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1.
Cell ; 166(4): 920-934, 2016 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-27499022

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/química
2.
Biophys J ; 120(9): 1692-1704, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33730552

RESUMO

To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. The physics of this key immunological process are poorly understood, but it is important to understand them because leukocytes have been shown to react to their mechanical environment. Using an innovative micropipette rheometer, we show in three different types of leukocytes that, when stimulated by microbeads mimicking target cells, leukocytes become up to 10 times stiffer and more viscous. These mechanical changes start within seconds after contact and evolve rapidly over minutes. Remarkably, leukocyte elastic and viscous properties evolve in parallel, preserving a well-defined ratio that constitutes a mechanical signature specific to each cell type. Our results indicate that simultaneously tracking both elastic and viscous properties during an active cell process provides a new, to our knowledge, way to investigate cell mechanical processes. Our findings also suggest that dynamic immunomechanical measurements can help discriminate between leukocyte subtypes during activation.


Assuntos
Leucócitos , Elasticidade , Viscosidade
3.
Cell Mol Biol Lett ; 25: 37, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32647530

RESUMO

The plasma membrane (PM) spatiotemporal organization is one of the major factors controlling cell signaling and whole-cell homeostasis. The PM lipids, including cholesterol, determine the physicochemical properties of the membrane bilayer and thus play a crucial role in all membrane-dependent cellular processes. It is known that lipid content and distribution in the PM are not random, and their transversal and lateral organization is highly controlled. Mainly sphingolipid- and cholesterol-rich lipid nanodomains, historically referred to as rafts, are extremely dynamic "hot spots" of the PM controlling the function of many cell surface proteins and receptors. In the first part of this review, we will focus on the recent advances of PM investigation and the current PM concept. In the second part, we will discuss the importance of several classes of ABC transporters whose substrates are lipids for the PM organization and dynamics. Finally, we will briefly present the significance of lipid ABC transporters for immune responses.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Animais , Transporte Biológico/fisiologia , Humanos , Imunidade/fisiologia , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo
4.
Nano Lett ; 18(10): 6544-6550, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30179011

RESUMO

There are very few techniques to reconstruct the shape of a cell at nanometric resolution, and those that exist are almost exclusively based on fluorescence, implying limitations due to staining constraints and artifacts. Reflection interference contrast microscopy (RICM), a label-free technique, permits the measurement of nanometric distances between refractive objects. However, its quantitative application to cells has been largely limited due to the complex interferometric pattern caused by multiple reflections on internal or thin structures like lamellipodia. Here we introduce 3D reflection interference contrast nanoscopy, 3D-RICN, which combines information from multiple illumination wavelengths and aperture angles to characterize the lamellipodial region of an adherent cell in terms of its distance from the surface and its thickness. We validate this new method by comparing data obtained on fixed cells imaged with atomic force microscopy and quantitative phase imaging. We show that as expected, cells adhering to micropatterns exhibit a radial symmetry for the lamellipodial thickness. We demonstrate that the substrate-lamellipod distance may be as high as 100 nm. We also show how the method applies to living cells, opening the way for label-free dynamical study of cell structures with nanometric resolution.

5.
Biophys J ; 115(3): 565-576, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30029772

RESUMO

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 ∼600 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-Ruído
6.
Immunology ; 150(2): 199-212, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27716898

RESUMO

Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A35-43 peptide associated with H-2Ld , although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2Ld . The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+ ]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.


Assuntos
Antígenos de Neoplasias/metabolismo , Exocitose , Mastocitoma/imunologia , Melanoma/imunologia , Fragmentos de Peptídeos/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/genética , Sinalização do Cálcio , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Antígeno de Histocompatibilidade H-2D/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Especificidade do Receptor de Antígeno de Linfócitos T
7.
PLoS Comput Biol ; 9(9): e1003245, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086124

RESUMO

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells.


Assuntos
Cálcio/metabolismo , Transdução de Sinais , Software , Linfócitos T/metabolismo , Animais , Células COS , Chlorocebus aethiops , Humanos , Sondas Moleculares
8.
Cell Rep ; 43(2): 113744, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38329874

RESUMO

Peroxisome biogenesis disorders (PBDs) represent a group of metabolic conditions that cause severe developmental defects. Peroxisomes are essential metabolic organelles, present in virtually every eukaryotic cell and mediating key processes in immunometabolism. To date, the full spectrum of PBDs remains to be identified, and the impact PBDs have on immune function is unexplored. This study presents a characterization of the hepatic immune compartment of a neonatal PBD mouse model at single-cell resolution to establish the importance and function of peroxisomes in developmental hematopoiesis. We report that hematopoietic defects are a feature in a severe PBD murine model. Finally, we identify a role for peroxisomes in the regulation of the major histocompatibility class II expression and antigen presentation to CD4+ T cells in dendritic cells. This study adds to our understanding of the mechanisms of PBDs and expands our knowledge of the role of peroxisomes in immunometabolism.


Assuntos
Transtornos Peroxissômicos , Síndrome de Zellweger , Animais , Camundongos , Síndrome de Zellweger/metabolismo , Peroxissomos/metabolismo , Apresentação de Antígeno , Transtornos Peroxissômicos/metabolismo
9.
Front Mol Neurosci ; 16: 1170313, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37138705

RESUMO

Microglial cells ensure essential roles in brain homeostasis. In pathological condition, microglia adopt a common signature, called disease-associated microglial (DAM) signature, characterized by the loss of homeostatic genes and the induction of disease-associated genes. In X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disease, microglial defect has been shown to precede myelin degradation and may actively contribute to the neurodegenerative process. We previously established BV-2 microglial cell models bearing mutations in peroxisomal genes that recapitulate some of the hallmarks of the peroxisomal ß-oxidation defects such as very long-chain fatty acid (VLCFA) accumulation. In these cell lines, we used RNA-sequencing and identified large-scale reprogramming for genes involved in lipid metabolism, immune response, cell signaling, lysosome and autophagy, as well as a DAM-like signature. We highlighted cholesterol accumulation in plasma membranes and observed autophagy patterns in the cell mutants. We confirmed the upregulation or downregulation at the protein level for a few selected genes that mostly corroborated our observations and clearly demonstrated increased expression and secretion of DAM proteins in the BV-2 mutant cells. In conclusion, the peroxisomal defects in microglial cells not only impact on VLCFA metabolism but also force microglial cells to adopt a pathological phenotype likely representing a key contributor to the pathogenesis of peroxisomal disorders.

10.
Front Mol Neurosci ; 16: 1299314, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38164407

RESUMO

Microglia are crucial for brain homeostasis, and dysfunction of these cells is a key driver in most neurodegenerative diseases, including peroxisomal leukodystrophies. In X-linked adrenoleukodystrophy (X-ALD), a neuroinflammatory disorder, very long-chain fatty acid (VLCFA) accumulation due to impaired degradation within peroxisomes results in microglial defects, but the underlying mechanisms remain unclear. Using CRISPR/Cas9 gene editing of key genes in peroxisomal VLCFA breakdown (Abcd1, Abcd2, and Acox1), we recently established easily accessible microglial BV-2 cell models to study the impact of dysfunctional peroxisomal ß-oxidation and revealed a disease-associated microglial-like signature in these cell lines. Transcriptomic analysis suggested consequences on the immune response. To clarify how impaired lipid degradation impacts the immune function of microglia, we here used RNA-sequencing and functional assays related to the immune response to compare wild-type and mutant BV-2 cell lines under basal conditions and upon pro-inflammatory lipopolysaccharide (LPS) activation. A majority of genes encoding proinflammatory cytokines, as well as genes involved in phagocytosis, antigen presentation, and co-stimulation of T lymphocytes, were found differentially overexpressed. The transcriptomic alterations were reflected by altered phagocytic capacity, inflammasome activation, increased release of inflammatory cytokines, including TNF, and upregulated response of T lymphocytes primed by mutant BV-2 cells presenting peptides. Together, the present study shows that peroxisomal ß-oxidation defects resulting in lipid alterations, including VLCFA accumulation, directly reprogram the main cellular functions of microglia. The elucidation of this link between lipid metabolism and the immune response of microglia will help to better understand the pathogenesis of peroxisomal leukodystrophies.

11.
Membranes (Basel) ; 11(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209140

RESUMO

Extensive studies showed the crucial role of ATP binding cassette (ABC) transporter ABCA1 in organizing the lipid microenvironment at the plasma membrane (PM) of living cells. However, the exact role of this protein in terms of lipid redistribution and lateral reorganization of the PM is still being discussed. Here, we took advantage of the spot variation fluorescence correlation spectroscopy (svFCS) to investigate the molecular dynamics of the ABCA1 expressed at the PM of Chinese hamster ovary cells (CHO-K1). We confirmed that this protein is strongly confined into the raft nanodomains. Next, in agreement with our previous observations, we showed that amphotericin B does not affect the diffusion properties of an active ABCA1 in contrary to inactive mutant ABCA1MM. We also evidenced that ApoA1 influences the molecular diffusion properties of ABCA1. Finally, we showed that the molecular confinement of ABCA1 depends on the cholesterol content in the PM, but presumably, this is not the only factor responsible for that. We concluded that the molecular dynamics of ABCA1 strongly depends on its activity and the PM composition. We hypothesize that other factors than lipids (i.e., proteins) are responsible for the strong confinement of ABCA1 in PM nanodomains which possibility has to be elucidated.

12.
Sci Rep ; 11(1): 6783, 2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33762632

RESUMO

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Cálcio/metabolismo , Adesão Celular/imunologia , Linhagem Celular Tumoral , Forma Celular , Expressão Ectópica do Gene , Humanos , Antígenos Comuns de Leucócito/metabolismo , Imagem Molecular
13.
Nat Chem Biol ; 4(9): 538-47, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18641634

RESUMO

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.


Assuntos
Microdomínios da Membrana , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Células COS , Chlorocebus aethiops , Colesterol/biossíntese , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Camundongos , Transdução de Sinais/fisiologia , Espectrometria de Fluorescência , Esfingolipídeos/antagonistas & inibidores , Esfingolipídeos/biossíntese , Linfócitos T/metabolismo
14.
FASEB J ; 23(6): 1775-85, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19151332

RESUMO

The ABCA1 transporter orchestrates cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors. The molecular mechanism is, however, unknown. We report here on the biophysical analysis in living HeLa cells of the ABCA1 lipid microenvironment at the plasma membrane. The modifications of membrane attributes induced by ABCA1 were assessed at both the outer and inner leaflet by monitoring either the lifetime of membrane inserted fluorescent lipid analogues by fluorescence lifetime imaging microscopy (FLIM) or, respectively, the membrane translocation of cationic sensors. Analysis of the partitioning of dedicated probes in plasma membrane blebs vesiculated from these cells allowed visualization of ABCA1 partitioning into the liquid disordered-like phase and corroborated the idea that ABCA1 destabilizes the lipid arrangement at the membrane. Specificity was demonstrated by comparison with cells expressing an inactive transporter. The physiological relevance of these modifications was finally demonstrated by the reduced membrane mobility and function of transferrin receptors under the influence of an active ABCA1. Collectively, these data assess that the control of both transversal and lateral lipid distribution at the membrane is the primary function of ABCA1 and positions the effluxes of cholesterol from cell membranes downstream to the redistribution of the sterol into readily extractable membrane pools.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular , Metabolismo dos Lipídeos , Lipídeos/química , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Membrana Celular/química , Membrana Celular/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Células HeLa , Humanos , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Potenciais da Membrana/fisiologia , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Receptores da Transferrina/metabolismo
16.
Front Immunol ; 9: 2864, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30564247

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/imunologia
17.
Sci Rep ; 8(1): 4966, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563576

RESUMO

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/citologia
18.
Biochim Biophys Acta ; 1585(2-3): 64-71, 2002 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-12531538

RESUMO

Programmed cell death is one of the major devices controlling cellular homeostasis. However, the generation of cell debris that follows the execution phase of apoptosis has to be backed up by their efficient removal by phagocyte. This highly dynamic process requires the concerted action of a number of surface molecules able to recognize early signals of membrane modifications on the apoptotic prey. Among those, the loss of phospholipid asymmetry and exposure of phosphatidylserine on the prey to be is determinant to engage phagocyte receptors and trigger the removal of corpses. A loss of membrane lipid asymmetry occurs also on the phagocyte determining its efficiency as an undertaker. Here we will discuss how, in our mind, the ATP binding cassette transporter, ABCA1, by its action on the arrangement of lipids at the phagocyte membrane, may actively promote their competence to engulf.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apoptose/fisiologia , Lipídeos de Membrana/metabolismo , Fagócitos/fisiologia , Proteínas de Transferência de Fosfolipídeos , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Animais , Caenorhabditis elegans , Proteínas de Transporte/metabolismo , Mamíferos , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Transdução de Sinais
19.
Methods Enzymol ; 519: 277-302, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23280115

RESUMO

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/métodos
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