Kinesin Motor Research Articles

The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton

Completion of neuronal migration is critical for brain development. Kif21b is a plus-end-directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex. Invivo analysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of newborn neurons independently of its motility on microtubules. We show that Kif21b directly binds and regulates the actin cytoskeleton both invitro and invivo in migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.

The kinesin Kif21b binds myosin Va and mediates changes in actin dynamics underlying homeostatic synaptic downscaling

Homeostatic synaptic plasticity adjusts the strength of synapses to restrain neuronal activity within a physiological range. Postsynaptic guanylate kinase-associated protein (GKAP) controls the bidirectional synaptic scaling of AMPA receptors (AMPARs); however, mechanisms by which chronic activity triggers cytoskeletal remodeling to downscale synaptic transmission are barely understood. Here, we report that the microtubule-dependent kinesin motor Kif21b binds GKAP and likewise is located in dendritic spines in a myosin Va- and neuronal-activity-dependent manner. Kif21b depletion unexpectedly alters actin dynamics in spines, and adaptation of actin turnover following chronic activity is lost in Kif21b-knockout neurons. Consistent with a role of the kinesin in regulating actin dynamics, Kif21b overexpression promotes actin polymerization. Moreover, Kif21b controls GKAP removal from spines and the decrease of GluA2-containing AMPARs from the neuronal surface, thereby inducing homeostatic synaptic downscaling. Our data highlight a critical role of Kif21b at the synaptic actin cytoskeleton underlying homeostatic scaling of neuronal firing.

Differential modification of the C-terminal tails of different α-tubulins and their importance for microtubule function in vivo.

Microtubules (MTs) are built from α-/β-tubulin dimers and used as tracks by kinesin and dynein motors to transport a variety of cargos, such as mRNAs, proteins, and organelles, within the cell. Tubulins are subjected to several post-translational modifications (PTMs). Glutamylation is one of them, and it is responsible for adding one or more glutamic acid residues as branched peptide chains to the C-terminal tails of both α- and β-tubulin. However, very little is known about the specific modifications found on the different tubulin isotypes in vivo and the role of these PTMs in MT transport and other cellular processes in vivo. In this study, we found that in Drosophila ovaries, glutamylation of α-tubulin isotypes occurred clearly on the C-terminal ends of αTub84B and αTub84D (αTub84B/D). In contrast, the ovarian α-tubulin, αTub67C, is not glutamylated. The C-terminal ends of αTub84B/D are glutamylated at several glutamyl sidechains in various combinations. Drosophila TTLL5 is required for the mono- and poly-glutamylation of ovarian αTub84B/D and with this for the proper localization of glutamylated microtubules. Similarly, the normal distribution of kinesin-1 in the germline relies on TTLL5. Next, two kinesin-1-dependent processes, the precise localization of Staufen and the fast, bidirectional ooplasmic streaming, depend on TTLL5, too, suggesting a causative pathway. In the nervous system, a mutation of TTLL5 that inactivates its enzymatic activity decreases the pausing of anterograde axonal transport of mitochondria. Our results demonstrate in vivo roles of TTLL5 in differential glutamylation of α-tubulins and point to the in vivo importance of α-tubulin glutamylation for cellular functions involving microtubule transport.

Molecular mechanism of interaction between kinesin motors affecting their residence times on microtubule lattice and end

Kinesin superfamily can be classified into 14 subfamilies. Some families of kinesin motors such as kinesin-1 are responsible for long-distance intracellular transports and thus the motors are required to reside on the microtubule (MT) lattice for a longer time than at the end. Some families such as kinesin-8 Kip3 and kinesin-5 Eg5 are responsible for the regulation of MT length by depolymerizing or polymerizing the MT from the plus end and thus the motors are required to reside at the MT end for a long time. Under the crowded condition of the motors, it was found experimentally that the residence times of the kinesin-8 Kip3 and kinesin-5 Eg5 at the MT end are reduced greatly compared to the single-motor case. However, the underlying mechanism of different families of kinesin motors having different MT-end residence times is unknown. The molecular mechanism by which the interaction between the two motors greatly reduces the residence time of the motor at the MT end is elusive. In addition, during the processive stepping on the MT lattice, when two kinesin motors meet it is unknown how the interaction between them affects their dissociation rates. To address the above unclear issues, here we make a consistent and theoretical study of the residence times of the kinesin-1, kinesin-8 Kip3 and kinesin-5 Eg5 motors on the MT lattice and at the end under both the single-motor condition and multiple-motors or crowded condition.

Glycogen synthase kinase 3β (GSK3β) and presenilin (PS) are key regulators of kinesin-1-mediated cargo motility within axons.

It has been a quarter century since the discovery that molecular motors are phosphorylated, but fundamental questions still remain as to how specific kinases contribute to particular motor functions, particularly in vivo, and to what extent these processes have been evolutionarily conserved. Such questions remain largely unanswered because there is no cohesive strategy to unravel the likely complex spatial and temporal mechanisms that control motility in vivo. Since diverse cargoes are transported simultaneously within cells and along narrow long neurons to maintain intracellular processes and cell viability, and disruptions in these processes can lead to cancer and neurodegeneration, there is a critical need to better understand how kinases regulate molecular motors. Here, we review our current understanding of how phosphorylation can control kinesin-1 motility and provide evidence for a novel regulatory mechanism that is governed by a specific kinase, glycogen synthase kinase 3β (GSK3β), and a scaffolding protein presenilin (PS).

Chaperonin CCT controls extracellular vesicle production and cell metabolism through kinesin dynamics.

Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post-translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell-content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid-dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule-based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT.

FEZ1 Plays Dual Roles in Early HIV-1 Infection by Independently Regulating Capsid Transport and Host Interferon-Stimulated Gene Expression.

Fasciculation and elongation factor zeta 1 (FEZ1), a multifunctional kinesin-1 adaptor, binds human immunodeficiency virus type 1 (HIV-1) capsids and is required for efficient translocation of virus particles to the nucleus to initiate infection. However, we recently found that FEZ1 also acts as a negative regulator of interferon (IFN) production and interferon-stimulated gene (ISG) expression in primary fibroblasts and human immortalized microglial cell line clone 3 (CHME3) microglia, a natural target cell type for HIV-1 infection. This raises the question of whether depleting FEZ1 negatively affects early HIV-1 infection through effects on virus trafficking or IFN induction or both. Here, we address this by comparing the effects of FEZ1 depletion or IFN-β treatment on early stages of HIV-1 infection in different cell systems with various IFN-β responsiveness. In either CHME3 microglia or HEK293A cells, depletion of FEZ1 reduced the accumulation of fused HIV-1 particles around the nucleus and suppressed infection. In contrast, various doses of IFN-β had little to no effect on HIV-1 fusion or the translocation of fused viral particles to the nucleus in either cell type. Moreover, the potency of IFN-β's effects on infection in each cell type reflected the level of induction of MxB, an ISG that blocks subsequent stages of HIV-1 nuclear import. Collectively, our findings demonstrate that loss of FEZ1 function impacts infection through its roles in two independent processes, as a direct regulator of HIV-1 particle transport and as a regulator of ISG expression. IMPORTANCE As a hub protein, fasciculation and elongation factor zeta 1 (FEZ1) interacts with a range of other proteins involved in various biological processes, acting as an adaptor for the microtubule (MT) motor kinesin-1 to mediate outward transport of intracellular cargoes, including viruses. Indeed, incoming HIV-1 capsids bind to FEZ1 to regulate the balance of inward/outward motor activity to ensure net forward movement toward the nucleus to initiate infection. However, we recently showed that FEZ1 depletion also induces interferon (IFN) production and interferon-stimulated gene (ISG) expression. As such, it remains unknown whether modulating FEZ1 activity affects HIV-1 infection through its ability to regulate ISG expression or whether FEZ1 functions directly, or both. Using distinct cell systems that separate the effects of IFN and FEZ1 depletion, here we demonstrate that the kinesin adaptor FEZ1 regulates HIV-1 translocation to the nucleus independently of its effects on IFN production and ISG expression.

Abstract LB017: Potential role of kinesin family member C1 (KIFC1) in melanoma

Abstract According to the Surveillance, Epidemiology, and End Results (SEER) analyses, based on 2017-2019 data, approximately ~2.1 percent of men and women will be diagnosed with melanoma at some point during their lifetime. Melanoma can be fatal if not surgically removed at an early stage, with the 5-year survival being a dismal 29.8% for a stage IV melanoma. Melanoma mortality is primarily due to the distant organ metastases and its resistance to treatment, even with newly developed immunotherapy and/or targeted therapies (BRAF and MEK inhibitors). Thus, the discovery of novel melanoma targets is crucial as they may be able to provide alternative treatment options or be used alone or in combination with current therapies. KIFC1 (kinesin family member C1; also known as HSET, an ortholog in Drosophila melanogaster), a nonessential kinesin motor protein, has an established role in centrosome clustering which is utilized by cancer cells to evade apoptosis and mitotic arrest. This occurs when replicating cells harbor additional centrosomes, a hallmark of cancer. The requirement of KIFC1 in mitosis of cancer cells harboring abnormal centrosomes, but not that of normal cells, makes it a potential drug target for cancer management. The goal of this study was to determine the role and significance of KIFC1 in melanoma. Differential expression analysis of GTEX and TCGA RNA-seq data identified significantly higher levels of KIFC1 in both primary and metastatic melanoma as compared to normal skin. In addition, higher expression of KIFC1 was found to be associated with poorer overall survival of melanoma patients. Furthermore, a quantitative protein estimation with the automated ProteinSimple Jess capillary western blotting system demonstrated higher KIFC1 expression in several melanoma cell lines when compared to normal melanocytes. In addition, we also found significantly increased expression of KIFC1 mRNA in mouse melanoma tumors obtained from the genetically engineered BrafV600E/PtenNULL mice, as compared to the normal skin. Next, we determined the effect of a small molecule KIFC1 inhibitor, AZ82 on A375, G361 and Hs294T melanoma cells. AZ82 is an ATP-competitive inhibitor that binds the KIFC1-microtubule complex, blocking the release of ADP and negating its role as a motor protein. Employing RealTime-Glo MT cell viability assay as well as the endpoint XTT growth assay, we found that AZ82 treatment resulted in a significant growth inhibition of human melanoma cells at 2-3.5 µM concentrations. Additionally, AZ82 treatments resulted in a significant decrease in clonogenic survival in all three human melanoma cell lines. Collectively, our data suggests a potential role of KIFC1 in melanoma progression, and merits further investigation to determine the roles and functional and therapeutic significance of KIFC1 in melanoma. Citation Format: Davis S. Mau, Mary A. Ndiaye, Gabriella R. Zaemisch, Chandra K. Singh, Jess Vera, Sean R. McIlwain, Nihal Ahmad. Potential role of kinesin family member C1 (KIFC1) in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB017.

Spatio-temporal patterning of extensile active stresses in microtubule-based active fluids.

Microtubule-based active fluids exhibit turbulent-like autonomous flows, which are driven by the molecular motor powered motion of filamentous constituents. Controlling active stresses in space and time is an essential prerequisite for controlling the intrinsically chaotic dynamics of extensile active fluids. We design single-headed kinesin molecular motors that exhibit optically enhanced clustering and thus enable precise and repeatable spatial and temporal control of extensile active stresses. Such motors enable rapid, reversible switching between flowing and quiescent states. In turn, spatio-temporal patterning of the active stress controls the evolution of the ubiquitous bend instability of extensile active fluids and determines its critical length dependence. Combining optically controlled clusters with conventional kinesin motors enables one-time switching from contractile to extensile active stresses. These results open a path towards real-time control of the autonomous flows generated by active fluids.

Gigaxonin is required for intermediate filament transport.

Gigaxonin is an adaptor protein for E3 ubiquitin ligase substrates. It is necessary for ubiquitination and degradation of intermediate filament (IF) proteins. Giant axonal neuropathy is a pathological condition caused by mutations in the GAN gene that encodes gigaxonin. This condition is characterized by abnormal accumulation of IFs in both neuronal and non-neuronal cells; however, it is unclear what causes IF aggregation. In this work, we studied the dynamics of IFs using their subunits tagged with a photoconvertible protein mEOS 3.2. We have demonstrated that the loss of gigaxonin dramatically inhibited transport of IFs along microtubules by the microtubule motor kinesin-1. This inhibition was specific for IFs, as other kinesin-1 cargoes, with the exception of mitochondria, were transported normally. Abnormal distribution of IFs in the cytoplasm can be rescued by direct binding of kinesin-1 to IFs, demonstrating that transport inhibition is the primary cause for the abnormal IF distribution. Another effect of gigaxonin loss was a more than 20-fold increase in the amount of soluble vimentin oligomers in the cytosol of gigaxonin knock-out cells. We speculate that these oligomers saturate a yet unidentified adapter that is required for kinesin-1 binding to IFs, which might inhibit IF transport along microtubules causing their abnormal accumulation.

Horizontal Magnetic Tweezers to Directly Measure the Force-Velocity Relationship for Multiple Kinesin Motors.

Transport of intracellular cargo along cytoskeletal filaments is often achieved by the concerted action of multiple motor molecules. While single-molecule studies have provided profound insight into the mechano-chemical principles and force generation of individual motors, studies on multi-motor systems are less advanced. Here, a horizontal magnetic-tweezers setup is applied, capable of producing up to 150 pN of horizontal force onto 2.8µm superparamagnetic beads, to motor-propelled cytoskeletal filaments. It is found that kinesin-1 driven microtubules decorated with individual beads display frequent transitions in their gliding velocities whichweattribute to dynamic changes in the number of engaged motors. Applying defined temporal force-ramps the force-velocity relationship is directly measured for multi-motor transport. It is found that the stall forces of individual motors are approximately additive and collective backward motion of the transport system under super-stall forces is observed. The magnetic-tweezers apparatus is expected to be readily applicable to a wide range of molecular and cellular motility assays.

Abstract 516: Discovery and preclinical characterization of AMG 650, a first-in-class inhibitor of kinesin KIF18A motor protein with potent activity against chromosomally unstable cancers

Abstract Chromosomal instability (CIN) is a hallmark of human cancers and is caused by persistent errors in chromosome segregation during mitosis. Aggressive types of human cancer such as high-grade serous ovarian cancer and triple-negative breast cancer have elevated levels of CIN and frequently harbor TP53 gene alterations and are poorly served by current treatment options. These two CIN+ cancer types also share mutually exclusive genetic alterations in BRCA1 and CCNE1 cancer genes. KIF18A is a mitotic kinesin motor protein that localizes to the plus-end tips of kinetochore microtubule (MT) spindle fibers, where it regulates chromosome alignment during cell division. KIF18A is overexpressed in a subset of human cancers, and its elevated expression is associated with tumor aggressiveness. Recent reports provide compelling evidence that genetic ablation of KIF18A reduced the viability of CIN cancer cells (Marquis et al. Nature Com 2021, Quinton et al. Nature 2021, Cohen-Sharir et al. Nature 2021). Here, we report the discovery and preclinical characterization of AMG 650, a potent and selective inhibitor of KIF18A with a compelling anti-cancer profile distinct from other cell cycle and anti-mitotic drug targets. Structural insights gained by cryo-EM illustrate the unique binding mode of AMG 650 to an allosteric pocket at the interface of KIF18A motor α4 and α6 helices and α-tubulin. AMG 650 selectively inhibits KIF18A MT-ATPase motor activity (IC50 = 48 nM) and exhibits specificity against a panel of diverse motor proteins. In cells, AMG 650 is active at double-digit nM concentrations and phenocopies KIF18A genetic KD/KO dependencies across a panel of DNA-barcoded cancer cell lines. AMG 650 selectively activates the mitotic checkpoint resulting in multipolarity and apoptosis in breast and ovarian cancer cell lines enriched with CIN features. Notably, AMG 650 has minimal effects on proliferating human bone marrow mononuclear cells in culture at concentrations active on sensitive cancer cells (>100X window). In vivo, AMG 650 has low clearance, long half-life, and good oral bioavailability across preclinical species. In mice, oral administration of AMG 650 induces a dose-dependent pharmacodynamic response (pH3 mitotic marker) that is sustained for 24 hours in OVCAR-3 tumor model. Importantly, continuous once-daily dosing with AMG 650 shows robust anti-cancer activity with evidence of durable tumor regressions in a subset of human ovarian and breast CDX/PDX tumor models at well-tolerated doses. Lastly, the combination of AMG 650 with PARP inhibitor Olaparib enhances anti-cancer activity relative to single agent alone in BRCA1- and CCNE1-altered tumor models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via AMG 650, a first-in-class KIF18A inhibitor. Citation Format: Brian Belmontes, Jodi Moriguchi, Grace Chung, Jan Sun, Maria Stefania S. Ninniri, Kelly Hanestad, Kui Chen, John D. McCarter, Upendra P. Dahal, Sudipa Ghimire-Rijal, Yue Hao, Christopher P. Mohr, Xinchao Yu, Matthew G. Rees, Melissa Ronan, Jennifer Roth, Sheroy Minocherhomji, Jennifer R. Allen, Matthew P. Bourbeau, Paul E. Hughes, Nuria A Tamayo, Marc N. Payton. Discovery and preclinical characterization of AMG 650, a first-in-class inhibitor of kinesin KIF18A motor protein with potent activity against chromosomally unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 516.

Abstract 4482: Discovery of GH2616, a potent and selective KIF18A inhibitor with robust in vivo efficacy in p53 mutant cancer

Abstract Chromosomal instability (CIN) is one of the hallmarks of cancer. Cancer cells often undergo whole-genome doubling (WGD) and P53 represents a major barrier to the proliferation of WGD+ cells. Therefore, cancer cells with either CIN or WGD often harbor the p53 mutation. Recently, KIF18A has emerged as a potential therapeutic target in CIN or WGD cancer cells. We have identified a potent and selective KIF18A inhibitor, GH2616. In vitro, GH2616 inhibited the kinesin motor activity with an IC50 less than 50 nM, and with an EC50 less than 50 nM in the mitotic index assay using HT29 cells (WGD). In vivo, GH2616 inhibited the growth of WGD CDX models with a dose dependent manner. GH2616 has good oral bioavailability in different species and metabolic stability for QD dosing. This compound also showed low potential of DDI thus suitable for drug combinations. In a cell panel with more than 200 cell lines, GH2616 selectively inhibit the growth of cells with p53 mutant and some other specific biomarkers in multiple tumor types. In conclusion, we discovered GH2616 as a novel KIF18A inhibitor with potent in vivo efficacy. It is currently under evaluation in IND-enabling studies and IND filing is expected in Q4, 2023. Citation Format: Jie Jack Li, Guiping Zhang, Jiapeng Li. Discovery of GH2616, a potent and selective KIF18A inhibitor with robust in vivo efficacy in p53 mutant cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4482.

Abstract 1565: Targeting of KIF11 and AURKA to improve outcomes in Ewing sarcoma

Abstract Ewing Sarcoma (EWS) is a rare and aggressive pediatric malignancy of the bone and soft tissues. Despite intensive systemic combination chemotherapy and surgery/radiotherapy for local control, patients with metastatic disease have dismal prognosis (<30% overall survival rate). To identify better therapies for patients with refractory, recurrent and metastatic disease, we previously conducted in silico and drug re-purposing screens (PMID: 27863422; 31025088) that identified proteins essential for mitotic spindle formation and cell cycle progression such as Aurora Kinase A (AURKA), Kinesin Family Member 11 (KIF11), Kinesin Family Member 15 (KIF15) and its binding partner; targeting protein for Xklp2 (TPX2) that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, indicating their potential as therapeutic targets. KIF11 and KIF15 are mitotic motor kinesins essential for bipolar spindle formation, whereas AURKA is a mitotic kinase essential for centrosome maturation and mitotic spindle assembly. Though KIF11 and AURKA inhibitors have been extensively used in clinical trials before but have not been tested together in combination for any type of cancer. We hypothesized that targeting these two key proteins essential for mitotic progression provides a therapeutic vulnerability for Ewing Sarcoma by inducing G2/M arrest and mitotic catastrophe. Specific KIF11 (SB-743921) and AURKA (VIC-1911, VITRAC Therapeutics) inhibitors were used for targeting these proteins. We evaluated the in vitro activity of VIC-1911 and SB-743921 and synergy was measured by percentage inhibition of growth via SynergyFinder, that employs reference models (Bliss) to score synergy (scores >10 is synergistic). EWS showed strong synergistic interaction at physiologically relevant doses compared to control cell lines (e.g., mesenchymal stem cells and non-EWS tumor cells). This drug combination was effective in refractory EWS cells established post-chemotherapy (synergy score >24) and at much lower doses in cells established from pre-treatment tumor tissue (synergy score >27) indicating effectiveness at various clinical stages of EWS disease. Importantly, this therapy appeared preferential to EWS cells with an EWS-ETS fusion as compared to non-EWS cells. We observed zero colony formation in vitro with the combination treatment and increased percentage accumulation of cells in G2/M phase upon cell-cycle analysis indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed sustained tumor regression (NED at 30 days after ending therapy). The combination arm showed significant survival versus control (p≤ 0.001, HR=0.42, 95% CI=0.14-1.25), VIC-1911 (p≤ 0.001, HR=0.48, 95% CI=0.16-1.42) or SB-743921 (p ≤ 0.01, p≤ HR=0.71, 95% CI=0.21-2.34) alone. Efforts are underway to translate these preclinical studies into an early phase clinical trial of patients with histologically proven EWS who have failed standard of care therapy. Citation Format: Soumya M. Turaga, Vikalp Vishwakarma, Stacey Hembruff, Rajni Puri, Priya Sabu, Glenson Samuel, Andrew K. Godwin. Targeting of KIF11 and AURKA to improve outcomes in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1565.

Key essential oil components delocalize Candida albicans Kar3p and impact microtubule structure

BackgroundTreatment of Candida albicans associated infections is often ineffective in the light of resistance, with an urgent need to discover novel antimicrobials. Fungicides require high specificity and can contribute to antifungal resistance, so inhibition of fungal virulence factors is a good strategy for developing new antifungals. ObjectivesExamine the impact of four plant-derived essential oil components (1,8-cineole, α-pinene, eugenol, and citral) on C. albicans microtubules, kinesin motor protein Kar3 and morphology. MethodsMicrodilution assays were used to determine minimal inhibitory concentrations, microbiological assays assessed germ tube, hyphal and biofilm formation, confocal microscopy probed morphological changes and localization of tubulin and Kar3p, and computational modelling was used to examine the theoretical binding of essential oil components to tubulin and Kar3p. ResultsWe show for the first time that essential oil components delocalize the Kar3p, ablate microtubules, and induce psuedohyphal formation with reduced biofilm formation. Single and double deletion mutants of kar3 were resistant to 1,8-cineole, sensitive to α-pinene and eugenol, but unimpacted by citral. Strains with homozygous and heterozygous Kar3p disruption had a gene‐dosage effect for all essential oil components, resulting in enhanced resistance or susceptibility patterns that were identical to that of cik1 mutants. The link between microtubule (αβ-tubulin) and Kar3p defects was further supported by computational modeling, showing preferential binding to αβ-tubulin and Kar3p adjacent to their Mg2+-binding sites. ConclusionThis study highlights how essential oil components interfere with the localization of the kinesin motor protein complex Kar3/Cik1 and disrupt microtubules, leading to their destabilization which results in hyphal and biofilm defects.

Why slow axonal transport is bidirectional – can axonal transport of tau protein rely only on motor-driven anterograde transport?

Slow axonal transport (SAT) moves multiple proteins from the soma, where they are synthesized, to the axon terminal. Due to the great lengths of axons, SAT almost exclusively relies on active transport, which is driven by molecular motors. The puzzling feature of slow axonal transport is its bidirectionality. Although the net direction of SAT is anterograde, from the soma to the terminal, experiments show that it also contains a retrograde component. One of the proteins transported by SAT is the microtubule-associated protein tau. To better understand why the retrograde component in tau transport is needed, we used the perturbation technique to analyze how the full tau SAT model can be simplified for the specific case when retrograde motor-driven transport and diffusion-driven transport of tau are negligible and tau is driven only by anterograde (kinesin) motors. The solution of the simplified equations shows that without retrograde transport the tau concentration along the axon length stays almost uniform (decreases very slightly), which is inconsistent with the experimenal tau concentration at the outlet boundary (at the axon tip). Thus kinesin-driven transport alone is not enough to explain the empirically observed distribution of tau, and the retrograde motor-driven component in SAT is needed.

Kinesin Family Member C1 Overexpression Exerts Tumor-Promoting Properties in Head and Neck Squamous Cell Carcinoma via the Rac1/Wnt/β-catenin Pathway

Kinesin family member C1 (KIFC1) is a kinesin-14 motor protein, and its abnormal upregulation promotes the malignant behavior of cancer cells. N6-methyladenosine (m6A) RNA methylation is a common modification of eukaryotic messenger RNA and affects RNA expression. In this study, we explored how KIFC1 regulated head and neck squamous cell carcinoma (HNSCC) tumorigenesis and how m6A modification affected KIFC1 expression. A bioinformatics analysis was performed to screen for genes of interest, and in vitro and in vivo studies were carried out to investigate the function and mechanism of KIFC1 in HNSCC tissues. We observed that the expression of KIFC1 in HNSCC tissues was significantly higher than that in normal or adjacent normal tissues. Patients with cancer with higher KIFC1 expression have a lower tumor differentiation status. Demethylase alkB homolog 5, a cancer-promoting factor in HNSCC tissues, could interact with KIFC1 messenger RNA and posttranscriptionally activate KIFC1 through m6A modification. KIFC1 downregulation suppressed HNSCC cell growth and metastasis in vivo and in vitro. However, overexpression of KIFC1 promoted these malignant behaviors. We demonstrated that KIFC1 overexpression activated the oncogenic Wnt/β-catenin pathway. KIFC1 interacted with the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1) at the protein level and increased its activity. The Rho GTPase Rac1 was indicated to be an upstream activator of the Wnt/β-catenin signaling pathway, and its Rac1 inhibitor, NSC-23766, treatment reversed the effects caused by KIFC1 overexpression. Those observations demonstrate that abnormal expression of KIFC1 may be regulated by demethylase alkB homolog 5 in an m6A-dependent manner and promote HNSCC progression via the Rac1/Wnt/β-catenin pathway.

Unraveling on kinesin acceleration in intracellular environments: A theory for active bath

A single-molecular motor kinesin harnesses thermal and nonthermal fluctuations to transport various cargoes along microtubules, converting chemical energy to directed movements. To describe the nonthermal fluctuations generated by the complex environment in living cells, we propose a bottom-up model which combines a Markov model of the kinesin and a mean-field model for the active bath, to mimic the kinesin movements in the intracellular environment. Simulations of the model system show that the kinesin and the probe attached to it are accelerated by such active bath. Further, we provide a theoretical insight into the simulation result by deriving a generalized Langevin equation (GLE) for the probe with a mean-field method, wherein an effective friction kernel and fluctuating noise terms are obtained explicitly. Numerical solutions of the GLE show very good agreement with simulation results. We sample such noises, calculate their variances and non-Gaussian parameters, and reveal that the dominant contribution to probe acceleration is attributed to noise variance.

Cellular cartography: Towards an atlas of the neuronal microtubule cytoskeleton.

Microtubules, one of the major components of the cytoskeleton, play a crucial role during many aspects of neuronal development and function, such as neuronal polarization and axon outgrowth. Consequently, the microtubule cytoskeleton has been implicated in many neurodevelopmental and neurodegenerative disorders. The polar nature of microtubules is quintessential for their function, allowing them to serve as tracks for long-distance, directed intracellular transport by kinesin and dynein motors. Most of these motors move exclusively towards either the plus- or minus-end of a microtubule and some have been shown to have a preference for either dynamic or stable microtubules, those bearing a particular post-translational modification or those decorated by a specific microtubule-associated protein. Thus, it becomes important to consider the interplay of these features and their combinatorial effects on transport, as well as how different types of microtubules are organized in the cell. Here, we discuss microtubule subsets in terms of tubulin isotypes, tubulin post-translational modifications, microtubule-associated proteins, microtubule stability or dynamicity, and microtubule orientation. We highlight techniques used to study these features of the microtubule cytoskeleton and, using the information from these studies, try to define the composition, role, and organization of some of these subsets in neurons.

Cargo transport properties are enhanced by cylindrical microtubule geometry and elliptical contact zone on cargo surface

Molecular motors are responsible for carrying cellular transport of various membranous vesicles or organelles along cytoskeletal tracks. Transport of cellular cargos require high forces that are generated by motors working in groups. Hence, the properties of cargo transport can be modulated by varying various parameters such as cargo size and shape, microtubule geometry, motor number and their arrangement on cargo surface. Only those motors which are present in the contact zone on cargo surface have potential to bind to microtubule. Although earlier studies revealed the importance of cargo size, total motors attached to microtubule and their arrangement on cargo transport, yet how the contact zone influences binding of motors to microtubule largely remains unexplored. Here, it has been shown that contact zone is elliptical in shape for a spherical cargo and increases with cargo size for Kinesin-1 motors. To further understand the combined effect of elliptical contact zone and microtubule geometry on cargo transport, 3D mean-field model with uniform and clustered arrangement of motors for different cargo sizes and motor number has been used. Our findings indicate that cylindrical microtubule geometry maximizes the microtubule-bound motors which enhances the runlength and velocity of cargo transport. Our results show that microtubule-bound motors decrease with cargo size for uniform arrangement of motors on cargo thus decreasing its runlength and velocity, whereas in clustered arrangement, the number of microtubule-bound motors increase with cargo size which leads to increase in runlength and velocity.