RESUMEN
Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.
Asunto(s)
Caveolas/metabolismo , Caveolas/ultraestructura , Caveolinas/metabolismo , Escherichia coli , Mamíferos/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , HumanosRESUMEN
Association with microtubules inhibits the fission of mitochondria in Schizosaccharomyces pombe. Here, we show that this attachment of mitochondria to microtubules is an important cell-intrinsic factor in determining cell division symmetry. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1Δ and Mmb1Δ, respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell division asymmetry in a significant proportion of both Dnm1Δ and Mmb1Δ cells. Asymmetric division in Dnm1Δ and Mmb1Δ cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter cell. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and, thereby, cell growth. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Humanos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , División Celular/genética , Mitocondrias/genéticaRESUMEN
Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.
Asunto(s)
Chaetomium/química , Chaetomium/ultraestructura , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/ultraestructura , Chaetomium/metabolismo , Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/ultraestructura , Humanos , Modelos Moleculares , Unión Proteica , Transporte de Proteínas , Nexinas de Clasificación/química , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/ultraestructura , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Retromer core complex is an endosomal scaffold that plays a critical role in orchestrating protein trafficking within the endosomal system. Here we characterized the effect of the Parkinson's disease-linked Vps35 D620N in the endo-lysosomal system using Vps35 D620N rescue cell models. Vps35 D620N fully rescues the lysosomal and autophagy defects caused by retromer knock-out. Analogous to Vps35 knock out cells, the endosome-to-trans-Golgi network transport of cation-independent mannose 6-phosphate receptor (CI-M6PR) is impaired in Vps35 D620N rescue cells because of a reduced capacity to form endosome transport carriers. Cells expressing the Vps35 D620N variant have altered endosomal morphology, resulting in smaller, rounder structures with less tubule-like branches. At the molecular level retromer incorporating Vps35 D620N variant has a decreased binding to retromer associated proteins wiskott-aldrich syndrome protein and SCAR homologue (WASH) and SNX3 which are known to associate with retromer to form the endosome transport carriers. Hence, the partial defects on retrograde protein trafficking carriers in the presence of Vps35 D620N represents an altered cellular state able to cause Parkinson's disease.
Asunto(s)
Enfermedad de Parkinson , Endosomas/metabolismo , Humanos , Lisosomas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Transporte de Proteínas , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias , Tropomiosina , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proliferación Celular , Seudópodos/metabolismo , Tropomiosina/metabolismoRESUMEN
The estrone ligand is used for modifying nanoparticle surfaces to improve their targeting effect on cancer cell lines. However, to date, there is no common agreement on the ideal linker length to be used for the optimum targeting performance. In this study, we aimed to investigate the impact of poly(poly ethylene glycol methyl ether methacrylate) (PPEGMEMA) linker length on the cellular uptake behavior of polymer-coated upconverting nanoparticles (UCNPs). Different triblock terpolymers, poly(poly (ethylene glycol) methyl ether methacrylate)-block-polymethacrylic acid-block-polyethylene glycol methacrylate phosphate (PPEGMEMAx-b-PMAAy-b-PEGMP3: x = 7, 15, 33, and 80; y = 16, 20, 18, and 18), were synthesized with different polymer linker chain lengths between the surface and the targeting ligand by reversible addition-fragmentation chain transfer polymerization. The estrone ligand was attached to the polymer via specific terminal conjugation. The cellular association of polymer-coated UCNPs with linker chain lengths was evaluated in MCF-7 cells by flow cytometry. Our results showed that the bioactivity of ligand modification is dependent on the length of the polymer linker. The shortest polymer PPEGMEMA7-b-PMAA16-b-PEGMP3 with estrone at the end of the polymer chain was found to have the best cellular association behavior in the estrogen receptor (ER)α-positive expression cell line MCF-7. Additionally, the anticancer drug doxorubicinâ¢HCl was encapsulated in the nanocarrier to evaluate the 2D and 3D cytotoxicity. The results showed that estrone modification could efficiently improve the cellular uptake in ERα-positive expression cell lines and in 3D spheroid models.
Asunto(s)
Éteres Metílicos , Nanopartículas , Estrona/farmacología , Humanos , Ligandos , Metacrilatos , Polietilenglicoles , Polímeros/farmacologíaRESUMEN
Single molecule experiments have recently attracted enormous interest. Many of these studies involve the encapsulation of a single molecule into nanoscale containers (such as vesicles, droplets and nanowells). In such cases, the single molecule encapsulation efficiency is a key parameter to consider in order to get a statistically significant quantitative information. It has been shown that such encapsulation typically follows a Poisson distribution and such theory of encapsulation has only been applied to the encapsulation of single molecules into perfectly sized monodispersed containers. However, experimentally nanocontainers are usually characterized by a size distribution, and often just a single binding pair (rather than a single molecule) is required to be encapsulated. Here the use of Poisson distribution is extended to predict the encapsulation efficiency of two different molecules in an association equilibrium. The Poisson distribution is coupled with a log-normal distribution in order to consider the effect of the container size distribution, and the effect of adsorption to the container is also considered. This theory will allow experimentalists to determine what single molecule encapsulation efficiency can be expected as a function of the experimental conditions. Two case studies, based on experimental data, are given to support the theoretical predictions.
Asunto(s)
NanotecnologíaRESUMEN
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.
Asunto(s)
Miosina Tipo IIA no Muscular/metabolismo , Fibras de Estrés/metabolismo , Tropomiosina/metabolismo , Línea Celular Tumoral , Humanos , Miosina Tipo IIA no Muscular/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fibras de Estrés/genética , Tropomiosina/genéticaRESUMEN
We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.
Asunto(s)
Ascorbato Peroxidasas/genética , Células Epiteliales/ultraestructura , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Anticuerpos de Dominio Único/química , Animales , Ascorbato Peroxidasas/metabolismo , Línea Celular , Sistema Libre de Células , Cricetulus , Células Epiteliales/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/genética , Proteína Fluorescente RojaRESUMEN
Microbial adhesion to host cells represents the initial step in the infection process. Several methods have been explored to inhibit microbial adhesion including the use of glycopolymers based on mannose, galactose, sialic acid and glucose. These sugar receptors are, however, abundant in the body, and are not unique to bacteria. Trehalose, in contrast, is a unique disaccharide that is widely expressed by microbes. This carbohydrate has not yet been explored as an anti-adhesive agent. Herein, gold nanoparticles (AuNPs) coated with trehalose-based polymers were prepared and compared to glucose-functionalized AuNPs and examined for their ability to prevent binding to endothelial cells. Acting as anti-adhesive agents, trehalose-functionalized NPs decreased the binding of S.â aureus to HUVECs, while outperforming the control NPs. Microscopy revealed that trehalose-coated NPs bound strongly to S.â aureus compared to the controls. In conclusion, nanoparticles based on trehalose could be a non-toxic alternative to inhibit S.â aureus infection.
Asunto(s)
Antibacterianos/farmacología , Glucosa/química , Oro/química , Nanopartículas del Metal/química , Staphylococcus aureus/efectos de los fármacos , Trehalosa/química , Antibacterianos/síntesis química , Antibacterianos/química , Adhesión Bacteriana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Staphylococcus aureus/fisiologíaRESUMEN
Visualization of scientific data is crucial not only for scientific discovery but also to communicate science and medicine to both experts and a general audience. Until recently, we have been limited to visualizing the three-dimensional (3D) world of biology in 2 dimensions. Renderings of 3D cells are still traditionally displayed using two-dimensional (2D) media, such as on a computer screen or paper. However, the advent of consumer grade virtual reality (VR) headsets such as Oculus Rift and HTC Vive means it is now possible to visualize and interact with scientific data in a 3D virtual world. In addition, new microscopic methods provide an unprecedented opportunity to obtain new 3D data sets. In this perspective article, we highlight how we have used cutting edge imaging techniques to build a 3D virtual model of a cell from serial block-face scanning electron microscope (SBEM) imaging data. This model allows scientists, students and members of the public to explore and interact with a "real" cell. Early testing of this immersive environment indicates a significant improvement in students' understanding of cellular processes and points to a new future of learning and public engagement. In addition, we speculate that VR can become a new tool for researchers studying cellular architecture and processes by populating VR models with molecular data.
Asunto(s)
Células/ultraestructura , Comprensión/fisiología , Programas Informáticos , Análisis y Desempeño de Tareas , Realidad Virtual , Humanos , Imagenología Tridimensional , Interfaz Usuario-ComputadorRESUMEN
The human immunodeficiency virus (HIV) capsid is a cone-shaped capsule formed from the viral capsid protein (CA), which is arranged into a lattice of hexamers and pentamers. The capsid comprises multiple binding interfaces for the recruitment of host proteins and macromolecules used by the virus to establish infection. Here, we coassembled CA proteins engineered for pentamer cross-linking and fluorescence labeling, into spherical particles. The CA spheres, which resemble the pentamer-rich structure of the end caps of the native HIV capsid, were immobilized onto surfaces as biorecognition elements for fluorescence microscopy-based quantification of host protein binding. The capsid-binding host protein cyclophilin A (CypA) is bound to CA spheres with the same affinity as CA tubes but at a higher CypA/CA stoichiometry, suggesting that the level of recruitment of CypA to the HIV capsid is dependent on curvature.
Asunto(s)
Cápside , Infecciones por VIH , VIH-1 , Proteínas de la Cápside , Ciclofilina A , HumanosRESUMEN
Herein, a glucose meter-based immunosensing platform is developed that allows the quantification of procalcitonin (PCT) in whole blood samples. PCT is a biomarker for sepsis and its early detection would improve the safety of the patient, as the diagnostic process will be easier and faster. The method employs liposomes with encapsulated glucose as a signal generation tag, which are then used in a sandwich immunoassay by conjugating an antibody to the liposome. The optimal liposomes' size and concentration of encapsulated glucose is determined experimentally to be 200 nm and 27.8 mM, respectively. Upon the addition of a surfactant (Triton X-100), the glucose is released and a signal is detected with a personal glucose meter (PGM). This signal is directly proportional to the concentration of the PCT in the sample. The dynamic range of the assay developed was 0.153-15.38 nM, and could allow the detection of PCT as low as 0.15 nM. The assay showed a high selectivity toward PCT against other proteins such as C-reactive protein and human serum albumin and good reproducibility. This assay was able to quantitatively determine the amount of PCT in whole blood samples at clinically-relevant concentrations.
Asunto(s)
Automonitorización de la Glucosa Sanguínea/instrumentación , Glucosa/química , Inmunoensayo/instrumentación , Liposomas/química , Polipéptido alfa Relacionado con Calcitonina/sangre , Cápsulas , Humanos , Cinética , Límite de DetecciónRESUMEN
Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W-containing retromer results in the accumulation of intracellular α-synuclein-positive aggregates, a hallmark of Parkinson disease. Overall, the Vps35 R524W-containing retromer has a decreased endosomal association, which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson disease.
Asunto(s)
Endosomas , Mutación Missense , Enfermedad de Parkinson , Agregación Patológica de Proteínas , Proteínas de Transporte Vesicular , alfa-Sinucleína , Sustitución de Aminoácidos , Células HeLa , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Caveolae are an abundant feature of the plasma membrane in many cells. Until recently, they were generally considered to be membrane invaginations whose formation primarily driven by integral membrane proteins called caveolins. However, the past decade has seen the emergence of the cavin family of peripheral membrane proteins as essential coat components and regulators of caveola biogenesis. In this Commentary, we summarise recent data on the role of cavins in caveola formation, highlighting structural studies that provide new insights into cavin coat assembly. In mammals, there are four cavin family members that associate through homo- and hetero-oligomerisation to form distinct subcomplexes on caveolae, which can be released into the cell in response to stimuli. Studies from several labs have provided a better understanding of cavin stoichiometry and the molecular basis for their oligomerisation, as well as identifying interactions with membrane phospholipids that may be important for caveola function. We propose a model in which coincident, low-affinity electrostatically controlled protein-protein and protein-lipid interactions allow the formation of caveolae, generating a meta-stable structure that can respond to plasma membrane stress by release of cavins.
Asunto(s)
Caveolas/metabolismo , Familia de Multigenes , Proteínas de Unión al ARN/metabolismo , Animales , Caveolas/química , Humanos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events.
Asunto(s)
Caveolina 1/metabolismo , Membrana Celular/metabolismo , Secuencia de Aminoácidos , Animales , Caveolina 1/química , Membrana Celular/química , Perros , Endopeptidasa K/metabolismo , Células de Riñón Canino Madin Darby , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Terciaria de Proteína , ProteolisisRESUMEN
Enzymatic ascorbate peroxidase (APEX) tagging allows for high-resolution, three-dimensional protein distribution analyses in cells and tissues. This chapter describes the application of APEX-tagging to visualize the trafficking of the epidermal growth factor receptor (EGFR) during epidermal growth factor-mediated receptor activation. Here, we describe the preparation of cells, methods to validate the stimulation of the EGFR, and visualization of the APEX-resolved distribution of the EGFR in the transmission electron microscope.
Asunto(s)
Receptores ErbB , Microscopía Electrónica de Transmisión , Transporte de Proteínas , Humanos , Ascorbato Peroxidasas/química , Ascorbato Peroxidasas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Microscopía Electrónica de Transmisión/métodosRESUMEN
Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only â¼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of â¼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.
Asunto(s)
Dineínas Citoplasmáticas , Microtúbulos , Imagen Individual de Molécula , Dineínas Citoplasmáticas/genética , Dineínas Citoplasmáticas/metabolismo , Complejo Dinactina/metabolismo , Endosomas/metabolismo , Microtúbulos/metabolismoRESUMEN
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.