RESUMO
Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.
Assuntos
Caveolina 3 , Cisteína , Músculo Esquelético , Processamento de Proteína Pós-Traducional , Isoformas de ProteínasRESUMO
To build a just, equitable, and diverse academy, scientists and institutions must address systemic barriers that sex and gender minorities face. This Commentary summarizes (1) critical context informing the contemporary oppression of transgender people, (2) how this shapes extant research on sex and gender, and (3) actions to build an inclusive and rigorous academy for all.
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Minorias Sexuais e de Gênero , Pessoas Transgênero , Masculino , Feminino , Humanos , Identidade de GêneroRESUMO
Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human HeLa cell line. When combined with the super-resolution technique expansion microscopy (ExM) involving swellable acrylamide hydrogels, fluorescent esters reveal nanoscale features including cytoplasmic membrane-bound compartments and nucleolar densities. We observe differential labelling patterns linked to the biochemical properties of the conjugated dye. Alterations in staining density and compartment specificity were seen depending on the timepoint of application in the ExM protocol. Additional complexity in labelling patterns was detected arising from inter-ester interactions. Our findings raise a number of considerations for the use of fluorescent esters. We demonstrate esters as a useful addition to the repertoire of stains of the cellular proteome, whether applied either on their own to visualise overall cellular morphology, or as counterstains providing ultrastructural context alongside specific target markers like antibodies.
Assuntos
Corantes Fluorescentes , Microscopia , Humanos , Corantes Fluorescentes/química , Células HeLa , Ésteres , AnticorposRESUMO
Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.
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Sinalização do Cálcio , Cálcio , Animais , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina , Coração , MiocárdioRESUMO
Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid-liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule in C. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, Mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid-liquid phase separation.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Grânulos de Ribonucleoproteínas de Células Germinativas , Proteínas de Caenorhabditis elegans/genética , Microscopia , Organelas/metabolismoRESUMO
Coordinated events of calcium (Ca2+) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8-5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca2+ released at each Ca2+ spark.
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Cálcio , Gânglios Espinais , Animais , Ratos , Sistemas do Segundo Mensageiro , Retículo Endoplasmático , Neurônios , Canal de Liberação de Cálcio do Receptor de RianodinaRESUMO
Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery of intracellular calcium signalling in cardiomyocytes. While a range of optical super-resolution microscopy techniques have revealed the nanoscale structure of these clusters, the three-dimensional (3D) nanoscale topologies of the clusters have remained mostly unresolved. In this paper, we demonstrate the exploitation of molecular-scale resolution in enhanced expansion microscopy (EExM) along with various 2D and 3D visualization strategies to observe the topological complexities, geometries and molecular sub-domains within the RyR clusters. Notably, we observed sub-domains containing RyR-binding protein junctophilin-2 (JPH2) occupying the central regions of RyR clusters in the deeper interior of the myocytes (including dyads), while the poles were typically devoid of JPH2, lending to a looser RyR arrangement. By contrast, peripheral RyR clusters exhibited variable co-clustering patterns and ratios between RyR and JPH2. EExM images of dyadic RyR clusters in right ventricular (RV) myocytes isolated from rats with monocrotaline-induced RV failure revealed hallmarks of RyR cluster fragmentation accompanied by breaches in the JPH2 sub-domains. Frayed RyR patterns observed adjacent to these constitute new evidence that the destabilization of the RyR arrays inside the JPH2 sub-domains may seed the primordial foci of dyad remodelling observed in heart failure. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.
Assuntos
Insuficiência Cardíaca , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Imageamento Tridimensional , Monocrotalina , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
February is LGBT+ history month, and to celebrate, Journal of Cell Science Editorial Advisory Board member David Bryant organised a conversation with a selection of scientists to explore their experiences of being LGBT+ in academia.
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Liderança , Minorias Sexuais e de Gênero , Mobilidade Ocupacional , Comunicação , HumanosRESUMO
In celebration of Pride Month, we asked transgender, genderqueer, and nonbinary scientists to tell us about what fascinates them, their ambitions and achievements, and how their gender identities have shaped their experiences in STEM. We owe a special thanks to 500 Queer Scientists (https://500queerscientists.com/), whose network and efforts at increasing LGBTQ+ scientists' visibility made this article possible.
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Engenharia , Matemática , Pesquisadores , Ciência , Minorias Sexuais e de Gênero , Tecnologia , Pessoas Transgênero , Feminino , Humanos , MasculinoRESUMO
Resolution is a key feature in microscopy which allows the visualization of the fine structure of cells. Much of the life processes within these cells depend on the three-dimensional (3D) complexity of these structures. Optical super-resolution microscopies are currently the preferred choice of molecular and cell biologists who seek to visualize the organization of specific protein species at the nanometer scale. Traditional super-resolution microscopy techniques have often been limited by sample thickness, axial resolution, specialist optical instrumentation and computationally-demanding software for assembling the images. In this chapter we detail the protocol, "enhanced expansion microscopy" (EExM), which combines X10 expansion microscopy with Airyscan confocal microscopy. EExM enables 15nm lateral (and 35nm axial) resolution, and is a relatively cheap, accessible option allowing single protein resolution for the non-specialist optical microscopists. We illustrate how EExM has been utilized for mapping the 3D topology of intracellular protein arrays at sample depths which are not always compatible with some of the traditional super-resolution techniques. We demonstrate that antibody markers can recognize and map post-translational modifications of individual proteins in addition to their 3D positions. Finally, we discuss the current uncertainties and validations in EExM which include the isotropy in gel expansion and assessment of the expansion factor (of resolution improvement).
Assuntos
Microscopia ConfocalRESUMO
Computational models of the heart at multiple spatial scales, from sub-cellular nanodomains to the whole-organ, are a powerful tool for the simulation of cardiac electrophysiology. Application of these models has provided remarkable insight into the normal and pathological functioning of the heart. In these two articles, we present methods for modelling cardiac electrophysiology at all of these spatial scales. In part one, presented here, we discuss methods and approaches for modelling sub-cellular calcium dynamics at the whole-cell and organ scales, valuable for modelling excitation-contraction coupling and mechanisms of arrhythmia triggers.
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Potenciais de Ação , Cálcio/metabolismo , Simulação por Computador , Coração/fisiologia , Modelos Cardiovasculares , Miócitos Cardíacos/fisiologia , Cálcio/fisiologia , Fenômenos Eletrofisiológicos , Humanos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Citosol/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fatores de TempoRESUMO
Endothelial cells selectively release cargo stored in Weibel-Palade bodies (WPBs) to regulate vascular function, but the underlying mechanisms are poorly understood. Here we show that histamine evokes the release of the proinflammatory ligand, P-selectin, while diverting WPBs carrying non-inflammatory cargo away from the plasma membrane to the microtubule organizing center. This differential trafficking is dependent on Rab46 (CRACR2A), a newly identified Ca2+-sensing GTPase, which localizes to a subset of P-selectin-negative WPBs. After acute stimulation of the H1 receptor, GTP-bound Rab46 evokes dynein-dependent retrograde transport of a subset of WPBs along microtubules. Upon continued histamine stimulation, Rab46 senses localized elevations of intracellular calcium and evokes dispersal of microtubule organizing center-clustered WPBs. These data demonstrate for the first time that a Rab GTPase, Rab46, integrates G protein and Ca2+ signals to couple on-demand histamine signals to selective WPB trafficking.
Assuntos
Sinalização do Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Receptores Histamínicos H1/genética , Corpos de Weibel-Palade/genética , Membrana Celular/genética , Dineínas/genética , Exocitose/genética , Proteínas de Ligação ao GTP/genética , Histamina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Microtúbulos/genética , Selectina-P/genética , Transporte Proteico/genética , Transdução de Sinais/genética , Corpos de Weibel-Palade/metabolismoRESUMO
Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of â¼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a ß-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.
Assuntos
Canais de Cálcio/metabolismo , Nanoestruturas/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Cálcio/metabolismo , Humanos , Microscopia , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
Remodelling of the membranes and protein clustering patterns during the pathogenesis of cardiomyopathies has renewed the interest in spatial visualisation of these structures in cardiomyocytes. Coincidental emergence of single molecule (super-resolution) imaging and tomographic electron microscopy tools in the last decade have led to a number of new observations on the structural features of the couplons, the primary sites of excitation-contraction coupling in the heart. In particular, super-resolution and tomographic electron micrographs have revised and refined the classical views of the nanoscale geometries of couplons, t-tubules and the organisation of the principal calcium handling proteins in both healthy and failing hearts. These methods have also allowed the visualisation of some features which were too small to be detected with conventional microscopy tools. With new analytical capabilities such as single-protein mapping, in situ protein quantification, correlative and live cell imaging we are now observing an unprecedented interest in adapting these research tools across the cardiac biophysical research discipline. In this article, we review the depth of the new insights that have been enabled by these tools toward understanding the structure and function of the cardiac couplon. We outline the major challenges that remain in these experiments and emerging avenues of research which will be enabled by these technologies.
RESUMO
Signaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites of cardiomyocytes where ryanodine receptors (RyRs) are clustered with their molecular partners. Localization microscopy has been crucial to visualizing these nanodomains but has been limited by brightness of markers, restricting the resolution and quantification of individual proteins clustered within. Harnessing the remarkable localization precision of DNA-PAINT (<10 nm), we visualized punctate labeling within these nanodomains, confirmed as single RyRs. RyR positions within sub-plasmalemmal nanodomains revealed how they are organized randomly into irregular clustering patterns leaving significant gaps occupied by accessory or regulatory proteins. RyR-inhibiting protein junctophilin-2 appeared highly concentrated adjacent to RyR channels. Analyzing these molecular maps showed significant variations in the co-clustering stoichiometry between junctophilin-2 and RyR, even between nearby nanodomains. This constitutes an additional level of complexity in RyR arrangement and regulation of calcium signaling, intrinsically built into the nanodomains.
Assuntos
Sinalização do Cálcio/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Análise por Conglomerados , HumanosRESUMO
High-force eccentric exercise results in sustained increases in cytoplasmic Ca2+ levels ([Ca2+]cyto), which can cause damage to the muscle. Here we report that a heavy-load strength training bout greatly alters the structure of the membrane network inside the fibres, the tubular (t-) system, causing the loss of its predominantly transverse organization and an increase in vacuolation of its longitudinal tubules across adjacent sarcomeres. The transverse tubules and vacuoles displayed distinct Ca2+-handling properties. Both t-system components could take up Ca2+ from the cytoplasm but only transverse tubules supported store-operated Ca2+ entry. The retention of significant amounts of Ca2+ within vacuoles provides an effective mechanism to reduce the total content of Ca2+ within the fibre cytoplasm. We propose this ability can reduce or limit resistance exercise-induced, Ca2+-dependent damage to the fibre by the reduction of [Ca2+]cyto to help maintain fibre viability during the period associated with delayed onset muscle soreness.
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Cálcio/metabolismo , Membrana Celular/metabolismo , Exercício Físico/fisiologia , Músculo Esquelético/metabolismo , Adulto , Citoplasma/metabolismo , Humanos , Masculino , Microscopia Confocal , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Sarcolema/metabolismo , Sarcômeros/metabolismo , Vacúolos/metabolismo , Adulto JovemRESUMO
Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as 'junctions', are fast communication hubs within excitable cells such as neurones and muscle. Here, we have examined two transgenic murine models probing the role of junctophilin-2, a membrane-tethering protein crucial for the formation and molecular organisation of sub-microscopic junctions in ventricular muscle cells of the heart. Quantitative single-molecule localisation microscopy showed that junctions in animals producing above-normal levels of junctophilin-2 were enlarged, allowing the re-organisation of the primary functional protein within it, the ryanodine receptor (RyR; in this paper, we use RyR to refer to the myocardial isoform RyR2). Although this change was associated with much enlarged RyR clusters that, due to their size, should be more excitable, functionally it caused a mild inhibition in the Ca2+ signalling output of the junctions (Ca2+ sparks). Analysis of the single-molecule densities of both RyR and junctophilin-2 revealed an â¼3-fold increase in the junctophilin-2 to RyR ratio. This molecular rearrangement is compatible with direct inhibition of RyR opening by junctophilin-2 to intrinsically stabilise the Ca2+ signalling properties of the junction and thus the contractile function of the cell.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Nanoestruturas/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais , Animais , Sinalização do Cálcio , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described.