize the CENP-E-specific inhibitors, mechanisms of drug resistances and their applications.

The mesenchymal morphology of cells expressing the EML4–ALK V3 oncogene is dependent on phosphorylation of Eg5 by NEK7

Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancers. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as variant 1 (V1). Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis, and have poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3-NEK9-NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain unknown. Here, we show that the microtubule-based kinesin, Eg5, is recruited to interphase microtubules in cells expressingEML4-ALK V3, whereas chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of NEK7 interferes withEg5 recruitment to microtubules in cells expressing EML4-ALK V3 and cell length is reduced, but this is reversed by coexpression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Together, we propose that Eg5 acts as a substrate of NEK7 in cells expressing EML4-ALK V3 andEg5 phosphorylation promotes the mesenchymal morphology typical of these cells.

Loss of EIF4G2 mediates aggressiveness in distinct human endometrial cancer subpopulations with poor survival outcome in patients

The non-canonical translation initiation factor EIF4G2 plays essential roles in cellular stress responses via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding its involvement in cancer development and progression. Here we assessed its role in endometrial cancer (EC), in a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall- and recurrence-free survival in Grade 2 EC patients, monitored over a period of up to 12 years. To establish a causative connection between low EIF4G2 expression and cancer progression, we stably knocked-down EIF4G2 in two human EC cell lines in parallel. EIF4G2 depletion resulted in increased resistance to conventional therapies and increased the prevalence of molecular markers for aggressive cell subsets, altering their transcriptional and proteomic landscapes. Prominent among the proteins with decreased abundance were Kinesin-1 motor proteins, KIF5B and KLC1, 2, 3. Multiplexed imaging of the EC patient tumor cohort showed a correlation between decreased expression of the kinesin proteins, and poor survival in patients with tumors of certain grades and stages. These findings reveal potential novel biomarkers for Grade 2 EC with ramifications for patient stratification and therapeutic interventions.

A Model for Chemomechanical Coupling of Kinesin-3 Motor.

Kinesin-3 motor, which is in the monomeric and inactive form in solution, after cargo-induced dimerization can step on microtubules towards the plus end with a high velocity and a supperprocessivity, which is responsible for transporting the cargo in axons and dendrites. The kinesin-3 motor has a large initial landing rate to microtubules and spends the majority of its stepping cycle in a one-head-bound state. Under the load the kinesin-3 motor can dissociate more readily than the kinesin-1 motor. To understand the physical origin of the peculiar features for the kinesin-3 motor, a model is presented here for its chemomechanical coupling. Based on the model the dynamics of the motor under no load, under the ramping load and under the constant load is studied analytically. The theoretical results explain well the available experimental data under no load and under the ramping load. For comparison, the corresponding available experimental data for the kinesin-1 motor under the ramping load are also explained. The predicted results of the velocity, dissociation rate and run length versus the constant load for the kinesin-3 motor are provided. The study has strong implications for the chemomechanical coupling mechanism of the kinesin-3 dimer. The origin of the kinesin-3 dimer in the predominant one-head-bound state is due to the fact that the rate of ATP transition to ADP in the trailing head is much larger than that of ADP release from the MT-bound head. The study shows that the kinesin-3 ADP-head has an evidently longer interaction distance with microtubule than the kinesin-1 ADP-head, explaining why in the initial ADP state the kinesin-3 motor has the much larger landing rate than the kinesin-1 motor and why under the load the kinesin-3 motor can dissociate more readily than the kinesin-1 motor. The online version contains supplementary material available at 10.1007/s12195-024-00795-1.

Loss-of-function of kinesin-5 KIF11 causes microcephaly, chorioretinopathy, and developmental disorders through chromosome instability and cell cycle arrest

Kinesin motors play a fundamental role in development by controlling intracellular transport, spindle assembly, and microtubule organization. In humans, patients carrying mutations in KIF11 suffer from an autosomal dominant inheritable disease called microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR). While mitotic functions of KIF11 proteins have been well documented in centrosome separation and spindle assembly, cellular mechanisms underlying KIF11 dysfunction and MCLMR remain unclear. In this study, we generate KIF11-inhibition chick and zebrafish models and find that KIF11 inhibition results in microcephaly, chorioretinopathy, and severe developmental defects in vivo. Notably, loss-of-function of KIF11 causes the formation of monopolar spindle and chromosome misalignment, which finally contribute to cell cycle arrest, chromosome instability, and cell death. Our results demonstrate that KIF11 is crucial for spindle assembly, chromosome alignment, and cell cycle progression of progenitor stem cells, indicating a potential link between polyploidy and MCLMR. Our data have revealed that KIF11 inhibition cause microcephaly, chorioretinopathy, and development disorders through the formation of monopolar spindle, polyploid, and cell cycle arrest.

Preferential transport of synaptic vesicles across neuronal branches is regulated by the levels of the anterograde motor UNC-104/KIF1A in vivo.

Asymmetric transport of cargo across axonal branches is a field of active research. Mechanisms contributing to preferential cargo transport along specific branches in vivo in wild type neurons are poorly understood. We find that anterograde synaptic vesicles preferentially enter the synaptic branch or pause at the branch point in Caenorhabditis elegans Posterior Lateral Mechanosensory neurons. The synaptic vesicle anterograde kinesin motor UNC-104/KIF1A regulates this vesicle behavior at the branch point. Reduced levels of functional UNC-104 cause vesicles to predominantly pause at the branch point and lose their preference for turning into the synaptic branch. SAM-4/Myrlysin, which aids in recruitment/activation of UNC-104 on synaptic vesicles, regulates vesicle behavior at the branch point similar to UNC-104. Increasing the levels of UNC-104 increases the preference of vesicles to go straight toward the asynaptic end. This suggests that the neuron optimizes UNC-104 levels on the cargo surface to maximize the fraction of vesicles entering the branch and minimize the fraction going to the asynaptic end.

Extreme-value analysis of intracellular cargo transport by motor proteins

The mechanisms underlying the chemo-mechanical coupling of motor proteins is usually described by a set of force-velocity relations that reflect the different mechanisms responsible for the walking behavior of such proteins on microtubules. However, the convexity of such relations remains controversial depending on the species, and in vivo experiments are inaccessible due to the complexity of intracellular environments. As alternative tool to investigate such mechanism, Extreme-value analysis (EVA) can offer insight on the deviations in the data from the median of the probability distributions. Here, we rely on EVA to investigate the motility functions of nanoscale motor proteins in neurons of the living worm Caenorhabditis elegans (C. elegans), namely the motion of kinesin and dynein along microtubules. While the essential difference between the two motors cannot be inferred from the mean velocities, such becomes evident in the EVA plots. Our findings extend the possibility and applicability of EVA for analysing motility data of nanoscale proteins in vivo.

Noncanonical interaction with microtubules via the N-terminal nonmotor domain is critical for the functions of a bidirectional kinesin.

Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end-directed kinesins. Here, we combined in vivo, in vitro, and cryo-electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of β-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. Last, we showed that NTnmd mutants are defective in plus-end-directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.

Further insights into kinesin motility using MINFLUX: A super-resolution nanoscopic method (kinesin motility: characterizing the half-steps and back-steps)

Further insights into kinesin motility using MINFLUX: A super-resolution nanoscopic method (kinesin motility: characterizing the half-steps and back-steps)

Robust behavior of kinesin and dynein motors in reconstituted bidirectional transport

Robust behavior of kinesin and dynein motors in reconstituted bidirectional transport

Kinesin light chain 1 stabilizes insulin receptor substrate 1 to regulate the IGF-1-AKT signaling pathway during myoblast differentiation.

The IGF signaling pathway plays critical role in regulating skeletal myogenesis. We have demonstrated that KIF5B, the heavy chain of kinesin-1 motor, promotes myoblast differentiation through regulating IGF-p38MAPK activation. However, the roles of the kinesin light chain (Klc) in IGF pathway and myoblast differentiation remain elusive. In this study, we found that Klc1 was upregulated during muscle regeneration and downregulated in senescence mouse muscles and dystrophic muscles from mdx (X-linked muscular dystrophic) mice. Gain- and loss-of-function experiments further displayed that Klc1 promotes AKT-mTOR activity and positively regulates myogenic differentiation. We further identified that the expression levels of IRS1, the critical node of IGF-1 signaling, are downregulated in Klc1-depleted myoblasts. Coimmunoprecipitation study revealed that IRS1 interacted with the 88-154 amino acid sequence of Klc1 via its PTB domain. Notably, the reduced Klc1 levels were found in senescence and osteoporosis skeletal muscle samples from both mice and human. Taken together, our findings suggested a crucial role of Klc1 in the regulation of IGF-AKT pathway during myogenesis through stabilizing IRS1, which might ultimately influence the development of muscle-related disorders.

Tropomyosin 1-I/C coordinates kinesin-1 and dynein motors during oskar mRNA transport.

Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. We show through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Our work reveals a new strategy for ensuring sequential activity of microtubule motors.

Wave-like oscillations of clamped microtubules driven by collective dynein transport

Microtubules (MTs) are observed to move and buckle driven by ATP-dependent molecular motors in both mitotic and interphasic eukaryotic cells as well as in specialized structures such as flagella and cilia with a stereotypical geometry. In previous work, clamped MTs driven by a few kinesin motors were seen to buckle and occasionally flap in what was referred to as flagella-like motion. Theoretical models of active-filament dynamics and a following force have predicted that, with sufficient force and binding-unbinding, such clamped filaments should spontaneously undergo periodic buckling oscillations. However, a systematic experimental test of the theory and reconciliation to a model was lacking. Here, we have engineered a minimal system of MTs clamped at their plus ends and transported by a sheet of dynein motors that demonstrate the emergence of spontaneous traveling-wave oscillations along single filaments. The frequencies of tip oscillations are in the millihertz range and are statistically indistinguishable in the onset and recovery phases. We develop a 2D computational model of clamped MTs binding and unbinding stochastically to motors in a “gliding-assay” geometry. The simulated MTs oscillate with a frequency comparable to experiment. The model predicts the effect of MT length and motor density on qualitative transitions between distinct phases of flapping, regular oscillations, and looping. We develop an effective “order parameter” based on the relative deflection along the filament and orthogonal to it. The transitions predicted in simulations are validated by experimental data. These results demonstrate a role for geometry, MT buckling, and collective molecular motor activity in the emergence of oscillatory dynamics.

Motor protein Kif6 regulates cilia motility and polarity in brain ependymal cells.

Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how kinesin motors contribute to cilia motility. Here, we show that Kif6 is a slow processive motor (12.2±2.0 nm/s) on microtubules in vitro and localizes to both the apical cytoplasm and the axoneme in ependymal cells, although it does not display processive movement in vivo. Using a mouse mutant that models a human Kif6 mutation in a proband displaying macrocephaly, hypotonia and seizures, we found that loss of Kif6 function causes decreased ependymal cilia motility and, subsequently, decreases fluid flow on the surface of brain ventricular walls. Disruption of Kif6 also disrupts orientation of cilia, formation of robust apical actin networks and stabilization of basal bodies at the apical surface. This suggests a role for the Kif6 motor protein in the maintenance of ciliary homeostasis within ependymal cells.

A Semi-Markov Approach to Study a Group of Kinesin Motors.

We propose a general mathematical and computational approach to study cellular transport driven by a group of kinesin motors. It is a framework for multi-scale modeling that integrates kinetic models of single kinesin motors, including detachment and reattachment events, to study group behaviors of several motors. By formulating the problem as a semi-Markov process and applying a central limit theorem, asymptotic velocity and diffusivity can be readily calculated, which offers considerable computational advantage over Monte Carlo simulations in tasks such as parameter sensitivity analysis and model selection. We demonstrate the method with some examples. The importance of incorporating the intrinsic microscopic-level dynamics of individual motors is illustrated by showing how changes at the microscopic level propagate to the motor-cargo complex at a mesoscopic level. Particularly, we showcase an example in which changes in the second moment of single-motor characteristics gives rise to different first moment characteristics of the motor group.