RESUMO
Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.
Assuntos
Fibroblastos/metabolismo , Mutação de Sentido Incorreto , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Transdução de Sinais , Actinas/química , Actinas/metabolismo , Animais , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Difusão , Endocitose , Ativação Enzimática , Glicosilação , Humanos , Interferon gama/metabolismo , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Receptores de Interferon/químicaRESUMO
Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/métodos , Modelos Químicos , Espectrometria de Fluorescência/métodos , Citoesqueleto de Actina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Difusão , Fluorescência , Microscopia Intravital/instrumentação , Citometria de Varredura a Laser/instrumentação , Citometria de Varredura a Laser/métodos , Lasers , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Software , Espectrometria de Fluorescência/instrumentaçãoRESUMO
Single-molecule localization microscopy (SMLM) enables the production of high-resolution images by imaging spatially isolated fluorescent particles. Although challenging, the result of SMLM analysis lists the position of individual molecules, leading to a valuable quantification of the stoichiometry and spatial organization of molecular actors. Both the signal/noise ratio and the density (Dframe), i.e., the number of fluorescent particles per µm2 per frame, have previously been identified as determining factors for reaching a given SMLM precision. Establishing a comprehensive theoretical study relying on these two parameters is therefore of central interest to delineate the achievable limits for accurate SMLM observations. Our study reports that in absence of prior knowledge of the signal intensity α, the density effect on particle localization is more prominent than that anticipated from theoretical studies performed at known α. A first limit appears when, under a low-density hypothesis (i.e., one-Gaussian fitting hypothesis), any fluorescent particle distant by less than â¼600 nm from the particle of interest biases its localization. In fact, all particles should be accounted for, even those dimly fluorescent, to ascertain unbiased localization of any surrounding particles. Moreover, even under a high-density hypothesis (i.e., multi-Gaussian fitting hypothesis), a second limit arises because of the impossible distinction of particles located too closely. An increase in Dframe is thus likely to deteriorate the localization precision, the image reconstruction, and more generally the quantification accuracy. Our study firstly provides a density-signal/noise ratio space diagram for use as a guide in data recording toward reaching an achievable SMLM resolution. Additionally, it leads to the identification of the essential requirements for implementing UNLOC, a parameter-free and fast computing algorithm approaching the Cramér-Rao bound for particles at high-density per frame and without any prior knowledge of their intensity. UNLOC is available as an ImageJ plugin.
Assuntos
Algoritmos , Nanotecnologia , Imagem Individual de Molécula , Razão Sinal-RuídoRESUMO
To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode's surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.
Assuntos
Caenorhabditis elegans/microbiologia , Comunicação Celular , Hypocreales/citologia , Hypocreales/fisiologia , Microscopia Confocal/métodos , Pinças Ópticas , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Lentes , Microscopia Confocal/instrumentação , Movimento , Esporos Fúngicos/citologia , Esporos Fúngicos/fisiologiaRESUMO
Both classical and quantum electrodynamics predict the existence of dipole-dipole long-range electrodynamic intermolecular forces; however, these have never been hitherto experimentally observed. The discovery of completely new and unanticipated forces acting between biomolecules could have considerable impact on our understanding of the dynamics and functioning of the molecular machines at work in living organisms. Here, using two independent experiments, on the basis of different physical effects detected by fluorescence correlation spectroscopy and terahertz spectroscopy, respectively, we demonstrate experimentally the activation of resonant electrodynamic intermolecular forces. This is an unprecedented experimental proof of principle of a physical phenomenon that, having been observed for biomacromolecules and with long-range action (up to 1000 Å), could be of importance for biology. In addition to thermal fluctuations that drive molecular motion randomly, these resonant (and thus selective) electrodynamic forces may contribute to molecular encounters in the crowded cellular space.
RESUMO
Fas interaction at the plasma membrane with its lipid and protein environment plays a crucial role in the early steps of Fas signalling induced by Fas ligand binding. Particularly, Fas localisation in the raft nanodomains, ezrin-mediated interaction with the actin cytoskeleton and subsequent internalization are critical steps in Fas-mediated cell death. We identified a lysine-rich region (LRR) in the cytoplasmic, membrane-proximal region of Fas as a key determinant modulating these initial events. Through a genetic approach, we demonstrate that Fas LRR represents another signal additional to palmitoylation targeting Fas to the raft nanodomains, and modulates Fas interaction with the cytoskeleton.
Assuntos
Apoptose , Lisina/metabolismo , Microdomínios da Membrana/metabolismo , Transdução de Sinais , Receptor fas/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteína Ligante Fas , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Lipoilação , Lisina/química , Lisina/genética , Camundongos , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptor fas/genéticaRESUMO
Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.
Assuntos
Membrana Celular/metabolismo , Espectrometria de Fluorescência , Animais , Células COS , Calibragem , Sobrevivência Celular , Chlorocebus aethiops , Colesterol/metabolismo , Difusão , Proteínas de Fluorescência Verde/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismoRESUMO
The skin protects animals from infection and physical damage. In Caenorhabditis elegans, wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity (Chuang et al., 2016). Here, by establishing a simple wounding system, we show that wounding provokes a reorganisation of plasma membrane subdomains. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerisation. We show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signalling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.
Assuntos
Membrana Celular , Epiderme , Imunidade Inata , Microtúbulos , Actinas/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/imunologia , Membrana Celular/metabolismo , Epiderme/imunologia , Epiderme/lesões , Epiderme/metabolismo , Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Microtúbulos/química , Microtúbulos/imunologia , Microtúbulos/metabolismo , Simportadores/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4+ T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation.
Assuntos
Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Sinalização do Cálcio/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Técnicas de Cocultura , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.
Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Animais , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Hibridomas , Células Jurkat , Camundongos , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Linfócitos T/citologiaRESUMO
Testicular macrophages (tMφ) are the principal immune cells of the mammalian testis. Beyond classical immune functions, they have been shown to be important for organogenesis, spermatogenesis, and male hormone production. In the adult testis, two different macrophage populations have been identified based on their distinct tissue localization and morphology, but their developmental origin and mode of homeostatic maintenance are unknown. In this study, we use genetic lineage-tracing models and adoptive transfer protocols to address this question. We show that embryonic progenitors give rise to the interstitial macrophage population, whereas peritubular macrophages are exclusively seeded postnatally in the prepuberty period from bone marrow (BM)-derived progenitors. As the proliferative capacity of interstitial macrophages declines, BM progenitors also contribute to this population. Once established, both the peritubular and interstitial macrophage populations exhibit a long life span and a low turnover in the steady state. Our observations identify distinct developmental pathways for two different tMφ populations that have important implications for the further dissection of their distinct roles in organ homeostasis and testicular function.
Assuntos
Macrófagos/fisiologia , Testículo/citologia , Animais , Proliferação de Células/fisiologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Imunidade Celular/fisiologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Espermatogênese/fisiologia , Células-Tronco/fisiologia , Testículo/imunologia , Testículo/metabolismoRESUMO
In the present paper, an experimental feasibility study on the detection of long-range intermolecular interactions through three-dimensional molecular diffusion in solution is performed. This follows recent theoretical and numerical analyses reporting that long-range electrodynamic forces between biomolecules could be identified through deviations from Brownian diffusion. The suggested experimental technique was fluorescence correlation spectroscopy (FCS). By considering two oppositely charged molecular species in aqueous solution, namely, lysozymes and fluorescent dye molecules (Alexa488), the diffusion coefficient of the dyes has been measured for different values of the concentration of lysozyme, that is, for different average distances between the oppositely charged molecules. For our model, long-range interactions are of electrostatic origin, suggesting that their action radius can be varied by changing the ionic strength of the solution. The experimental outcomes clearly prove the detectability of long-range intermolecular interactions by means of the FCS technique. Molecular dynamics simulations provide a clear and unambiguous interpretation of the experimental results.
Assuntos
Corantes Fluorescentes/química , Fluorbenzenos/química , Muramidase/química , Espectrometria de Fluorescência/métodos , Algoritmos , Animais , Galinhas , Difusão , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Desenho de Equipamento , Íons/química , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Muramidase/metabolismo , Soluções , Espectrometria de Fluorescência/instrumentação , Eletricidade Estática , Água/químicaRESUMO
Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.
Assuntos
Sinalização do Cálcio , Fator de Crescimento Epidérmico/metabolismo , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Receptores ErbB/metabolismo , Humanos , CinéticaRESUMO
While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τ(d) and the squared waist of observation w(2). Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.
Assuntos
Espectrometria de Fluorescência/métodos , Calibragem , Adesão Celular , Linhagem Celular , Humanos , Microscopia/métodosRESUMO
Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT). Single-molecule videomicroscopy, offering millisecond and nanometric resolution, allows a detailed representation of membrane organization by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions. We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution. The nanometric accuracy obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods such as STORM, PALM, RESOLFT or STED, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.
Assuntos
Membrana Celular/química , Pontos Quânticos , Algoritmos , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Difusão , Microscopia de Vídeo/métodos , Método de Monte Carlo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismoRESUMO
Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.