RESUMO
The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family.
Assuntos
Cinesinas , Microtúbulos , Tetrahymena thermophila , Cinesinas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Tetrahymena thermophila/metabolismo , Tetrahymena thermophila/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Trifosfato de Adenosina/metabolismo , Cílios/metabolismo , Tetrahymena/metabolismo , Tetrahymena/genéticaRESUMO
In the realm of this study, obtaining a comprehensive understanding of ischemic brain injury and its molecular foundations is of paramount importance. Our study delved into single-cell data analysis, with a specific focus on sub-celltypes and differentially expressed genes in the aftermath of ischemic injury. Notably, we observed a significant enrichment of the "ATP METABOLIC PROCESS" and "ATP HYDROLYSIS ACTIVITY" pathways, featuring pivotal genes such as Pbx3, Dguok, and Kif21b. A remarkable finding was the consistent upregulation of genes like Fabp7 and Bcl11a within the MCAO group, highlighting their crucial roles in regulating the pathway of mitochondrial ATP synthesis coupled proton transport. Furthermore, our network analysis unveiled pathways like "Neuron differentiation" and "T cell differentiation" as central in the regulatory processes of sub-celltypes. These findings provide valuable insights into the intricate molecular responses and regulatory mechanisms that govern brain injury. The shared differentially expressed genes among sub-celltypes emphasize their significance in orchestrating responses post-ischemic injury. Our research, viewed from the perspective of a medical researcher, contributes to the evolving understanding of the molecular landscape underlying ischemic brain injury, potentially paving the way for targeted therapeutic strategies and improved patient outcomes.
Assuntos
Trifosfato de Adenosina , Infarto da Artéria Cerebral Média , Cinesinas , Mitocôndrias , Células Precursoras de Oligodendrócitos , Transdução de Sinais , Animais , Transdução de Sinais/genética , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/biossíntese , Cinesinas/genética , Cinesinas/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Humanos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Ratos , Proteínas Proto-OncogênicasRESUMO
Despite the importance of cellular senescence in human health, how damaged cells undergo senescence remains elusive. We have previously shown that promyelocytic leukemia nuclear body (PML-NBs) translocation of the ciliary FBF1 is essential for senescence induction in stressed cells. Here we discover that an early cellular event occurring in stressed cells is the transient assembly of stress-induced nucleus-to-cilium microtubule arrays (sinc-MTs). The sinc-MTs are distinguished by unusual polyglutamylation and unique polarity, with minus-ends nucleating near the nuclear envelope and plus-ends near the ciliary base. KIFC3, a minus-end-directed kinesin, is recruited to plus-ends of sinc-MTs and interacts with the centrosomal protein CENEXIN1. In damaged cells, CENEXIN1 co-translocates with FBF1 to PML-NBs. Deficiency of KIFC3 abolishes PML-NB translocation of FBF1 and CENEXIN1, as well as senescence initiation in damaged cells. Our study reveals that KIFC3-mediated nuclear transport of FBF1 along polyglutamylated sinc-MTs is a prerequisite for senescence induction in mammalian cells.
Assuntos
Núcleo Celular , Senescência Celular , Cílios , Cinesinas , Microtúbulos , Humanos , Cinesinas/metabolismo , Cinesinas/genética , Núcleo Celular/metabolismo , Microtúbulos/metabolismo , Cílios/metabolismo , Animais , Transporte Ativo do Núcleo Celular , CamundongosRESUMO
Given the infiltrative nature of human glioblastoma (GBM), cocktail drug therapy will remain a vital tool for the treatment of the disease. We investigated fluspirilene, perphenazine, and sulpiride, three classic anti-schizophrenic drugs, as possible anti-GBM agents. The CCK-8 assay demonstrated that fluspirilene possesses the most outstanding anti-GBM effect. We performed molecular mechanisms studies in vitro and an orthotopic xenograft model in mice. Fluspirilene inhibited proliferation and migration in vitro in U87MG and U251 GBM cell lines. Flow cytometry demonstrated that treatment increased apoptosis and cells accumulated in the G2/M phase. Our analysis of publicly available expression data for several cell lines treated with the drug led to the identification of several genes, including KIF20A, that are downregulated by fluspirilene and lead to growth inhibition/apoptosis. We also demonstrated that siRNA knockdown of KIF20A, a member of the kinesin family, attenuated cell proliferation in GBM cells and an orthotopic xenograft model in mice. A regulator of KIF20A, the oncogenic transcription factor FOXM1, was identified using the String database, which harbors protein interaction networks. In fluspirilene-treated cells, FOXM1 protein was decreased, indicating that KIF20A was downregulated in the presence of the drug due to decreased FOXM1 protein. These results demonstrate that fluspirilene is an effective anti-GBM agent that works by suppressing the FOXM1-KIF20A oncogenic axis.
Assuntos
Apoptose , Proliferação de Células , Proteína Forkhead Box M1 , Glioblastoma , Cinesinas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Forkhead Box M1/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Humanos , Animais , Camundongos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Cinesinas/antagonistas & inibidores , Cinesinas/metabolismo , Movimento Celular/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Mutations targeting distinct domains of the neuron-specific kinesin KIF5A associate with different neurodegenerative/neurodevelopmental disorders, but the molecular bases of this clinical heterogeneity are unknown. We characterised five key mutants covering the whole spectrum of KIF5A-related phenotypes: spastic paraplegia (SPG, R17Q and R280C), Charcot-Marie-Tooth disease (CMT, R864*), amyotrophic lateral sclerosis (ALS, N999Vfs*40), and neonatal intractable myoclonus (NEIMY, C975Vfs*73) KIF5A mutants. CMT-R864*-KIF5A and ALS-N999Vfs*40-KIF5A showed impaired autoinhibition and peripheral localisation accompanied by altered mitochondrial distribution, suggesting transport competence disruption. ALS-N999Vfs*40-KIF5A formed SQSTM1/p62-positive inclusions sequestering WT-KIF5A, indicating a gain of toxic function. SPG-R17Q-KIF5A and ALS-N999Vfs*40-KIF5A evidenced a shorter half-life compared to WT-KIF5A, and proteasomal blockage determined their accumulation into detergent-insoluble inclusions. Interestingly, SPG-R280C-KIF5A and ALS-N999Vfs*40-KIF5A both competed for degradation with proteasomal substrates. Finally, NEIMY-C975Vfs*73-KIF5A displayed a similar, but more severe aberrant behaviour compared to ALS-N999Vfs*40-KIF5A; these two mutants share an abnormal tail but cause disorders on the opposite end of KIF5A-linked phenotypic spectrum. Thus, our observations support the pathogenicity of novel KIF5A mutants, highlight abnormalities of recurrent variants, and demonstrate that both unique and shared mechanisms underpin KIF5A-related diseases.
Assuntos
Cinesinas , Mutação , Transtornos do Neurodesenvolvimento , Cinesinas/metabolismo , Cinesinas/genética , Humanos , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Mutação/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Animais , Mitocôndrias/metabolismo , Mitocôndrias/genética , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologiaRESUMO
Microplastics and nanoplastics (MNPs) originating from plastic pollution pose potential threats to cardiovascular health, with prior studies linking MNPs to atherosclerosis. Our earlier research elucidated how nanoplastics enhance macrophages' phagocytic activity, leading to the formation of foam cells and an elevated risk of atherosclerosis. However, the specific influence of MNPs on smooth muscle cells (SMCs) in the context of MNP-induced atherosclerosis remains poorly understood. In this study, ApoE knockout (ApoE-/-) male mice with a high-fat diet were orally exposed to environmentally realistic concentrations of 2.5-250â¯mg/kg polystyrene nanoplastics (PS-NPs, 50â¯nm) for consecutive 19 weeks. Cardiovascular toxicity was comprehensively assessed through histopathological, transcriptomic, and proteomic analyses, while mechanisms underlying this toxicity were explored through in vitro studies. Herein, hematoxylin and eosin staining revealed accelerated atherosclerotic plaque development in ApoE-/- mice exposed to PS-NPs. Multi-omics analysis identified kinesin family member 15 (KIF15) as a pivotal target molecule. Both in vitro and in vivo experiments affirmed the specific upregulation of KIF15 in mouse aortic SMCs exposed to PS-NPs. Furthermore, in vitro experiments demonstrated that PS-NPs can promote the migration ability of MOVAS cells. Knockdown of Kif15 revealed its role in reducing MOVAS cell migration, with subsequent exposure to PS-NPs reversing the increased migration ability. This suggests that PS-NPs promote SMC migration by upregulating KIF15, and the migration of SMCs is closely associated with atherosclerosis outcomes. This study significantly advances our understanding of MNP-induced cardiovascular toxicity, providing valuable insights for risk assessment of human MNP exposure.
Assuntos
Aterosclerose , Movimento Celular , Cinesinas , Miócitos de Músculo Liso , Poliestirenos , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/patologia , Camundongos , Masculino , Cinesinas/metabolismo , Movimento Celular/efeitos dos fármacos , Poliestirenos/toxicidade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Microplásticos/toxicidade , Nanopartículas/toxicidade , Apolipoproteínas E/genética , Camundongos Knockout , Camundongos Knockout para ApoE , Camundongos Endogâmicos C57BLRESUMO
Kinesin-5 inhibitors offer cancer cell-targeted approach, thus securing reduced systemic toxicity compared to other antimitotic agents. By modifying the 1,4-dihydropyridine-based kinesin-5 inhibitor CPUYL064 with a ferrocenyl moiety (Fc), we designed and prepared a series of organometallic hybrids that show high antiproliferative activity, with the best compounds exhibiting up to 19-fold increased activity. This enhanced activity can be attributed to the presence of the ferrocenyl moiety.
Assuntos
Antineoplásicos , Proliferação de Células , Di-Hidropiridinas , Desenho de Fármacos , Compostos Ferrosos , Cinesinas , Di-Hidropiridinas/química , Di-Hidropiridinas/farmacologia , Di-Hidropiridinas/síntese química , Cinesinas/antagonistas & inibidores , Cinesinas/metabolismo , Humanos , Compostos Ferrosos/química , Compostos Ferrosos/farmacologia , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Metalocenos/química , Metalocenos/farmacologiaRESUMO
Cancer stem cells (CSCs) have the potential to self-renew and induce cancer, which may contribute to a poor prognosis by enabling metastasis, recurrence, and therapy resistance. Hence, this study was performed to identify the association between CSC-related genes and triple-negative breast cancer (TNBC) development. Stemness gene sets were downloaded from StemChecker. Based on the online databases, a consensus clustering algorithm was conducted for unsupervised classification of TNBC samples. The variations between subtypes were assessed with regard to prognosis, tumor immune microenvironment (TIME), and chemotherapeutic sensitivity. The stemness-related gene signature was established and random survival forest analysis was employed to identify the core gene for validation experiments and tumor sphere formation assays. 499 patients with TNBC were classified into three subgroups and the Cluster 1 had a better OS than others. After that, WGCNA study was performed to identify genes important for Cluster 1 subtype. Out of all 8 modules, the subtype of Cluster 1 and the yellow module with 103 genes demonstrated the largest positive association. After that, a four-gene stemness-related signature was established. Based on the yellow module, the 39 potential pivotal genes were subjected to the random forest survival analysis to find out the gene that was relatively important for OS. KIF15 was confirmed as the targeted gene by LASSO and random survival forest analyses. In vitro experiments, the downregulation of KIF15 promoted the stemness of TNBC cells. The expression levels of stem cell markers Nanog, SOX2, and OCT4 were found to be elevated in TNBC cell lines after KIF15 inhibition. A stemness-associated risk model was constructed to forecast the clinical outcomes of TNBC patients. The downregulation of KIF15 expression in a subpopulation of TNBC stem cells may promote stemness and possibly TNBC progression.
Assuntos
Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Cinesinas , Aprendizado de Máquina , Células-Tronco Neoplásicas , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Cinesinas/genética , Cinesinas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Prognóstico , Microambiente Tumoral/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , AlgoritmosRESUMO
KIFC3 is a member of the Kinesin superfamily proteins (KIFs). The role of KIFC3 in non-small cell lung cancer (NSCLC) is unknown. This study aimed to elucidate the function of KIFC3 in NSCLC and the underlying mechanism. Immunohistochemistry indicated that KIFC3 was highly expressed in NSCLC tissues and correlated with the degree of differentiation, tumor size, lymph node metastasis and TNM stage. MTT, colony formation and Transwell assays demonstrated that KIFC3 overexpression promoted the proliferation, migration and invasion of NSCLC cells in vitro, while KIFC3 knockdown led to the opposite results. The protein expression levels of PI3Kp85α and p-Akt were increased after KIFC3 overexpression, meanwhile the downstream protein expression levels such as cyclin D1, CDK4, CDK6, RhoA, RhoC and MMP2 were increased. This promotion effect could be inhibited by a specific inhibitor of the PI3K/Akt pathway, LY294002. Co-immunoprecipitation assays confirmed the interaction between endogenous/exogenous KIFC3 and PI3Kp85α. Tumor formation experiments in nude mice confirmed that KIFC3 overexpression promoted the proliferation, migration and invasion of NSCLC cells in vivo and performed its biological function through the PI3K/Akt signaling pathway.In conclusion, KIFC3 promotes the malignant behavior of NSCLC cells through the PI3K/Akt signaling pathway.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Movimento Celular , Proliferação de Células , Neoplasias Pulmonares , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Movimento Celular/genética , Animais , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Feminino , Masculino , Camundongos , Linhagem Celular Tumoral , Pessoa de Meia-Idade , Cinesinas/metabolismo , Cinesinas/genética , Camundongos Nus , Regulação Neoplásica da Expressão GênicaRESUMO
B-Myb, also known as MYB proto-oncogene like 2 (MYBL2), is an important transcription factor implicated in transcription regulation, cell cycle and tumorigenesis. However, the molecular mechanism underlying B-Myb-controlled transactivation in different cell contexts as well as its functional implication in cancers remains elusive. In this study, we have conducted a comprehensive genome-wide analysis of B-Myb binding sites in multiple immortalized or cancer cell lines and identified its critical target genes. The results revealed that B-Myb regulates a common set of core cell cycle genes and cell type-specific genes through collaboration with other important transcription factors (e.g. NFY and MuvB complex) and binding to cell type-invariant promoters and cell type-specific enhancers and super-enhancers. KIF2C, UBE2C and MYC were further validated as B-Myb target genes. Loss-of-function analysis demonstrated that KIF2C knockdown inhibited tumor cell growth both in vitro and in vivo, suppressed cell motility and cell cycle progression, accompanied with defects in microtubule organization and mitosis, strongly suggesting that KIF2C is a critical regulator of cancer cell growth and mitosis, and maintains high cancer cell motility ability and microtubule dynamics. Pan-cancer transcriptomic analysis revealed that the overexpression of both B-Myb and KIF2C presents as independent prognostic markers in various types of cancer. Notably, B-Myb associates with NFYB, binds to target gene promoters, enhancers and super-enhancers, and provokes a cascade of oncogenic gene expression profiles in cancers. Overall, our results highly suggest the critical implication of B-Myb-mediated gene regulation in cancers, and the promising therapeutic and prognostic potentials of B-Myb and KIF2C for cancer diagnosis and treatment.
Assuntos
Ativação Transcricional , Humanos , Ativação Transcricional/genética , Linhagem Celular Tumoral , Neoplasias/genética , Neoplasias/metabolismo , Proto-Oncogene Mas , Regulação Neoplásica da Expressão Gênica , Cinesinas/metabolismo , Cinesinas/genética , Transativadores/metabolismo , Transativadores/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Camundongos , Estudo de Associação Genômica Ampla , Regiões Promotoras Genéticas , Movimento Celular/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Cinesinas , Neurônios Motores , Animais , Cinesinas/genética , Cinesinas/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Mutação , Humanos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Drosophila melanogaster/genética , Transmissão Sináptica/genética , Modelos Animais de Doenças , Axônios/metabolismo , Axônios/patologia , Larva/genética , Larva/metabolismoRESUMO
Primary cilia are antenna-like organelles which sense extracellular cues and act as signalling hubs. Cilia dysfunction causes a heterogeneous group of disorders known as ciliopathy syndromes affecting most organs. Cilia disassembly, the process by which cells lose their cilium, is poorly understood but frequently observed in disease and upon cell transformation. Here, we uncover a role for the PI3Kα signalling enzyme in cilia disassembly. Genetic PI3Kα-hyperactivation, as observed in PIK3CA-related overgrowth spectrum (PROS) and cancer, induced a ciliopathy-like phenotype during mouse development. Mechanistically, PI3Kα and PI3Kß produce the PIP3 lipid at the cilia transition zone upon disassembly stimulation. PI3Kα activation initiates cilia disassembly through a kinase signalling axis via the PDK1/PKCι kinases, the CEP170 centrosomal protein and the KIF2A microtubule-depolymerising kinesin. Our data suggest diseases caused by PI3Kα-activation may be considered 'Disorders with Ciliary Contributions', a recently-defined subset of ciliopathies in which some, but not all, of the clinical manifestations result from cilia dysfunction.
Assuntos
Cílios , Classe I de Fosfatidilinositol 3-Quinases , Transdução de Sinais , Cílios/metabolismo , Animais , Camundongos , Humanos , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Ciliopatias/metabolismo , Ciliopatias/genética , Ciliopatias/patologia , Cinesinas/metabolismo , Cinesinas/genéticaRESUMO
The acentrosomal spindle apparatus has kinetochore fibers organized and converged toward opposite poles; however, mechanisms underlying the organization of these microtubule fibers into an orchestrated bipolar array were largely unknown. Kinesin-14D is one of the four classes of Kinesin-14 motors that are conserved from green algae to flowering plants. In Arabidopsis thaliana, three Kinesin-14D members displayed distinct cell cycle-dependent localization patterns on spindle microtubules in mitosis. Notably, Kinesin-14D1 was enriched on the midzone microtubules of prophase and mitotic spindles and later persisted in the spindle and phragmoplast midzones. The kinesin-14d1 mutant had kinetochore fibers disengaged from each other during mitosis and exhibited hypersensitivity to the microtubule-depolymerizing herbicide oryzalin. Oryzalin-treated kinesin-14d1 mutant cells had kinetochore fibers tangled together in collapsed spindle microtubule arrays. Kinesin-14D1, unlike other Kinesin-14 motors, showed slow microtubule plus end-directed motility, and its localization and function were dependent on its motor activity and the novel malectin-like domain. Our findings revealed a Kinesin-14D1-dependent mechanism that employs interpolar microtubules to regulate the organization of kinetochore fibers for acentrosomal spindle morphogenesis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cinesinas , Microtúbulos , Fuso Acromático , Arabidopsis/metabolismo , Arabidopsis/genética , Cinesinas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fuso Acromático/metabolismo , Mitose , Morfogênese , Cinetocoros/metabolismo , Dinitrobenzenos/farmacologia , Sulfanilamidas/farmacologiaRESUMO
The microtubule-kinesin biomolecular motor system, which is vital for cellular function, holds significant promise for nanotechnological applications. In vitro gliding assays have demonstrated the ability to transport microcargo by propelling microtubules across kinesin-coated surfaces. However, the uncontrolled directional motion of microtubules has posed significant challenges, limiting the system's application for precise cargo delivery. Microfluidic devices provide a means to direct microtubule movement through their geometric features. Norland Optical Adhesive (NOA) is valued for its mold-free application in microfluidic device fabrication; however, microtubules often climb up channel walls, limiting controlled movement. In this study, a surface passivation method for NOA is introduced, using polyethylene glycol via a thiol-ene click reaction. This technique significantly improved the directional control and concentration of microtubules within NOA microchannels. This approach presents new possibilities for the precise application of biomolecular motors in nanotechnology, enabling advancements in the design of microfluidic systems for complex biomolecular manipulations.
Assuntos
Adesivos , Cinesinas , Microtúbulos , Propriedades de Superfície , Microtúbulos/química , Microtúbulos/metabolismo , Adesivos/química , Cinesinas/química , Cinesinas/metabolismo , Nanotecnologia/métodos , Polietilenoglicóis/química , Técnicas Analíticas Microfluídicas , Dispositivos Lab-On-A-ChipRESUMO
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.
Assuntos
Cinesinas , Microtúbulos , Modelos Moleculares , Cinesinas/metabolismo , Cinesinas/química , Microtúbulos/metabolismo , Mutação , Multimerização Proteica , Humanos , Animais , DrosophilaRESUMO
Fast axonal transport is crucial for neuronal function and is driven by kinesins and cytoplasmic dynein. Here, we investigated the role of kinesin-1 in dense core vesicle (DCV) transport in C. elegans, using mutants in the kinesin light chains (klc-1 and klc-2) and the motor subunit (unc-116) expressing an ida-1::gfp transgene that labels DCVs. DCV transport in both directions was greatly impaired in an unc-116 mutant and had reduced velocity in a klc-2 mutant. In contrast, the speed of retrograde DCV transport was increased in a klc-1 mutant whereas anterograde transport was unaffected. We identified striking differences between the klc mutants in their effects on worm locomotion and responses to drugs affecting neuromuscular junction activity. We also determined lifespan, finding that unc-116 mutant was short-lived whereas the klc single mutant lifespan was wild type. The ida-1::gfp transgenic strain was also short-lived, but surprisingly, klc-1 and klc-2 extended the ida-1::gfp lifespan beyond that of wild type. Our findings suggest that kinesin-1 not only influences anterograde and retrograde DCV transport but is also involved in regulating lifespan and locomotion, with the two kinesin light chains playing distinct roles.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Cinesinas , Locomoção , Longevidade , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Cinesinas/metabolismo , Cinesinas/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Locomoção/genética , Longevidade/genética , Neurônios/metabolismo , Mutação/genética , Vesículas Secretórias/metabolismo , Animais Geneticamente Modificados , Transporte Axonal , Junção Neuromuscular/metabolismo , Proteínas de Ciclo CelularRESUMO
Each cargo in a cell employs a unique set of motor proteins for its transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of endogenous kinesin-1, -2, -3 and dynein motors and examined their effect on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, -3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. Transient optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. Kinesin-2 and -3 support long-range transport, and optogenetic inhibition reduces the distances that their cargoes move. These results suggest that the directionality of transport is controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, modulating the activity of a single type of motor on the cargo is sufficient to direct motility.
Assuntos
Dineínas , Cinesinas , Optogenética , Cinesinas/metabolismo , Optogenética/métodos , Dineínas/metabolismo , Humanos , Animais , Endossomos/metabolismo , Lisossomos/metabolismo , Transporte Biológico , Células HeLa , EndocitoseRESUMO
Ciliary beat and intraflagellar transport depend on dynein and kinesin motors. The kinesin-9 family members Kif6 and Kif9 are implicated in motile cilia motilities across protists and mammals. How they function and whether they act redundantly, however, remain unclear. Here, we show that Kif6 and Kif9 play distinct roles in mammals. Kif6 forms puncta that move bidirectionally along axonemes, whereas Kif9 appears to oscillate regionally on the ciliary central apparatus. Consistently, only Kif6 displays microtubule-based motor activity in vitro, and its ciliary localization requires its ATPase activity. Kif6 deficiency in mice disrupts coordinated ciliary beat across ependymal tissues and impairs cerebrospinal fluid flow, resulting in severe hydrocephalus and high mortality. Kif9 deficiency causes mild hydrocephalus without obviously affecting the ciliary beat or the lifespan. Kif6-/- and Kif9-/- males are infertile but exhibit oligozoospermia with poor sperm motility and defective forward motion of sperms, respectively. These results suggest Kif6 as a motor for cargo transport and Kif9 as a central apparatus regulator.
Assuntos
Cílios , Cinesinas , Camundongos Knockout , Animais , Cinesinas/metabolismo , Cinesinas/genética , Cílios/metabolismo , Masculino , Camundongos , Transporte Proteico , Motilidade dos Espermatozoides/genética , Hidrocefalia/metabolismo , Hidrocefalia/genética , Hidrocefalia/patologia , Camundongos Endogâmicos C57BL , Axonema/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Humanos , Microtúbulos/metabolismoRESUMO
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.
Assuntos
Cinesinas , Microtúbulos , Fuso Acromático , Cinesinas/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Humanos , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
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.