RESUMO
The actin cytoskeleton is composed of both branched and unbranched actin filaments. In mammals, the unbranched actin filaments are primarily copolymers of actin and tropomyosin. Biochemical and imaging studies indicate that different tropomyosin isoforms are segregated to different actin filament populations in cells and tissues, providing isoform-specific functionality to the actin filament. Intrinsic to this model is the prediction that single-molecule imaging of tropomyosin isoforms would confirm homopolymer formation along the length of single actin filaments, a knowledge gap that remains unaddressed in the cellular environment. We combined chemical labeling of genetically engineered tropomyosin isoforms with electron tomography to locate individual tropomyosin molecules in fibroblasts. We find that the organization of two non-muscle tropomyosins, Tpm3.1 with Tpm4.2, can be distinguished from each other using light and electron microscopy. Visualization of single tropomyosin molecules associated with actin filaments supports the hypothesis that tropomyosins form continuous homopolymers, instead of heteropolymers, in the presence of all physiologically native actin-binding proteins. This is true for both isoforms tested. Furthermore, the data suggest that the tropomyosin molecules on one side of an actin filament may not be in register with those on the opposite side, indicating that each tropomyosin polymer may assembly independently.
RESUMO
Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions.
Assuntos
Neoplasias Ovarianas , Humanos , Feminino , Movimento Celular , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Via de Sinalização Wnt , Transição Epitelial-Mesenquimal , Proteínas de Ligação a RNA/metabolismoRESUMO
Genome wide association studies (GWAS) have identified thousands of single nucleotide polymorphisms (SNPs) associated with the risk of common disorders. However, since the large majority of these risk SNPs reside outside gene-coding regions, GWAS generally provide no information about causal mechanisms regarding the specific gene(s) that are affected or the tissue(s) in which these candidate gene(s) exert their effect. The 'gold standard' method for understanding causal genes and their mechanisms of action are laborious basic science studies often involving sophisticated knockin or knockout mouse lines, however, these types of studies are impractical as a high-throughput means to understand the many risk variants that cause complex diseases like coronary artery disease (CAD). As a solution, we developed a streamlined, data-driven informatics pipeline to gain mechanistic insights on complex genetic loci. The pipeline begins by understanding the SNPs in a given locus in terms of their relative location and linkage disequilibrium relationships, and then identifies nearby expression quantitative trait loci (eQTLs) to determine their relative independence and the likely tissues that mediate their disease-causal effects. The pipeline then seeks to understand associations with other disease-relevant genes, disease sub-phenotypes, potential causality (Mendelian randomization), and the regulatory and functional involvement of these genes in gene regulatory co-expression networks (GRNs). Here, we applied this pipeline to understand a cluster of SNPs associated with CAD within and immediately adjacent to the gene encoding HDAC9. Our pipeline demonstrated, and validated, that this locus is causal for CAD by modulation of TWIST1 expression levels in the arterial wall, and by also governing a GRN related to metabolic function in skeletal muscle. Our results reconciled numerous prior studies, and also provided clear evidence that this locus does not govern HDAC9 expression, structure or function. This pipeline should be considered as a powerful and efficient way to understand GWAS risk loci in a manner that better reflects the highly complex nature of genetic risk associated with common disorders.
Assuntos
Doença da Artéria Coronariana , Estudo de Associação Genômica Ampla , Proteína 1 Relacionada a Twist/metabolismo , Animais , Doença da Artéria Coronariana/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Histona Desacetilases/metabolismo , Desequilíbrio de Ligação , Camundongos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , Proteínas Repressoras/metabolismoRESUMO
BACKGROUND: Hundreds of candidate genes have been associated with coronary artery disease (CAD) through genome-wide association studies. However, a systematic way to understand the causal mechanism(s) of these genes, and a means to prioritize them for further study, has been lacking. This represents a major roadblock for developing novel disease- and gene-specific therapies for patients with CAD. Recently, powerful integrative genomics analyses pipelines have emerged to identify and prioritize candidate causal genes by integrating tissue/cell-specific gene expression data with genome-wide association study data sets. METHODS: We aimed to develop a comprehensive integrative genomics analyses pipeline for CAD and to provide a prioritized list of causal CAD genes. To this end, we leveraged several complimentary informatics approaches to integrate summary statistics from CAD genome-wide association studies (from UK Biobank and CARDIoGRAMplusC4D) with transcriptomic and expression quantitative trait loci data from 9 cardiometabolic tissue/cell types in the STARNET study (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task). RESULTS: We identified 162 unique candidate causal CAD genes, which exerted their effect from between one and up to 7 disease-relevant tissues/cell types, including the arterial wall, blood, liver, skeletal muscle, adipose, foam cells, and macrophages. When their causal effect was ranked, the top candidate causal CAD genes were CDKN2B (associated with the 9p21.3 risk locus) and PHACTR1; both exerting their causal effect in the arterial wall. A majority of candidate causal genes were represented in cross-tissue gene regulatory co-expression networks that are involved with CAD, with 22/162 being key drivers in those networks. CONCLUSIONS: We identified and prioritized candidate causal CAD genes, also localizing their tissue(s) of causal effect. These results should serve as a resource and facilitate targeted studies to identify the functional impact of top causal CAD genes.
Assuntos
Aterosclerose , Doença da Artéria Coronariana , Aterosclerose/genética , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Locos de Características QuantitativasRESUMO
The actin cytoskeleton is the primary driver of cellular adhesion and mechanosensing due to its ability to generate force and sense the stiffness of the environment. At the cell's leading edge, severing of the protruding Arp2/3 actin network generates a specific actin/tropomyosin (Tpm) filament population that controls lamellipodial persistence. The interaction between these filaments and adhesion to the environment is unknown. Using cellular cryo-electron tomography we resolve the ultrastructure of the Tpm/actin copolymers and show that they specifically anchor to nascent adhesions and are essential for focal adhesion assembly. Re-expression of Tpm1.8/1.9 in transformed and cancer cells is sufficient to restore cell-substrate adhesions. We demonstrate that knock-out of Tpm1.8/1.9 disrupts the formation of dorsal actin bundles, hindering the recruitment of α-actinin and non-muscle myosin IIa, critical mechanosensors. This loss causes a force-generation and proliferation defect that is notably reversed when cells are grown on soft surfaces. We conclude that Tpm1.8/1.9 suppress the metastatic phenotype, which may explain why transformed cells naturally downregulate this Tpm subset during malignant transformation.
Assuntos
Neoplasias , Tropomiosina , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proliferação de Células , Pseudópodes/metabolismo , Tropomiosina/metabolismoRESUMO
BACKGROUND: Anti-microtubule agents are widely used to treat ovarian cancers, but the efficacy is often compromised by drug resistance. We investigated co-targeting the actin/tropomyosin cytoskeleton and microtubules to increase treatment efficacy in ovarian cancers and potentially overcome resistance. METHODS: The presence of tropomyosin-3.1 (Tpm3.1) was examined in clinical specimens from ovarian cancer patients using immunohistochemistry. Combinatorial effects of an anti-Tpm3.1 compound, ATM-3507, with vinorelbine and paclitaxel were evaluated in ovarian cancer cells via MTS and apoptosis assays. The mechanisms of action were established using live- and fixed-cell imaging and protein analysis. RESULTS: Tpm3.1 is overexpressed in 97% of tumour tissues (558 of 577) representing all histotypes of epithelial ovarian cancer. ATM-3507 displayed synergy with both anti-microtubule agents to reduce cell viability. Only vinorelbine synergised with ATM-3507 in causing apoptosis. ATM-3507 significantly prolonged vinorelbine-induced mitotic arrest with elevated activity of the spindle assembly checkpoint and mitotic cell death; however, ATM-3507 showed minor impact on paclitaxel-induced mitotic defects. Both combinations substantially increased post-mitotic G1 arrest with cyclin D1 and E1 downregulation and an increase of p21Cip and p27Kip. CONCLUSION: Combined targeting of Tpm3.1/actin and microtubules is a promising treatment strategy for ovarian cancer that should be further tested in clinical settings.
Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Cloretos/farmacologia , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Tropomiosina/metabolismo , Regulação para Cima , Vinorelbina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Epitelial do Ovário/tratamento farmacológico , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/tratamento farmacológico , Tropomiosina/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacosRESUMO
Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.
Assuntos
Tomografia com Microscopia Eletrônica/métodos , Técnicas Genéticas , Imageamento Tridimensional/métodos , Animais , Ascorbato Peroxidases , Congelamento , Ouro , Camundongos , ProteínasRESUMO
1- and 1,5-Aminoalkylamine substituted anthraquinones (AAQs, 1C3 and 1,5C3) were peptide coupled to 1-, 2-, and 3-pyrrole lexitropsins to generate compounds that incorporated both DNA minor groove and intercalating moieties. The corresponding platinum(II) amidine complexes were synthesized through a synthetically facile amine-to-platinum mediated nitrile 'Click' reaction. The precursors as well as the corresponding platinum(II) complexes were biologically evaluated in 2D monolayer cells and 3D tumour cell models. Despite having cellular accumulation levels that were up to five-fold lower than that of cisplatin, the platinum complexes had cytotoxicities that were only three-fold lower. Accumulation was lowest for the complexes with two or three pyrrole groups, but the latter was the most active of the complexes exceeding the activity of cisplatin in the MDA-MB-231 cell line. All compounds showed moderate to good penetration into spheroids of DLD-1 cells with the distributions being consistent with active uptake of the pyrrole containing complexes in regions of the spheroids starved of nutrients.
Assuntos
Amidinas/química , Antraquinonas/química , Fluorescência , Nylons/química , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Compostos Organoplatínicos/metabolismoRESUMO
Tropomyosins are elongated alpha-helical proteins that form co-polymers with most actin filaments within a cell and play important roles in the structural and functional diversification of the actin cytoskeleton. How the assembly of tropomyosins along an actin filament is regulated and the kinetics of tropomyosin association with an actin filament is yet to be fully determined. A recent series of publications have used total internal reflection fluorescence (TIRF) microscopy in combination with advanced surface and protein chemistry to visualise the molecular assembly of actin/tropomyosin filaments in vitro. Here, we review the use of the in vitro TIRF assay in the determination of kinetic data on tropomyosin filament assembly. This sophisticated approach has enabled generation of real-time single-molecule data to fill the gap between in vitro bulk assays and in vivo assays of tropomyosin function. The in vitro TIRF assays provide a new foundation for future studies involving multiple actin-binding proteins that will more accurately reflect the physiological protein-protein interactions in cells.
RESUMO
Antimicrotubule vinca alkaloids are widely used in the clinic but their toxicity is often dose limiting. Strategies that enhance their effectiveness at lower doses are needed. We show that combining vinca alkaloids with compounds that target a specific population of actin filaments containing the cancer-associated tropomyosin Tpm3.1 result in synergy against a broad range of tumor cell types. We discovered that low concentrations of vincristine alone induce supernumerary microtubule asters that form transient multi-polar spindles in early mitosis. Over time these asters can be reconstructed into functional bipolar spindles resulting in cell division and survival. These microtubule asters are organized by the nuclear mitotic apparatus protein (NuMA)-dynein-dynactin complex without involvement of centrosomes. However, anti-Tpm3.1 compounds at nontoxic concentrations inhibit this rescue mechanism resulting in delayed onset of anaphase, formation of multi-polar spindles, and apoptosis during mitosis. These findings indicate that drug targeting actin filaments containing Tpm3.1 potentiates the anticancer activity of low-dose vincristine treatment. IMPLICATIONS: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.
Assuntos
Citoesqueleto de Actina/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Piperazinas/administração & dosagem , Tropomiosina/metabolismo , Vincristina/administração & dosagem , Células A549 , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Células HeLa , Humanos , Neoplasias Pulmonares/metabolismo , Células MCF-7 , Camundongos , Piperazinas/farmacologia , Tropomiosina/antagonistas & inibidores , Vincristina/farmacologiaRESUMO
The physiological function of actin filaments is challenging to dissect because of the pleiotropic impact of global disruption of the actin cytoskeleton. Tropomyosin isoforms have provided a unique opportunity to address this issue. A substantial fraction of actin filaments in animal cells consist of co-polymers of actin with specific tropomyosin isoforms which determine the functional capacity of the filament. Genetic manipulation of the tropomyosins has revealed isoform specific roles and identified the physiological function of the different actin filament types based on their tropomyosin isoform composition. Surprisingly, there is remarkably little redundancy between the tropomyosins resulting in highly penetrant impacts of both ectopic overexpression and knockout of isoforms. The physiological roles of the tropomyosins cover a broad range from development and morphogenesis to cell migration and specialised tissue function and human diseases.
Assuntos
Citoesqueleto de Actina/metabolismo , Tropomiosina/metabolismo , Animais , Humanos , Isoformas de Proteínas/metabolismoRESUMO
Although F-actin has a large number of binding partners and regulators, the number of phenotypic states available to the actin cytoskeleton is unknown. Here, we quantified 74 features defining filamentous actin (F-actin) and cellular morphology in >25 million cells after treatment with a library of 114,400 structurally diverse compounds. After reducing the dimensionality of these data, only â¼25 recurrent F-actin phenotypes emerged, each defined by distinct quantitative features that could be machine learned. We identified 2,003 unknown compounds as inducers of actin-related phenotypes, including two that directly bind the focal adhesion protein, talin. Moreover, we observed that compounds with distinct molecular mechanisms could induce equivalent phenotypes and that initially divergent cellular responses could converge over time. These findings suggest a conceptual parallel between the actin cytoskeleton and gene regulatory networks, where the theoretical plasticity of interactions is nearly infinite, yet phenotypes in vivo are constrained into a limited subset of practicable configurations.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Adaptação Fisiológica/fisiologia , Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Sequência de Aminoácidos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Feminino , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ligação Proteica , Talina/metabolismoRESUMO
Co-polymers of tropomyosin and actin make up a major fraction of the actin cytoskeleton. Tropomyosin isoforms determine the function of an actin filament by selectively enhancing or inhibiting the association of other actin binding proteins, altering the stability of an actin filament and regulating myosin activity in an isoform-specific manner. Previous work has implicated specific roles for at least five different tropomyosin isoforms in stress fibres, as depletion of any of these five isoforms results in a loss of stress fibres. Despite this, most models of stress fibres continue to exclude tropomyosins. In this study, we investigate tropomyosin organisation in stress fibres by using super-resolution light microscopy and electron microscopy with genetically tagged, endogenous tropomyosin. We show that tropomyosin isoforms are organised in subdomains within the overall domain of stress fibres. The isoforms Tpm3.1 and 3.2 (hereafter Tpm3.1/3.2, encoded by TPM3) colocalise with non-muscle myosin IIa and IIb heads, and are in register, but do not overlap, with non-muscle myosin IIa and IIb tails. Furthermore, perturbation of Tpm3.1/3.2 results in decreased myosin IIa in stress fibres, which is consistent with a role for Tpm3.1 in maintaining myosin IIa localisation in stress fibres.
Assuntos
Miosina não Muscular Tipo IIA/metabolismo , Fibras de Estresse/metabolismo , Tropomiosina/metabolismo , Linhagem Celular Tumoral , Humanos , Miosina não Muscular Tipo IIA/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fibras de Estresse/genética , Tropomiosina/genéticaRESUMO
The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down; depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.
Assuntos
Citoesqueleto de Actina/metabolismo , Forminas/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Células Cultivadas , Forminas/genética , Células HEK293 , Humanos , Toxinas Marinhas/farmacologia , Microscopia Confocal , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Tiazolidinas/farmacologia , Tropomiosina/genética , Tropomiosina/metabolismoRESUMO
The actin cytoskeleton is dysregulated in cancer, yet this critical cellular machinery has not translated as a druggable clinical target due to cardio-toxic side-effects. Many actin regulators are also considered undruggable, being structural proteins lacking clear functional sites suitable for targeted drug design. In this review, we discuss opportunities and challenges associated with drugging the actin cytoskeleton through its structural regulators, taking tropomyosins as a target example. In particular, we highlight emerging data acquisition and analysis trends driving phenotypic, imaging-based compound screening. Finally, we consider how the confluence of these trends is now bringing functionally integral machineries such as the actin cytoskeleton, and associated structural regulatory proteins, into an expanded repertoire of druggable targets with previously unexploited clinical potential.
Assuntos
Citoesqueleto de Actina/metabolismo , Animais , Humanos , Fenótipo , Tropomiosina/metabolismoRESUMO
A series of peptides based on the prostate-specific antigen (PSA)-specific sequence histidine-serine-serine-lysine-leucine-glutamine were functionalized with an anthraquinone fluorophore at the C-terminal residue side chain using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. The effect of incorporating a negatively charged N-terminal tetra-glutamic acid group into the substrate and the effect of masking the negatively charged C-terminal carboxylic acid functionality of the substrate were investigated using confocal fluorescence microscopy in two cell lines, DLD-1 and LnCaP. The addition of a tetra-glutamic acid group to the N-terminus of the intact sequence was shown to reduce cellular uptake of the intact substrate prior to activation by PSA. In contrast, masking the C-terminal carboxylic acid group of the substrate as a methyl ester was shown to improve cellular uptake of the peptide fragment after activation by PSA. The synthesized C-terminal methyl ester substrates with the anthraquinone attached to the side chain were confirmed to be cleaved by PSA in LC-MS analysis, and the cytotoxicity of the substrates was shown to increase in the presence of PSA, consistent with cleavage and uptake of the C-terminal fragment. The results indicate that C- and N-terminal functionalization of peptide substrates targeting PSA can be used to modulate the cellular uptake of peptides before and after enzymatic activation, which may thus be an important consideration in the design of tumor-activated prodrugs.
Assuntos
Corantes Fluorescentes/metabolismo , Antígeno Prostático Específico/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
Tropomyosin proteins form stable coiled-coil dimers that polymerize along the α-helical groove of actin filaments [1]. The actin cytoskeleton consists of both co-polymers of actin and tropomyosin and polymers of tropomyosin-free actin [2]. The fundamental distinction between these two types of filaments is that tropomyosin determines the functional capability of actin filaments in an isoform-dependent manner [3-9]. However, it is unknown what portion of actin filaments are associated with tropomyosin. To address this deficit, we have measured the relative distribution between these two filament populations by quantifying tropomyosin and actin levels in a variety of human cell types, including bone (U2OS); breast epithelial (MCF-10A); transformed breast epithelial (MCF-7); and primary (BJpar), immortalized (BJeH), and Ras-transformed (BJeLR) BJ fibroblasts [10]. Our measurements of tropomyosin and actin predict the saturation of the actin cytoskeleton, implying that tropomyosin binding must be inhibited in order to generate tropomyosin-free actin filaments. We find the majority of actin filaments to be associated with tropomyosin in four of the six cell lines tested and the portion of actin filaments associated with tropomyosin to decrease with transformation. We also discover that high-molecular-weight (HMW), unlike low-molecular-weight (LMW), tropomyosin isoforms are primarily co-polymerized with actin in untransformed cells. This differential partitioning of tropomyosins is not due to a lack of N-terminal acetylation of LMW tropomyosins, but it is, in part, explained by the susceptibility of soluble HMW tropomyosins to proteasomal degradation. We conclude that actin-tropomyosin co-polymers make up a major fraction of the human actin cytoskeleton.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Polímeros/química , Tropomiosina/química , HumanosRESUMO
Many actin filaments in animal cells are co-polymers of actin and tropomyosin. In many cases, non-muscle myosin II associates with these co-polymers to establish a contractile network. However, the temporal relationship of these three proteins in the de novo assembly of actin filaments is not known. Intravital subcellular microscopy of secretory granule exocytosis allows the visualisation and quantification of the formation of an actin scaffold in real time, with the added advantage that it occurs in a living mammal under physiological conditions. We used this model system to investigate the de novo assembly of actin, tropomyosin Tpm3.1 (a short isoform of TPM3) and myosin IIA (the form of non-muscle myosin II with its heavy chain encoded by Myh9) on secretory granules in mouse salivary glands. Blocking actin polymerization with cytochalasin D revealed that Tpm3.1 assembly is dependent on actin assembly. We used time-lapse imaging to determine the timing of the appearance of the actin filament reporter LifeAct-RFP and of Tpm3.1-mNeonGreen on secretory granules in LifeAct-RFP transgenic, Tpm3.1-mNeonGreen and myosin IIA-GFP (GFP-tagged MYH9) knock-in mice. Our findings are consistent with the addition of tropomyosin to actin filaments shortly after the initiation of actin filament nucleation, followed by myosin IIA recruitment.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Tropomiosina/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Pesadas de Miosina , Miosina não Muscular Tipo IIA/genética , Ligação Proteica , Vesículas Secretórias/genética , Vesículas Secretórias/metabolismo , Tropomiosina/genéticaRESUMO
The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specific actin filament populations. We have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was significantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 ß-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confirming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs affect glucose metabolism in vivo by inhibiting Tpm3.1's function with few off-target effects.
Assuntos
Citoesqueleto de Actina/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Tropomiosina/antagonistas & inibidores , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Glucose/administração & dosagem , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Tropomiosina/fisiologiaRESUMO
Nanomedicine is an emerging field with great potential in disease theranostics. We generated sterically stabilized superparamagnetic iron oxide nanoparticles (s-SPIONs) with average core diameters of 10 and 25 nm and determined the in vivo biodistribution and clearance profiles. Healthy nude mice underwent an intraperitoneal injection of these s-SPIONs at a dose of 90 mg Fe/kg body weight. Tissue iron biodistribution was monitored by atomic absorption spectroscopy and Prussian blue staining. Histopathological examination was performed to assess tissue toxicity. The 10 nm s-SPIONs resulted in higher tissue-iron levels, whereas the 25 nm s-SPIONs peaked earlier and cleared faster. Increased iron levels were detected in all organs and body fluids tested except for the brain, with notable increases in the liver, spleen, and the omentum. The tissue-iron returned to control or near control levels within 7 days post-injection, except in the omentum, which had the largest and most variable accumulation of s-SPIONs. No obvious tissue changes were noted although an influx of macrophages was observed in several tissues suggesting their involvement in s-SPION sequestration and clearance. These results demonstrate that the s-SPIONs do not degrade or aggregate in vivo and intraperitoneal administration is well tolerated, with a broad and transient biodistribution. In an ovarian tumor model, s-SPIONs were shown to accumulate in the tumors, highlighting their potential use as a chemotherapy delivery agent.