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1.
Methods Mol Biol ; 2831: 1-9, 2024.
Article in English | MEDLINE | ID: mdl-39134839

ABSTRACT

Primary neuronal cultures allow for in vitro analysis of early developmental processes such as axon pathfinding and growth dynamics. When coupled with methods to visualize and measure microtubule dynamics, this methodology enables an inside look at how the cytoskeleton changes in response to extracellular signaling cues. Here, we describe the culturing conditions and tools required to extract primary cortical neurons from postnatal mouse brains and visualize cytoskeletal components.


Subject(s)
Cerebral Cortex , Neurons , Animals , Mice , Neurons/cytology , Neurons/metabolism , Cerebral Cortex/cytology , Cells, Cultured , Microtubules/metabolism , Primary Cell Culture/methods , Cell Culture Techniques/methods , Cytoskeleton/metabolism
2.
Methods Mol Biol ; 2831: 235-249, 2024.
Article in English | MEDLINE | ID: mdl-39134854

ABSTRACT

The study of microtubules arrangements and dynamics during axon outgrowth and pathfinding has gained scientific interest during the last decade, and numerous technical resources for its visualization and analysis have been implemented. In this chapter, we describe the cell culture protocols of embryonic cortical and retinal neurons, the methods for transfecting them with fluorescent reporters of microtubule polymerization, and the procedures for time-lapse imaging and quantification in order to study microtubule dynamics during axon morphogenesis.


Subject(s)
Axons , Microtubules , Microtubules/metabolism , Animals , Axons/metabolism , Polymerization , Time-Lapse Imaging/methods , Neuronal Outgrowth , Neurons/metabolism , Neurons/cytology , Mice , Cells, Cultured , Microtubule-Associated Proteins/metabolism
3.
Methods Mol Biol ; 2831: 219-234, 2024.
Article in English | MEDLINE | ID: mdl-39134853

ABSTRACT

The specialized function and extreme geometry of neurons necessitates a unique reliance upon long-distance microtubule-based transport. Appropriate trafficking of axonal cargos by motor proteins is essential for establishing circuitry during development and continuing function throughout a lifespan. Visualizing and quantifying cargo movement provides valuable insight into how axonal organelles are replenished, recycled, and degraded during the dynamic dance of outgoing and incoming axonal traffic. Long-distance axonal trafficking is of particular importance as it encompasses a pathway commonly disrupted in developmental and degenerative disease states. Here, we describe neuronal organelles and outline methods for live imaging and quantifying their movement throughout the axon via transient expression of fluorescently labeled organelle markers. This resource provides recommendations for target proteins/domains and appropriate acquisition time scales for visualizing distinct neuronal cargos in cultured neurons derived from human induced pluripotent stem cells (iPSCs) and primary rat neurons.


Subject(s)
Axonal Transport , Induced Pluripotent Stem Cells , Neurons , Organelles , Animals , Neurons/metabolism , Neurons/cytology , Rats , Organelles/metabolism , Cells, Cultured , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Axons/metabolism , Microtubules/metabolism
4.
Mol Med ; 30(1): 119, 2024 Aug 11.
Article in English | MEDLINE | ID: mdl-39129004

ABSTRACT

BACKGROUND: AGTPBP1 is a cytosolic carboxypeptidase that cleaves poly-glutamic acids from the C terminus or side chains of α/ß tubulins. Although its dysregulated expression has been linked to the development of non-small cell lung cancer, the specific roles and mechanisms of AGTPBP1 in pancreatic cancer (PC) have yet to be fully understood. In this study, we examined the role of AGTPBP1 on PC in vitro and in vivo. METHODS: Immunohistochemistry was used to examine the expression of AGTPBP1 in PC and non-cancerous tissues. Additionally, we assessed the malignant behaviors of PC cells following siRNA-mediated AGTPBP1 knockdown both in vitro and in vivo. RNA sequencing and bioinformatics analysis were performed to identify the differentially expressed genes regulated by AGTPBP1. RESULTS: We determined that AGTPBP1 was overexpressed in PC tissues and the higher expression of AGTPBP1 was closely related to the location of tumors. AGTPBP1 inhibition can significantly decrease cell progression in vivo and in vitro. Moreover, the knockdown of AGTPBP1 inhibited the expression of ERK1/2, P-ERK1/2, MYLK, and TUBB4B proteins via the ERK signaling pathway. CONCLUSION: Our research indicates that AGTPBP1 may be a putative therapeutic target for PC.


Subject(s)
Carboxypeptidases , Gene Expression Regulation, Neoplastic , MAP Kinase Signaling System , Microtubules , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Carboxypeptidases/metabolism , Carboxypeptidases/genetics , Cell Line, Tumor , Microtubules/metabolism , Animals , Mice , Male , Female , Cell Proliferation , Disease Progression , Middle Aged , Cell Movement/genetics
5.
Nat Commun ; 15(1): 6564, 2024 Aug 03.
Article in English | MEDLINE | ID: mdl-39095439

ABSTRACT

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/metabolism
6.
Bull Math Biol ; 86(9): 113, 2024 Aug 03.
Article in English | MEDLINE | ID: mdl-39096399

ABSTRACT

During cell division, the mitotic spindle moves dynamically through the cell to position the chromosomes and determine the ultimate spatial position of the two daughter cells. These movements have been attributed to the action of cortical force generators which pull on the astral microtubules to position the spindle, as well as pushing events by these same microtubules against the cell cortex and plasma membrane. Attachment and detachment of cortical force generators working antagonistically against centring forces of microtubules have been modelled previously (Grill et al. in Phys Rev Lett 94:108104, 2005) via stochastic simulations and mean-field Fokker-Planck equations (describing random motion of force generators) to predict oscillations of a spindle pole in one spatial dimension. Using systematic asymptotic methods, we reduce the Fokker-Planck system to a set of ordinary differential equations (ODEs), consistent with a set proposed by Grill et al., which can provide accurate predictions of the conditions for the Fokker-Planck system to exhibit oscillations. In the limit of small restoring forces, we derive an algebraic prediction of the amplitude of spindle-pole oscillations and demonstrate the relaxation structure of nonlinear oscillations. We also show how noise-induced oscillations can arise in stochastic simulations for conditions in which the mean-field Fokker-Planck system predicts stability, but for which the period can be estimated directly by the ODE model and the amplitude by a related stochastic differential equation that incorporates random binding kinetics.


Subject(s)
Computer Simulation , Mathematical Concepts , Microtubules , Models, Biological , Spindle Apparatus , Stochastic Processes , Spindle Apparatus/physiology , Microtubules/physiology , Microtubules/metabolism , Nonlinear Dynamics , Mitosis/physiology
7.
J Cell Biol ; 223(10)2024 Oct 07.
Article in English | MEDLINE | ID: mdl-39133213

ABSTRACT

Mitochondrial functions can be regulated by membrane contact sites with the endoplasmic reticulum (ER). These mitochondria-ER contact sites (MERCs) are functionally heterogeneous and maintained by various tethers. Here, we found that REEP5, an ER tubule-shaping protein, interacts with Mitofusins 1/2 to mediate mitochondrial distribution throughout the cytosol by a new transport mechanism, mitochondrial "hitchhiking" with tubular ER on microtubules. REEP5 depletion led to reduced tethering and increased perinuclear localization of mitochondria. Conversely, increasing REEP5 expression facilitated mitochondrial distribution throughout the cytoplasm. Rapamycin-induced irreversible REEP5-MFN1/2 interaction led to mitochondrial hyperfusion, implying that the dynamic release of mitochondria from tethering is necessary for normal mitochondrial distribution and dynamics. Functionally, disruption of MFN2-REEP5 interaction dynamics by forced dimerization or silencing REEP5 modulated the production of mitochondrial reactive oxygen species (ROS). Overall, our results indicate that dynamic REEP5-MFN1/2 interaction mediates cytosolic distribution and connectivity of the mitochondrial network by "hitchhiking" and this process regulates mitochondrial ROS, which is vital for multiple physiological functions.


Subject(s)
Endoplasmic Reticulum , GTP Phosphohydrolases , Mitochondria , Reactive Oxygen Species , Endoplasmic Reticulum/metabolism , Mitochondria/metabolism , Humans , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , Reactive Oxygen Species/metabolism , HeLa Cells , Microtubules/metabolism , HEK293 Cells , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Protein Binding , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Cytosol/metabolism , Mitochondrial Dynamics
8.
Sci Rep ; 14(1): 18286, 2024 08 07.
Article in English | MEDLINE | ID: mdl-39112501

ABSTRACT

Despite widespread public interest in the health impact of exposure to microwave radiation, studies of the influence of microwave radiation on biological samples are often inconclusive or contradictory. Here we examine the influence of microwave radiation of frequencies 3.5 GHz, 20 GHz and 29 GHz on the growth of microtubules, which are biological nanotubes that perform diverse functions in eukaryotic cells. Since microtubules are highly polar and can extend several micrometres in length, they are predicted to be sensitive to non-ionizing radiation. Moreover, it has been speculated that tubulin dimers within microtubules might rapidly toggle between different conformations, potentially participating in computational or other cooperative processes. Our data show that exposure to microwave radiation yields a microtubule growth curve that is distorted relative to control studies utilizing a homogeneous temperature jump. However, this apparent effect of non-ionizing radiation is reproduced by control experiments using an infrared laser or hot air to heat the sample and thereby mimic the thermal history of samples exposed to microwaves. As such, no non-thermal effects of microwave radiation on microtubule growth can be assigned. Our results highlight the need for appropriate control experiments in biophysical studies that may impact on the sphere of public interest.


Subject(s)
Microtubules , Microwaves , Microtubules/radiation effects , Microtubules/metabolism , Tubulin/metabolism , Animals , Temperature
9.
Eur Biophys J ; 53(5-6): 339-354, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39093405

ABSTRACT

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 Multimerization
10.
PLoS Biol ; 22(8): e3002751, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39137170

ABSTRACT

ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.


Subject(s)
Cell Cycle Proteins , Centrosome , Microtubules , Nerve Tissue Proteins , Neural Stem Cells , Neurogenesis , Animals , Microtubules/metabolism , Mice , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Neurogenesis/genetics , Neural Stem Cells/metabolism , Centrosome/metabolism , Cell Proliferation , Cell Movement , Cerebral Cortex/metabolism , Cerebral Cortex/embryology , Cerebral Cortex/growth & development , Tubulin/metabolism , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , ADP-Ribosylation Factors/metabolism , ADP-Ribosylation Factors/genetics
11.
Nat Commun ; 15(1): 6509, 2024 Aug 02.
Article in English | MEDLINE | ID: mdl-39095354

ABSTRACT

Microtubule organization in cells relies on targeting mechanisms. Cytoplasmic linker proteins (CLIPs) and CLIP-associated proteins (CLASPs) are key regulators of microtubule organization, yet the underlying mechanisms remain elusive. Here, we reveal that the C-terminal domain of CLASP2 interacts with a common motif found in several CLASP-binding proteins. This interaction drives the dynamic localization of CLASP2 to distinct cellular compartments, where CLASP2 accumulates in protein condensates at the cell cortex or the microtubule plus end. These condensates physically contact each other via CLASP2-mediated competitive binding, determining cortical microtubule targeting. The phosphorylation of CLASP2 modulates the dynamics of the condensate-condensate interaction and spatiotemporally navigates microtubule growth. Moreover, we identify additional CLASP-interacting proteins that are involved in condensate contacts in a CLASP2-dependent manner, uncovering a general mechanism governing microtubule targeting. Our findings not only unveil a tunable multiphase system regulating microtubule organization, but also offer general mechanistic insights into intricate protein-protein interactions at the mesoscale level.


Subject(s)
Microtubule-Associated Proteins , Microtubules , Protein Binding , Microtubules/metabolism , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/genetics , Humans , Phosphorylation , Binding, Competitive , HeLa Cells , Biomolecular Condensates/metabolism , HEK293 Cells , Animals
13.
Curr Biol ; 34(15): R728-R731, 2024 Aug 05.
Article in English | MEDLINE | ID: mdl-39106829

ABSTRACT

Cell migration through complex 3D environments relies on the interplay between actin and microtubules. A new study shows that, when cells pass through narrow constrictions, CLASP-dependent microtubule stabilisation at the cell rear controls actomyosin contractility to enable nuclear translocation and preserve cell integrity.


Subject(s)
Cell Movement , Microtubules , Microtubules/metabolism , Microtubules/physiology , Cell Movement/physiology , Actomyosin/metabolism , Actins/metabolism , Animals
14.
Curr Biol ; 34(15): R731-R734, 2024 Aug 05.
Article in English | MEDLINE | ID: mdl-39106830

ABSTRACT

Spindles are microtubule-based machines that segregate chromosomes during cell division. Spindle morphology and dynamics are malleable based on forces within the spindle, and a new study reveals the extreme plasticity of the Saccharomyces cerevisiae spindle to adapt and segregate engineered mega-chromosomes.


Subject(s)
Saccharomyces cerevisiae , Spindle Apparatus , Saccharomyces cerevisiae/genetics , Spindle Apparatus/metabolism , Chromosome Segregation , Microtubules/metabolism , Chromosomes, Fungal/genetics
15.
World J Gastroenterol ; 30(26): 3257-3260, 2024 Jul 14.
Article in English | MEDLINE | ID: mdl-39086641

ABSTRACT

Over the years immunotherapy has demonstrably improved the field of cancer treatment. However, achieving long-term survival for colorectal cancer (CRC) patients remains a significant unmet need. Combination immunotherapies incorporating targeted drugs like MEK or multi-kinase inhibitors have offered some palliative benefit. Nevertheless, substantial gaps remain in the current therapeutic armamentarium for CRC. In recent years, there has been a surge of interest in exploring novel treatment strategies, including the application of light-activated drugs in conjunction with optical devices. This approach holds promise for achieving localized and targeted delivery of cytotoxic agents, such as microtubule-targeting drugs, directly to cancerous cells within the colon.


Subject(s)
Colorectal Neoplasms , Microtubules , Humans , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Colorectal Neoplasms/immunology , Microtubules/drug effects , Microtubules/metabolism , Antineoplastic Agents/therapeutic use , Molecular Targeted Therapy/methods , Drug Delivery Systems/methods , Photosensitizing Agents/therapeutic use , Immunotherapy/methods , Photochemotherapy/methods , Tubulin Modulators/therapeutic use , Tubulin Modulators/pharmacology
16.
J Cell Biol ; 223(11)2024 Nov 04.
Article in English | MEDLINE | ID: mdl-39115447

ABSTRACT

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 , Humans
17.
Nat Commun ; 15(1): 6677, 2024 Aug 06.
Article in English | MEDLINE | ID: mdl-39107283

ABSTRACT

Clarification of the cytotoxic function of T cells is crucial for understanding human immune responses and immunotherapy procedures. Here, we report a high-throughput Bessel oblique plane microscopy (HBOPM) platform capable of 3D live imaging and phenotyping of chimeric antigen receptor (CAR)-modified T-cell cytotoxicity against cancer cells. The HBOPM platform has the following characteristics: an isotropic subcellular resolution of 320 nm, large-scale scouting over 400 interacting cell pairs, long-term observation across 5 hours, and quantitative analysis of the Terabyte-scale 3D, multichannel, time-lapse image datasets. Using this advanced microscopy platform, several key subcellular events in CAR-T cells are captured and comprehensively analyzed; these events include the instantaneous formation of immune synapses and the sustained changes in the microtubing morphology. Furthermore, we identify the actin retrograde flow speed, the actin depletion coefficient, the microtubule polarization and the contact area of the CAR-T/target cell conjugates as essential parameters strongly correlated with CAR-T-cell cytotoxic function. Our approach will be useful for establishing criteria for quantifying T-cell function in individual patients for all T-cell-based immunotherapies.


Subject(s)
Imaging, Three-Dimensional , Immunotherapy, Adoptive , Microtubules , Receptors, Chimeric Antigen , T-Lymphocytes , Humans , Receptors, Chimeric Antigen/metabolism , Receptors, Chimeric Antigen/immunology , Imaging, Three-Dimensional/methods , Immunotherapy, Adoptive/methods , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Microtubules/metabolism , Cell Line, Tumor , Immunological Synapses/immunology , Immunological Synapses/metabolism , Cytotoxicity, Immunologic , Actins/metabolism , Microscopy/methods , Phenotype
18.
Development ; 151(20)2024 Oct 15.
Article in English | MEDLINE | ID: mdl-39041335

ABSTRACT

The multicellular haploid stage of land plants develops from a single haploid cell produced by meiosis - the spore. Starting from a non-polar state, these spores develop polarity, divide asymmetrically and establish the first axis of symmetry. Here, we show that the nucleus migrates from the cell centroid to the basal pole during polarisation of the Marchantia polymorpha spore cell. A microtubule organising centre on the leading edge of the nucleus initiates a microtubule array between the nuclear surface and the cortex at the basal pole. Simultaneously, cortical microtubules disappear from the apical hemisphere but persist in the basal hemisphere. This is accompanied by the formation a dense network of fine actin filaments between the nucleus and the basal pole cortex. Experimental depolymerisation of either microtubules or actin filaments disrupts cellular asymmetry. These data demonstrate that the cytoskeleton reorganises during spore polarisation and controls the directed migration of the nucleus to the basal pole. The presence of the nucleus at the basal pole provides the cellular asymmetry for the asymmetric cell division that establishes the apical-basal axis of the plant.


Subject(s)
Actin Cytoskeleton , Cell Nucleus , Cell Polarity , Marchantia , Microtubules , Spores , Microtubules/metabolism , Cell Nucleus/metabolism , Actin Cytoskeleton/metabolism , Marchantia/metabolism , Marchantia/genetics , Marchantia/cytology , Cell Polarity/physiology
19.
Nat Commun ; 15(1): 5530, 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38956021

ABSTRACT

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 Conformation
20.
Cell Mol Biol Lett ; 29(1): 94, 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38956497

ABSTRACT

BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved. METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment. RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation. CONCLUSION: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.


Subject(s)
Actins , Aneuploidy , Neoplasm Invasiveness , Tubulin , Urinary Bladder Neoplasms , Animals , Humans , Mice , Actins/metabolism , Actins/genetics , Cell Line, Tumor , Cell Movement/genetics , Genomic Instability , Microtubules/metabolism , Protein Binding , Tubulin/metabolism , Tubulin/genetics , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , Urothelium/pathology , Urothelium/metabolism , Connexins/metabolism
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