ABSTRACT
Cisplatin (DDP) is commonly used in the treatment of non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD), and the primary cause for its clinical inefficacy is chemoresistance. Here, we aimed to investigate a novel mechanism of chemoresistance in LUAD cells, focusing on the calcium-sensing receptor (CaSR). In this study, high CaSR expression was detected in DDP-resistant LUAD cells, and elevated CaSR expression is strongly correlated with poor prognosis in LUAD patients receiving chemotherapy. LUAD cells with high CaSR expression exhibited decreased sensitivity to cisplatin, and the growth of DDP-resistant LUAD cells was inhibited by cisplatin treatment in combination with CaSR suppression, accompanied by changes in BRCA1 and cyclin B1 protein expression both in vitro and in vivo. Additionally, an interaction between CaSR and KIF11 was identified. Importantly, suppressing KIF11 resulted in decreased protein levels of BRCA1 and cyclin B1, enhancing the sensitivity of DDP-resistant LUAD cells to cisplatin with no obvious decrease in CaSR. Here, our findings established the critical role of CaSR in promoting cisplatin resistance in LUAD cells by modulating cyclin B1 and BRCA1 and identified KIF11 as a mediator, highlighting the potential therapeutic value of targeting CaSR to overcome chemoresistance in LUAD.
Subject(s)
Adenocarcinoma of Lung , BRCA1 Protein , Cisplatin , Cyclin B1 , Drug Resistance, Neoplasm , Kinesins , Lung Neoplasms , Receptors, Calcium-Sensing , Humans , Cisplatin/therapeutic use , Cisplatin/pharmacology , Receptors, Calcium-Sensing/metabolism , Receptors, Calcium-Sensing/genetics , Adenocarcinoma of Lung/metabolism , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/genetics , BRCA1 Protein/metabolism , BRCA1 Protein/genetics , Cyclin B1/metabolism , Cyclin B1/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Cell Line, Tumor , Kinesins/metabolism , Kinesins/genetics , Animals , Mice , Mice, Nude , Female , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Male , Mice, Inbred BALB CABSTRACT
Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.
Subject(s)
Cell Nucleus , Dyneins , Kinesins , Microtubule-Associated Proteins , Microtubules , Nerve Tissue Proteins , Neurons , Animals , Microtubules/metabolism , Neurons/metabolism , Kinesins/metabolism , Kinesins/genetics , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Dyneins/metabolism , Cell Nucleus/metabolism , Mice , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/genetics , Active Transport, Cell Nucleus , Dynactin Complex/metabolism , Dynactin Complex/genetics , Cell Movement , Microfilament Proteins/metabolism , Microfilament Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Cerebellum/metabolism , Cerebellum/cytology , Binding Sites , HumansABSTRACT
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. In this study, we investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA. Our findings reveal that both motors are non-processive, producing single load-dependent power strokes per MT encounter, with estimated load-free power strokes of ~30 and ~35 nm, respectively. Each homodimeric motor generates forces of ~0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Notably, the cooperative activity among multiple motors leads to increased MT-sliding velocities. These results quantitatively elucidate the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.
Subject(s)
Kinesins , Microtubules , Spindle Apparatus , Kinesins/metabolism , Microtubules/metabolism , Spindle Apparatus/metabolism , Animals , Humans , Microtubule-Associated Proteins/metabolismABSTRACT
Mitotic centromere-associated kinesin (MCAK) motor protein is a typical member of the kinesin-13 family, which can depolymerize microtubules from both plus and minus ends. A critical issue for the MCAK motor is how it performs the depolymerase activity. To address the issue, the pathway of the MCAK motor moving on microtubules and depolymerizing the microtubules is presented here. On the basis of the pathway, the dynamics of both the wild-type and mutant MCAK motors is studied theoretically, which include the full-length MCAK, the full-length MCAK with mutations in the α4-helix of the motor domain, the mutant full-length MCAK with a neutralized neck, the monomeric MCAK and the mutant monomeric MCAK with a neutralized neck. The studies show that a single dimeric MCAK motor can depolymerize microtubules in a processive manner, with either one tubulin or two tubulins being removed per times. The theoretical results are in agreement with the available experimental data. Moreover, predicted results are provided.
Subject(s)
Kinesins , Microtubules , Models, Molecular , Kinesins/metabolism , Kinesins/chemistry , Microtubules/metabolism , Mutation , Protein MultimerizationABSTRACT
We investigate the etiology of amyotrophic lateral sclerosis (ALS) in a 35-year-old woman presenting with progressive weakness in her left upper limb. Prior to sequencing, a comprehensive neurological work-up was performed, including neurological examination, electrophysiology, biomarker assessment, and brain and spinal cord MRI. Six months before evaluation, the patient experienced weakness and atrophy in her left hand, accompanied by brisk reflexes and Hoffman sign in the same arm. Electroneuromyography revealed lower motor neuron involvement in three body regions. Neurofilament light chains were elevated in her cerebrospinal fluid. Brain imaging showed asymmetrical T2 hyperintensity of the corticospinal tracts and T2 linear hypointensity of the precentral gyri. Trio genome sequencing identified a likely pathogenic de novo variant in the KIF1A gene (NM_001244008.2): c.574A>G, p.(Ile192Val). Pathogenic variants in KIF1A have been associated with a wide range of neurological manifestations called KIF1A-associated neurological diseases (KAND). This report describes a likely pathogenic de novo variant in KIF1A associated with ALS, expanding the phenotypic spectrum of KAND and our understanding of the pathophysiology of ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Kinesins , Mutation, Missense , Humans , Kinesins/genetics , Amyotrophic Lateral Sclerosis/genetics , Female , Adult , Upper Extremity/physiopathology , Upper Extremity/pathology , Magnetic Resonance ImagingABSTRACT
OBJECTIVE: This study aimed to describe the clinical and genetic characteristics of Chinese patients with congenital fibrosis of the extraocular muscles (CFEOM), and to evaluate the phenotype-genotype correlations in these patients. METHODS: This was a retrospective study. Patients with CFEOM underwent detailed ophthalmic examinations and magnetic resonance imaging (MRI). Panel-based next-generation sequencing was performed to identify pathogenic variants of disease-causing genes. RESULTS: Sixty-two patients with CFEOM were recruited into this study. Thirty-nine patients were diagnosed with CFEOM1 and 23 with CFEOM3. Forty-nine of the 62 patients with CFEOM carried either KIF21A (41/49) or TUBB3 variants (8/49). Six known missense variants in the KIF21A and TUBB3 genes, and a novel variant (c.3906T > A, p.D1302E) in the KIF21A gene were detected. Most patients with CFEOM1 carrying the KIF21A mutation displayed isolated CFEOM, whereas patients with CFEOM3 carrying the TUBB3 mutation had a wide range of clinical manifestations, either CFEOM alone or syndromes. Nystagmus was also present in 12 patients with CFEOM. Furthermore, the MRI findings varied, ranging from attenuation of the extraocular muscles to dysgenesis of the cranial nerves and brain structure. CONCLUSIONS: The novel variants identified in this study will further expand the spectrum of pathogenic variants in CFEOM-related genes. However, no phenotype-genotype correlations were established because of the diversity of the clinical characteristics of these patients.
Subject(s)
Fibrosis , Kinesins , Humans , Male , Female , Fibrosis/genetics , Fibrosis/pathology , Child , Kinesins/genetics , Retrospective Studies , Adolescent , Child, Preschool , Adult , Tubulin/genetics , Young Adult , Magnetic Resonance Imaging , Oculomotor Muscles/pathology , Oculomotor Muscles/diagnostic imaging , Asian People/genetics , Infant , Mutation/genetics , East Asian People , Congenital Cranial Dysinnervation Disorders , OphthalmoplegiaABSTRACT
Few efficacious treatment options are available for patients with small cell lung carcinoma (SCLC), indicating the need to develop novel therapeutic approaches. In this study, we explored kinesin family member 11 (KIF11), a potential therapeutic target in SCLC. An analysis of publicly available data suggested that KIF11 mRNA expression levels are significantly higher in SCLC tissues than in normal lung tissues. When KIF11 was targeted by RNA interference or a small-molecule inhibitor (SB743921) in two SCLC cell lines, Lu-135 and NCI-H69, cell cycle progression was arrested at the G2/M phase with complete growth suppression. Further work suggested that the two cell lines were more significantly affected when both KIF11 and BCL2L1, an anti-apoptotic BCL2 family member, were inhibited. This dual inhibition resulted in markedly decreased cell viability. These findings collectively indicate that SCLC cells are critically dependent on KIF11 activity for survival and/or proliferation, as well as that KIF11 inhibition could be a new strategy for SCLC treatment.
Subject(s)
Cell Survival , Kinesins , Lung Neoplasms , Small Cell Lung Carcinoma , Humans , Kinesins/metabolism , Kinesins/genetics , Kinesins/antagonists & inhibitors , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/metabolism , Small Cell Lung Carcinoma/pathology , Cell Line, Tumor , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/drug therapy , Cell Survival/drug effects , Cell Survival/genetics , Cell Proliferation , bcl-X Protein/metabolism , bcl-X Protein/genetics , Gene Expression Regulation, Neoplastic , Apoptosis/genetics , Benzamides , QuinazolinesABSTRACT
At present, the role of many long non-coding RNAs (lncRNAs) as tumor suppressors in the formation and development of cervical cancer (CC) has been studied. However, lncRNA prostate cancer gene expression marker 1 (PCGEM1), whose high expression not only aggravates ovarian cancer but also can induce tumorigenesis and endometrial cancer progression, has not been studied in CC. The objective of this study was to investigate the expression and the underlying role of PCGEM1 in CC. The relative expression of PCGEM1 in CC cells was detected by real-time PCR. After the suppression of PCGEM1 expression by shRNA, the changes in the proliferation, migration, and invasion capacities were detected via CCK-8 assay, EdU assay, and colony formation assay wound healing assay. Transwell assay and the changes in expressions of epithelial-to-mesenchymal transition (EMT) markers were determined by western blot and immunofluorescence. The interplay among PCGEM1, miR-642a-5p, and kinesin family member 5B (KIF5B) was confirmed by bioinformatics analyses and luciferase reporter assay. Results showed that PCGEM1 expressions were up-regulated within CC cells. Cell viabilities, migration, and invasion were remarkably reduced after the suppression of PCGEM1 expression by shRNA in Hela and SiHa cells. N-cadherin was silenced, but E-cadherin expression was elevated by sh-PCGEM1. Moreover, by sponging miR-642a-5p in CC, PCGEM1 was verified as a competitive endogenous RNA (ceRNA) that modulates KIF5B levels. MiR-642a-5p down-regulation partially rescued sh-PCGEM1's inhibitory effects on cell proliferation, migration, invasion, and EMT process. In conclusion, the PCGEM1/miR-642a-5p/KIF5B signaling axis might be a novel therapeutic target in CC. This study provides a research basis and new direction for targeted therapy of CC.
Subject(s)
Cell Movement , Cell Proliferation , Disease Progression , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Kinesins , MicroRNAs , RNA, Long Noncoding , Uterine Cervical Neoplasms , Humans , RNA, Long Noncoding/genetics , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/metabolism , MicroRNAs/genetics , Female , Kinesins/genetics , Kinesins/metabolism , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Cell Movement/genetics , Cell Line, Tumor , HeLa Cells , Neoplasm InvasivenessABSTRACT
Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans. We found that the status of ß-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.
Subject(s)
Caenorhabditis elegans , Microtubules , Protein Processing, Post-Translational , Tubulin , Animals , Tubulin/metabolism , Tubulin/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/genetics , Microtubules/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Acetylation , Axons/metabolism , Axons/physiology , Phosphorylation , Nerve Regeneration , Kinesins/metabolism , Kinesins/geneticsABSTRACT
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.
Subject(s)
Cryoelectron Microscopy , Kinesins , Microtubules , Tubulin , Kinesins/metabolism , Kinesins/genetics , Kinesins/chemistry , Microtubules/metabolism , Humans , Tubulin/metabolism , Tubulin/chemistry , Tubulin/genetics , Protein Binding , Mutation , Models, Molecular , Protein ConformationABSTRACT
The application of adjuvant treatment has significantly enhanced the survival of patients with resectable non-small cell lung cancer (NSCLC) carrying driver gene mutations. However, adjuvant-targeted therapy remains controversial for some NSCLC patients carrying rare gene mutations such as RET, as there is currently a lack of confirmed randomized controlled trials demonstrating efficacy. In this report, we describe the case of a 58-year-old man with stage IIIA NSCLC who underwent complete lobectomy with selective lymph node dissection. Postoperative next-generation sequencing revealed that the patient harbored a rare KIF13A-RET fusion. The patient elected to receive adjuvant treatment with pralsetinib monotherapy and underwent serial circulating tumor DNA (ctDNA) monitoring after surgery. During follow-up, despite experiencing dose reduction and irregular medication adherence, the patient still achieved a satisfactory disease-free survival (DFS) of 27 months. Furthermore, ctDNA predicted tumor recurrence 4 months earlier than imaging techniques. The addition of bevacizumab to the original regimen upon recurrence continued to be beneficial. Pralsetinib demonstrated promising efficacy as adjuvant therapy, while ctDNA analysis offered a valuable tool for early detection of tumor recurrence. By leveraging targeted therapies and innovative monitoring techniques, we aim to improve outcomes and quality of life for NSCLC patients in the future.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Kinesins , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Male , Middle Aged , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Kinesins/genetics , Pyrimidinones/therapeutic use , Proto-Oncogene Proteins c-ret/genetics , Circulating Tumor DNA/genetics , Pyrazoles/therapeutic use , Pyridines , PyrimidinesABSTRACT
After whole-genome duplication (WGD), tetraploid cells can undergo multipolar mitosis or pseudo-bipolar mitosis with clustered centrosomes. Kinesins play a crucial role in regulating spindle formation. However, the contribution of kinesin expression levels to the heterogeneity in centrosome clustering observed across different cell lines after WGD remains unclear. We identified two subsets of cell lines: "BP" cells efficiently cluster extra centrosomes for pseudo-bipolar mitosis, and "MP" cells primarily undergo multipolar mitosis after WGD. Diploid MP cells contained higher levels of KIF11 and KIF15 compared with BP cells and showed reduced sensitivity to centrosome clustering induced by KIF11 inhibitors. Moreover, partial inhibition of KIF11 or depletion of KIF15 converted MP cells from multipolar to bipolar mitosis after WGD. Multipolar spindle formation involved microtubules but was independent of kinetochore-microtubule attachment. Silencing KIFC1, but not KIFC3, promoted multipolar mitosis in BP cells, indicating the involvement of specific kinesin-14 family members in counteracting the forces from KIF11/KIF15 after WGD. These findings highlight the collective role of KIF11, KIF15, and KIFC1 in determining the polarity of the mitotic spindle after WGD.
Subject(s)
Centrosome , Kinesins , Mitosis , Spindle Apparatus , Kinesins/metabolism , Kinesins/genetics , Centrosome/metabolism , Humans , Mitosis/genetics , Spindle Apparatus/metabolism , Gene Duplication , Microtubules/metabolism , Cell Line , Kinetochores/metabolism , Genome, HumanABSTRACT
A better understanding of incidences at the cellular level in uterine cancer is necessary for its effective treatment and favourable prognosis. Till date, it lacks appropriate molecular target-based treatment because of unknown molecular mechanisms that proceed to cancer and no drug has shown the required results of treatment with less severe side effects. Uterine Cancer is one of the top five cancer diagnoses and among the ten most common death-causing cancer in the United States of America. There is no FDA-approved drug for it yet. Therefore, it became necessary to identify the molecular targets for molecular targeted therapy of this widely prevalent cancer type. For this study, we used a network-based approach to the list of the deregulated (both up and down-regulated) genes taking adjacent p-Value ≤ 0.05 as significance cut off for the mRNA data of uterine cancer. We constructed the protein-protein interaction (PPI) network and analyzed the degree, closeness, and betweenness centrality-like topological properties of the PPI network. Then we traced the top 30 genes listed from each topological property to find the key regulators involved in the endometrial cancer (ECa) network. We then detected the communities and sub-communities from the PPI network using the Cytoscape network analyzer and Louvain modularity optimization method. A set of 26 (TOP2A, CENPE, RAD51, BUB1, BUB1B, KIF2C, KIF23, KIF11, KIF20A, ASPM, AURKA, AURKB, PLK1, CDC20, CDKN2A, EZH2, CCNA2, CCNB1, CDK1, FGF2, PRKCA, PGR, CAMK2A, HPGDS, and CDCA8) genes were found to be key genes of ECa regulatory network altered in disease state and might be playing the regulatory role in complex ECa network. Our study suggests that among these genes, KIF11 and H PGDS appeared to be novel key genes identified in our research. We also identified these key genes interactions with miRNAs.
Subject(s)
Biomarkers, Tumor , Protein Interaction Maps , Uterine Neoplasms , Humans , Female , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Uterine Neoplasms/genetics , Uterine Neoplasms/pathology , Uterine Neoplasms/metabolism , Gene Regulatory Networks , Gene Expression Regulation, Neoplastic , Gene Expression Profiling/methods , KinesinsABSTRACT
Intervertebral disc degeneration (IVDD) is characterized by the senescence and declining vitality of nucleus pulposus cells (NPCs), often driven by mitochondrial dysfunction. This study elucidates that mesenchymal stem cells (MSCs) play a crucial role in attenuating NPC senescence by secreting mitochondria-containing microvesicles (mitoMVs). Moreover, it demonstrates that static magnetic fields (SMF) enhance the secretion of mitoMVs by MSCs. By distinguishing mitoMV generation from exosomes, this study shifts focus to understanding the molecular mechanisms of SMF intervention, emphasizing cargo transport and plasma membrane budding processes, with RNA sequencing indicating the potential involvement of the microtubule-based transport protein Kif5b. The study further confirms the interaction between Rab22a and Kif5b, revealing Rab22a's role in sorting mitoMVs into microvesicles (MVs) and potentially mediating subsequent plasma membrane budding. Subsequent construction of a gelatin methacrylate (GelMA) hydrogel delivery system further addresses the challenges of in vivo application and verifies the substantial potential of mitoMVs in delaying IVDD. This research not only sheds light on the molecular intricacies of SMF-enhanced mitoMV secretion but also provides innovative perspectives for future IVDD therapeutic strategies.
Subject(s)
Cell-Derived Microparticles , Intervertebral Disc Degeneration , Magnetic Fields , Mesenchymal Stem Cells , Mitochondria , Nucleus Pulposus , Mesenchymal Stem Cells/metabolism , Intervertebral Disc Degeneration/therapy , Intervertebral Disc Degeneration/metabolism , Mitochondria/metabolism , Animals , Cell-Derived Microparticles/metabolism , Nucleus Pulposus/metabolism , Humans , Rats , Kinesins/metabolism , Cells, Cultured , Rats, Sprague-Dawley , rab GTP-Binding Proteins/metabolism , MaleABSTRACT
Biological sciences are increasingly uncovering the foundations of life in greater detail, made possible by the development of research methods enabling exploration at the nanometer scale. Optical microscopy, a field with a significant contribution to current knowledge, is inherently limited by the Abbe limit, stemming from the fundamental wave properties of light. Through the efforts of scientists, this limit can be circumvented, as evidenced by STED and MINFLUX techniques. STED allows imaging with a resolution down to 40 nm, while MINFLUX enables resolution as fine as 2 nm. Both techniques require labelling of biological molecular targets with fluorescent markers and enable imaging in living cells, facilitating the study of dynamic biological processes. This article provides an introduction to super-resolution techniques STED and MINFLUX, demonstrating their utility through the example of studying kinesin movement along microtubules using the MINFLUX technique.
Subject(s)
Microscopy, Fluorescence , Microtubules , Microscopy, Fluorescence/methods , Microtubules/metabolism , Kinesins/metabolism , Humans , Fluorescent Dyes , AnimalsABSTRACT
Kinesin-1 ensembles maneuver vesicular cargoes through the three-dimensional (3D) intracellular microtubule (MT) network. To define how such cargoes navigate MT intersections, we first determined how many kinesins from an ensemble on a lipid-based cargo simultaneously engage a MT, and then determined the directional outcomes (straight, turn, terminate) for liposome cargoes at perpendicular MT intersections. Run lengths of 350-nm diameter liposomes decorated with up to 20, constitutively active, truncated kinesin-1 KIF5B (K543) were longer than single motor transported cargo, suggesting multiple motor engagement. However, detachment forces of lipid-coated beads with ~20 kinesins, measured using an optical trap, showed no more than three simultaneously engaged motors, with a single engaged kinesin predominating, indicating anticooperative MT binding. At two-dimensional (2D) and 3D in vitro MT intersections, liposomes frequently paused (~2 s), suggesting kinesins simultaneously bind both MTs and engage in a tug-of-war. Liposomes showed no directional outcome bias in 2D (1.1 straight:turn ratio) but preferentially went straight (1.8 straight:turn ratio) in 3D intersections. To explain these data, we developed a mathematical model of liposome transport incorporating the known mechanochemistry of kinesins, which diffuse on the liposome surface, and have stiff tails in both compression and extension that impact how motors engage the intersecting MTs. Our model predicts the ~3 engaged motor limit observed in the optical trap and the bias toward going straight in 3D intersections. The striking similarity of these results to our previous study of liposome transport by myosin Va suggests a "universal" mechanism by which cargoes navigate 3D intersections.
Subject(s)
Kinesins , Liposomes , Microtubules , Kinesins/metabolism , Kinesins/chemistry , Liposomes/chemistry , Liposomes/metabolism , Microtubules/metabolism , Biological Transport , Animals , Molecular Motor Proteins/metabolism , Molecular Motor Proteins/chemistry , Optical TweezersABSTRACT
The polarized nature of neurons depends on their microtubule dynamics and orientation determined by both microtubule-stabilizing and destabilizing factors. The role of destabilizing factors in developing and maintaining neuronal polarity is unclear. We investigated the function of KLP-7, a microtubule depolymerizing motor of the Kinesin-13 family, in axon-dendrite compartmentalization using PVD neurons in Caenorhabditis elegans. Loss of KLP-7 caused a mislocalization of axonal proteins, including RAB-3, SAD-1, and their motor UNC-104, to dendrites. This is rescued by cell-autonomous expression of the KLP-7 or colchicine treatment, indicating the involvement of KLP-7-dependent microtubule depolymerization. The high mobility of KLP-7 is correlated to increased microtubule dynamics in the dendrites, which restricts the enrichment of UNC-44, an integral component of Axon Initial Segment (AIS) in these processes. Due to the loss of KLP-7, ectopic enrichment of UNC-44 in the dendrite potentially redirects axonal traffic into dendrites that include plus-end out microtubules, axonal motors, and cargoes. These observations indicate that KLP-7-mediated depolymerization defines the microtubule dynamics conducive to the specific enrichment of AIS components in dendrites. This further compartmentalizes dendritic and axonal microtubules, motors, and cargoes, thereby influencing neuronal polarity.
Subject(s)
Axons , Caenorhabditis elegans Proteins , Caenorhabditis elegans , Cell Polarity , Dendrites , Kinesins , Microtubules , Animals , Caenorhabditis elegans/metabolism , Dendrites/metabolism , Caenorhabditis elegans Proteins/metabolism , Kinesins/metabolism , Microtubules/metabolism , Axons/metabolism , Cell Polarity/physiology , Neurons/metabolism , Protein Transport , Nerve Tissue Proteins/metabolismABSTRACT
Immune therapy has significantly improved the prognosis of hepatocellular carcinoma (HCC) patients, yet its efficacy remains limited, underscoring the urgency to identify new therapeutic targets and biomarkers. Here, we investigated the pathological and physiological roles of KIF20A and assess its potential in enhancing HCC treatment efficacy when combined with PD-1 inhibitors. We initially assess KIF20A's oncogenic function using liver-specific KIF20A knockout (Kif20a CKO) mouse models and orthotopic xenografts. Subsequently, we establish a regulatory axis involving KIF20A, FBXW7, and c-Myc, validated through construction of c-Myc splicing mutants. Large-scale clinical immunohistochemistry (IHC) analyses confirm the pathological relevance of the KIF20A-FBXW7-c-Myc axis in HCC. We demonstrate that KIF20A overexpression correlates with poor prognosis in HCC by competitively inhibiting FBXW7-mediated degradation of c-Myc, thereby promoting glycolysis and enhancing tumor proliferation. Conversely, KIF20A downregulation suppresses these effects, impairing tumor growth through c-Myc downregulation. Notably, KIF20A inhibition attenuates c-Myc-induced MMR expression, associated with improved prognosis in HCC patients receiving PD-1 inhibitor therapy. Furthermore, in Kif20a CKO HCC mouse models, we observe synergistic effects between Kif20a knockout and anti-PD-1 antibodies, significantly enhancing immunotherapeutic efficacy against HCC. Our findings suggest that targeting the KIF20A-c-Myc axis could identify HCC patients likely to benefit from anti-PD-1 therapy. In conclusion, we propose that combining KIF20A inhibitors with anti-PD-1 treatment represents a promising therapeutic strategy for HCC, offering new avenues for clinical development and patient stratification.
Subject(s)
Carcinoma, Hepatocellular , F-Box-WD Repeat-Containing Protein 7 , Kinesins , Liver Neoplasms , Proto-Oncogene Proteins c-myc , Ubiquitination , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/immunology , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Animals , Humans , Mice , Kinesins/genetics , Kinesins/antagonists & inhibitors , Kinesins/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , F-Box-WD Repeat-Containing Protein 7/genetics , F-Box-WD Repeat-Containing Protein 7/metabolism , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Cell Line, Tumor , Mice, Knockout , Xenograft Model Antitumor Assays , Cell Proliferation , Immunotherapy/methods , Male , Prognosis , Gene Expression Regulation, NeoplasticABSTRACT
Microtubule (MT)-dependent transport is a critical means of intracellular movement of cellular cargo by kinesin and dynein motors. MT-dependent transport is tightly regulated by cellular MT-associated proteins (MAPs) that directly bind to MTs and either promote or impede motor protein function. Viruses have been widely shown to usurp MT-dependent transport to facilitate their virion movement to sites of replication and/or for exit from the cell. However, it is unclear if viruses also negatively regulate MT-dependent transport. Using single-molecule motility and cellular transport assays, we show that the vaccinia virus (VV)-encoded MAP, A51R, inhibits kinesin-1-dependent transport along MTs in vitro and in cells. This inhibition is selective as the function of kinesin-3 is largely unaffected by VV A51R. Interestingly, we show that A51R promotes the perinuclear accumulation of cellular cargo transported by kinesin-1 such as lysosomes and mitochondria during infection. Moreover, A51R also regulates the release of specialized VV virions that exit the cell using kinesin-1-dependent movement. Using a fluorescently tagged rigor mutant of kinesin-1, we show that these motors accumulate on A51R-stabilized MTs, suggesting these stabilized MTs may form a "kinesin-1 sink" to regulate MT-dependent transport in the cell. Collectively, our findings uncover a new mechanism by which viruses regulate host cytoskeletal processes.
Subject(s)
Kinesins , Microtubules , Vaccinia virus , Kinesins/metabolism , Kinesins/genetics , Microtubules/metabolism , Humans , Vaccinia virus/metabolism , Vaccinia virus/physiology , Vaccinia virus/genetics , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/genetics , Biological Transport , HeLa CellsABSTRACT
Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans. If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans, we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes.