RESUMO
Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the major enzyme responsible for generating phosphatidylinositol (4)-phosphate [PI(4)P] at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-cysteine motif in EFR3B (Cys5, Cys7 and Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B 'lipoforms' affects both interactions between EFR3B and TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] resynthesis following phospholipase C signaling, and EFR3B partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code involved in controlling protein-protein and protein-lipid interactions that affect a plasma membrane-resident lipid biosynthetic pathway.
Assuntos
Lipoilação , Fosfatidilinositóis , Membrana Celular/metabolismo , Homeostase , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismoRESUMO
Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FcεRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FcεRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FcεRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FcεRI that it initially phosphorylates for amplified activity. Net phosphorylation above a nonfunctional threshold is achieved in the stimulated state but not in the resting state, and current evidence supports the hypothesis that this relies on Ag crosslinking to disrupt a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to suprathreshold phosphorylation of Ag-crosslinked IgE-FcεRI in live rat basophilic leukemia mast cells. Our high-precision diffusion measurements by imaging fluorescence correlation spectroscopy on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains in the membrane inner leaflet and concomitant sharpening of segregation from liquid disordered (Ld)-like regions. With other structural variants, we determine that lipid-based interactions are essential for access by Lyn, leading to phosphorylation of and protein-based binding to clustered FcεRI. By contrast, TM tyrosine phosphatase, PTPα, is excluded from these regions due to its Ld-preference and steric exclusion of TM segments. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells.
Assuntos
Antígenos/metabolismo , Membrana Celular/metabolismo , Lipídeos/fisiologia , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Animais , Antígenos/imunologia , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Cricetulus , Proteínas de Fluorescência Verde/metabolismo , Metabolismo dos Lipídeos , Mastócitos/imunologia , Nanoestruturas , Ratos , Quinases da Família src/metabolismoRESUMO
Interleaflet coupling-the influence of one leaflet on the properties of the opposing leaflet-is a fundamental plasma membrane organizational principle. This coupling is proposed to participate in maintaining steady-state biophysical properties of the plasma membrane, which in turn regulates some transmembrane signaling processes. A prominent example is antigen (Ag) stimulation of signaling by clustering transmembrane receptors for immunoglobulin E (IgE), FcεRI. This transmembrane signaling depends on the stabilization of ordered regions in the inner leaflet for sorting of intracellular signaling components. The resting inner leaflet has a lipid composition that is generally less ordered than the outer leaflet and that does not spontaneously phase separate in model membranes. We propose that interleaflet coupling can mediate ordering and disordering of the inner leaflet, which is poised in resting cells to reorganize upon stimulation. To test this in live cells, we first established a straightforward approach to evaluate induced changes in membrane order by measuring inner leaflet diffusion of lipid probes by imaging fluorescence correlation spectroscopy, by imaging fluorescence correlation spectroscopy (ImFCS), before and after methyl-α-cyclodexrin (mαCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated responses: recruitment of cytoplasmic Syk kinase to the inner leaflet and exocytosis of secretory granules (degranulation). Based on the ImFCS data in resting cells, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order- or disorder-promoting lipids, respectively. We find that the degree of both stimulated Syk recruitment and degranulation correlates positively with LEX-mediated changes of inner leaflet order in resting cells. Overall, our results show that resting-state lipid ordering of the outer leaflet influences the ordering of the inner leaflet, likely via interleaflet coupling. This imposed lipid reorganization modulates transmembrane signaling stimulated by Ag clustering of IgE-FcεRI.
RESUMO
Experiencing the honor of this international recognition in chemistry, I wonder how this came to be. I reflect on my imperfect but rewarding path to where I am now, and on those who have helped me along the way.
RESUMO
Infectious diseases, such as influenza, present a prominent global problem including the constant threat of pandemics that initiate in avian or other species and then pass to humans. We report a new sensor that can be specifically functionalized to detect antibodies associated with a wide range of infectious diseases in multiple species. This biosensor is based on electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the antibody catalyzed water oxidation pathway (ACWOP). Our platform includes a polymer brush-modified surface where specific antibodies bind to conjugated haptens with high affinity and specificity. Hydrogen peroxide provides an electrochemical signal that is mediated by Resorufin/Amplex Red. We characterize the biosensor platform, using model anti-DNP antibodies, with the ultimate goal of designing a versatile device that is inexpensive, portable, reliable, and fast. We demonstrate detection of antibodies at concentrations that fall well within clinically relevant levels.
Assuntos
Anticorpos Catalíticos/química , Técnicas Biossensoriais/métodos , Peróxido de Hidrogênio/análise , Imunoglobulina G/análise , Água/química , Acrilatos/química , Técnicas Biossensoriais/instrumentação , Catálise , Dinitrobenzenos/química , Limite de Detecção , Oxirredução , Polietilenoglicóis/química , Silício/química , Oxigênio Singlete/químicaRESUMO
Oral delivery, while a highly desirable form of nanoparticle-drug administration, is limited by challenges associated with overcoming several biological barriers. Here, the authors study how fluorescent and poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles sized 5 to 50 nm interact with major barriers including intestinal mucus, intestinal epithelium, and stomach acid. From imaging fluorescence correlation spectroscopy studies using quasi-total internal reflection fluorescence microscopy, diffusion of nanoparticles through highly scattering mucus is progressively hindered above a critical hydrodynamic size around 20 nm. By studying Caco-2 cell monolayers mimicking the intestinal epithelia, it is observed that ultrasmall nanoparticles below 10 nm diameter (Cornell prime dots, [C' dots]) show permeabilities correlated with high absorption in humans from primarily enhanced passive passage through tight junctions. Particles above 20 nm diameter exclusively show active transport through cells. After establishing C' dot stability in artificial gastric juice, in vivo oral gavage experiments in mice demonstrate successful passage through the body followed by renal clearance without protein corona formation. Results suggest C' dots as viable candidates for oral administration to patients with a proven pathway towards clinical translation and may generate renewed interest in examining silica as a food additive and its effects on nutrition and health.
Assuntos
Portadores de Fármacos , Nanopartículas , Humanos , Ratos , Camundongos , Animais , Portadores de Fármacos/química , Células CACO-2 , Ratos Sprague-Dawley , Dióxido de Silício/química , Nanopartículas/químicaRESUMO
The ability to spatially deposit multiple biomolecules onto a single surface with high-resolution while retaining biomolecule stability and integrity is critical to the development of micro- and nanoscale biodevices. While conventional lithographic patterning methods are attractive for this application, they typically require the use of UV exposure and/or harsh solvents and imaging materials, which may be damaging to fragile biomolecules. Here, we report the development of a new patterning process based on a fluorinated patterning material that is soluble in hydrofluoroether solvents, which we show to be benign to biomolecules, including proteins and DNA. We demonstrate the implementation of these materials into an orthogonal processing system for patterning multibiomolecule arrays by imprint lithography at room temperature. We further showcase this method's capacity for fabricating patterns of receptor-specific ligands for fundamental cell studies.
Assuntos
DNA/metabolismo , Impressão Molecular/métodos , Proteínas/química , Animais , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , DNA/química , Hibridização Genética , Leucemia Basofílica Aguda , Metacrilatos/química , Metacrilatos/metabolismo , Nanotecnologia , Proteínas/metabolismo , Ratos , Estreptavidina/metabolismo , Propriedades de SuperfícieRESUMO
Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson's disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress1. We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and found that a-syn prevents recovery of stimulated mitochondrial Ca2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.
RESUMO
Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson' disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress. 1 We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca 2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and found that a-syn prevents recovery of stimulated mitochondrial Ca 2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca 2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.
RESUMO
Cell surface receptors that bind the Fc segment of antibodies to initiate signaling play fundamental roles in immune responses. Multiple, diverse Fc receptors (e.g., Fc gamma, Fc-alpha, and Fc-epsilon) are expressed on different immune cells, including natural killer cells, macrophages, mast cells, and neutrophils. Fc receptors bind particular antibody isotypes (e.g., IgG, IgA, IgE, respectively) thereby sensitizing the cells to their specific antigens. Receptor clustering by antigen or other multivalent ligands induces a signaling cascade that leads to targeted secretion of chemical mediators (e.g., histamine, cytokines, and chemokines) and other cell-specific responses. Spatial targeting and compartmentalization are common mechanisms for regulating Fc receptor signaling. However, the tools for studying these dynamic interactions at cellular levels have been limited due to the nanoscale dimensions of the signaling complexes and their dispersal across the cell surface. To overcome these limitations in our model system, we use microfabricated surfaces containing spatially defined ligands to cluster and activate IgE receptors (FcεRI), which initiate allergic responses by mast cells. Micron-scale control of receptor assemblies allows investigation with conventional fluorescence microscopy of spatially regulated redistributions of intracellular signaling components. This approach in conjunction with biochemical techniques has proven valuable for investigating immune receptor signaling.
Assuntos
Receptores Fc/imunologia , Antígenos , Ligantes , Mastócitos , Fagocitose , Receptores de IgERESUMO
Plasma membranes host numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation within the plasma membrane has emerged as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the coexistence of liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions. However, this phase/domain separation is nanoscopic and transient in live cells. It has been recently proposed that transbilayer coupling between the inner and outer leaflets of the plasma membrane is driven by their asymmetric lipid distribution and by dynamic cytoskeleton-lipid composites that contribute to the formation and transience of Lo/Ld phase separation in live cells. In this Perspective, we highlight new approaches to investigate how transbilayer coupling may influence phase separation. For quantitative evaluation of the impact of these interactions, we introduce an experimental strategy centered around Imaging Fluorescence Correlation Spectroscopy (ImFCS), which measures membrane diffusion with very high precision. To demonstrate this strategy, we choose two well-established model systems for transbilayer interactions: cross-linking by multivalent antigen of immunoglobulin E bound to receptor FcεRI and cross-linking by cholera toxin B of GM1 gangliosides. We discuss emerging methods to systematically perturb membrane lipid composition, particularly exchange of outer leaflet lipids with exogenous lipids using methyl alpha cyclodextrin. These selective perturbations may be quantitatively evaluated with ImFCS and other high-resolution biophysical tools to discover novel principles of lipid-mediated phase separation in live cells in the context of their pathophysiological relevance.
Assuntos
Lipídeos de Membrana , Membrana Celular/química , Difusão , Lipídeos de Membrana/metabolismo , Espectrometria de FluorescênciaRESUMO
Alpha-synuclein is a presynaptic protein linked to Parkinson's disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1.
Assuntos
Doença de Parkinson , Membranas Sinápticas , Vesículas Sinápticas , alfa-Sinucleína , Humanos , alfa-Sinucleína/química , Lipídeos/química , Doença de Parkinson/metabolismo , Vesículas Sinápticas/metabolismo , Domínios Proteicos , Membranas Sinápticas/químicaRESUMO
We have developed a method of performing near-field fluorescence correlation spectroscopy via an array of planarized circular apertures of 50 nm diameter. This technique provides 1 µs and 60 nm resolution on proximal samples, including live cells, without incorporating a scanning probe or pulsed lasers or requiring penetration of the sample into the aperture. Millions of apertures are created in an array within a thin film of aluminum on a coverslip and planarized to achieve no height distinction between the apertures and the surrounding metal. Supported lipid bilayers and plasma membranes from live cells adhere to the top of this substrate. We performed fluorescence correlation spectroscopy to demonstrate the sub-diffraction-limited illumination with these devices.
Assuntos
Nanoestruturas/química , Espectrometria de Fluorescência/métodos , Animais , Membrana Celular/metabolismo , Difusão , Corantes Fluorescentes/metabolismo , Nanoestruturas/ultraestrutura , RatosRESUMO
We use patterned poly(acrylic acid) (PAA) polymer brushes to explore the effects of surface chemistry and topography on cell-surface interactions. Most past studies of surface topography effects on cell adhesion have focused on patterned feature sizes that are larger than the dimensions of a cell, and PAA brushes have been characterized as cell repellent. Here we report cell adhesion studies for RBL mast cells incubated on PAA brush surfaces patterned with a variety of different feature sizes. We find that when patterned at subcellular dimensions on silicon surfaces, PAA brushes that are 30 or 15 nm thick facilitate cell adhesion. This appears to be mediated by fibronectin, which is secreted by the cells, adsorbing to the brushes and then engaging cell-surface integrins. The result is detectable accumulation of plasma membrane within the brushes, and this involves cytoskeletal remodeling at the cell-surface interface. By decreasing brush thickness, we find that PAA can be 'tuned' to promote cell adhesion with down-modulated membrane accumulation. We exemplify the utility of patterned PAA brush arrays for spatially controlling the activation of cells by modifying brushes with ligands that specifically engage IgE bound to high-affinity receptors on mast cells.
Assuntos
Resinas Acrílicas/química , Resinas Acrílicas/farmacologia , Mastócitos/efeitos dos fármacos , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Dinitrobenzenos/química , Fibronectinas/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/citologia , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais/efeitos dos fármacos , Propriedades de Superfície , TemperaturaRESUMO
Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc epsilonRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-Fc epsilonRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-Fc epsilonRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-Fc epsilonRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (alpha5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-Fc epsilonRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc epsilonRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc epsilonRI beta subunit but enhances stimulated Ca(2+) release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc epsilonRI has a net positive effect on Fc epsilonRI signaling.
Assuntos
Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Paxilina/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais/fisiologia , Actinas/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Immunoblotting , Ligantes , Microscopia de Fluorescência , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Talina/metabolismo , Vinculina/metabolismoRESUMO
Stochastic optical reconstruction microscopy (STORM) is an optical super-resolution microscopy (SRM) technique that traditionally requires toxic and non-physiological imaging buffers and setups that are not conducive to live-cell studies. It is observed that ultrasmall (<10 nm) fluorescent core-shell aluminosilicate nanoparticles (aC' dots) covalently encapsulating organic fluorophores enable STORM with a single excitation source and in a regular (non-toxic) imaging buffer. It is shown that fourfold coordinated aluminum is responsible for dye blinking, likely via photoinduced redox processes. It is demonstrated that this phenomenon is observed across different dye families leading to probes brighter and more photostable than the parent free dyes. Functionalization of aC' dots with antibodies allows targeted fixed cell STORM imaging. Finally, aC' dots enable live-cell STORM imaging providing quantitative measures of the size of intracellular vesicles and the number of particles per vesicle. The results suggest the emergence of a powerful ultrasmall, bright, and photostable optical SRM particle platform with characteristics relevant to clinical translation for the quantitative assessment of cellular structures and processes from live-cell imaging.
Assuntos
Silicatos de Alumínio/química , Microscopia de Fluorescência/métodos , Nanopartículas , Tamanho da Partícula , Linhagem Celular , Sobrevivência Celular , Humanos , Processamento de Imagem Assistida por ComputadorRESUMO
Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcepsilonRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.
Assuntos
Membrana Celular/metabolismo , Imunoglobulina E/fisiologia , Receptores de IgE/metabolismo , Quinases da Família src/metabolismo , Animais , Degranulação Celular , Linhagem Celular , Difusão , Proteínas de Fluorescência Verde/genética , Humanos , Mastócitos/fisiologia , Microdomínios da Membrana/fisiologia , Camundongos , Fosforilação , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Espectrometria de Fluorescência , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
A myriad of transient, nanoscopic lipid- and protein-based interactions confer a steady-state organization of the plasma membrane in resting cells that is poised to orchestrate assembly of key signaling components upon reception of an extracellular stimulus. Although difficult to observe directly in live cells, these subtle interactions can be discerned by their impact on the diffusion of membrane constituents. Here, we quantified the diffusion properties of a panel of structurally distinct lipid, lipid-anchored, and transmembrane (TM) probes in RBL mast cells by imaging fluorescence correlation spectroscopy (ImFCS). We developed a statistical analysis of data combined from many pixels over multiple cells to characterize differences in diffusion coefficients as small as 10%, which reflect differences in underlying interactions. We found that the distinctive diffusion properties of lipid probes can be explained by their dynamic partitioning into Lo-like proteolipid nanodomains, which encompass a major fraction of the membrane and whose physical properties are influenced by actin polymerization. Effects on diffusion of functional protein modules in both lipid--anchored and TM probes reflect additional complexity in steady state membrane organization. The contrast we observe between different probes diffusing through the same membrane milieu represents the dynamic resting steady state, which serves as a baseline for monitoring plasma membrane remodeling that occurs upon stimulation.
Assuntos
Membrana Celular/metabolismo , Mastócitos/metabolismo , Espectrometria de Fluorescência , Actinas/metabolismo , Animais , Linhagem Celular , Difusão , Proteínas de Fluorescência Verde/metabolismo , Imageamento Tridimensional , Imunoglobulina E/metabolismo , Lipídeos/química , Polimerização , Ratos , Receptores de IgE/metabolismoRESUMO
Epidermal growth factor receptor (EGFR) dysregulation is observed in many human cancers and is both a cause of oncogenesis and a target for chemotherapy. We previously showed that partial charge neutralization of the juxtamembrane (JX) region of EGFR via the EGFR R1-6 mutant construct induces constitutive receptor activation and transformation of NIH 3T3 cells, both from the plasma membrane and from the ER when combined with the ER-retaining L417H mutation (Bryant et al. in J Biol Chem 288:34930-34942, 2013). Here, we use chemical crosslinking and immunoblotting to show that these mutant constructs form constitutive, phosphorylated dimers in both the plasma membrane and the ER. Furthermore, we combine this electrostatic perturbation with conformationally-restricted receptor mutants to provide evidence that activation of EGFR R1-6 dimers requires functional coupling both between the EGFR extracellular dimerization arms and between intracellular tyrosine kinase domains. These findings provide evidence that the electrostatic charge of the JX region normally serves as a negative regulator of functional dimerization of EGFR.