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We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 µm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.
Assuntos
Gotículas Lipídicas , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/química , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Recuperação de Fluorescência Após Fotodegradação/métodos , Humanos , Citometria de Fluxo/métodos , Espectrometria de Fluorescência/métodosRESUMO
The diffusion and reorganization of phospholipids and membrane-associated proteins are fundamental for cellular function. Fluorescence cross-correlation spectroscopy (FCCS) measures diffusion and molecular interactions at nanomolar concentration in biological systems. We have developed an economical method to simultaneously monitor diffusion and complexation with the use of super-continuum laser and spectral deconvolution from a single detector. Customizable excitation wavelengths were chosen from the wide-band source and spectral fitting of the emitted light revealed the interactions for up to four chromatically overlapping fluorophores simultaneously. This method was applied to perform four-color FCCS that we demonstrated with polystyrene nanoparticles, lipid vesicles, and membrane-bound molecules. Up to four individually customizable excitation channels were selected from the broad-spectrum fiber laser to excite the diffusers within a diffraction-limited spot. The fluorescence emission passed through a cleanup filter and a dispersive prism prior to being collected by a sCMOS or EMCCD camera with up to 1.8 kHz frame rates. The emission intensity versus time of each fluorophore was extracted through a linear least-square fitting of each camera frame and temporally correlated via custom software. Auto- and cross-correlation functions enabled the measurement of the diffusion rates and binding partners. We have measured the induced aggregation of nanobeads and lipid vesicles in solution upon increasing the buffer salinity. Because of the adaptability of investigating four fluorophores simultaneously with a cost-effective method, this technique will have wide application for examining macromolecular complex formation in model and living systems.
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The mechanisms by which the lipid droplet (LD) membrane is remodeled in concert with the activation of lipolysis incorporate a complex interplay of proteins, phospholipids, and neutral lipids. Model LDs (mLDs) provide an isolated, purified system for testing the mechanisms by which the droplet composition, size, shape, and tension affects triglyceride metabolism. Described here are methods of making and testing mLDs ranging from 0.1 to 40 µm diameter with known composition. Methods are described for imaging mLDs with high-resolution microscopy during buffer exchanges for the measurement of membrane binding, diffusion, and tension via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), pendant droplet tensiometry, and imaging flow cytometry. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes in triglyceride metabolism.
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Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. Here, we developed a fluorescence resonance energy transfer (FRET) sensor for LC-acyl-CoAs based on the allosterically regulated interaction between α/ß hydrolase domain-containing 5 (ABHD5) and Perilipin 5. The genetically encoded sensor rapidly detects intracellular LC-acyl-CoAs generated from exogenous and endogenous fatty acids (FAs), as well as synthetic ABHD5 ligands. Stimulation of lipolysis in brown adipocytes elevated intracellular LC-acyl-CoAs in a cyclic fashion, which was eliminated by inhibiting PNPLA2 (ATGL), the major triglyceride lipase. Interestingly, inhibition of LC-acyl-CoA transport into mitochondria elevated intracellular LC-acyl-CoAs and dampened their cycling. Together, these observations reveal an intimate feedback control between LC-acyl-CoA generation from lipolysis and utilization in mitochondria. We anticipate that this sensor will be an important tool to dissect intracellular LC-acyl-CoA dynamics as well to discover novel synthetic ABHD5 ligands.
Assuntos
Acil Coenzima A , Transferência Ressonante de Energia de Fluorescência , Acil Coenzima A/metabolismo , Lipólise/fisiologia , Lipase/genética , Ácidos GraxosRESUMO
The diffusion and reorganization of phospholipids and membrane-associated proteins are fundamental for cellular function. Fluorescence cross-correlation spectroscopy (FCCS) measures the diffusion and molecular interactions at nanomolar concentration in biological systems. We have developed a novel, economical method to simultaneously monitor diffusion and oligomerization with the use of super-continuum laser and spectral deconvolution from a single detector. Customizable excitation wavelengths were chosen from the wide-band source and spectral fitting of the emitted light revealed the interactions for up to four spectrally overlapping fluorophores simultaneously. This method was applied to perform four-color FCCS, as demonstrated with polystyrene nanoparticles, lipid vesicles, and membrane-bound molecules. Up to four individually customizable excitation channels were selected from the broad-spectrum fiber laser to excite the diffusers within a diffraction-limited spot. The fluorescence emission passed through a cleanup filter and a dispersive prism prior to being collected by a sCMOS or EMCCD camera with up to 10 kHz frame rates. The emission intensity versus time of each fluorophore was extracted through a linear least-square fitting of each camera frame and temporally correlated via custom software. Auto- and cross-correlation functions enabled the measurement of the diffusion rates and binding partners. We have measured the induced aggregation of nanobeads and lipid vesicles in solution upon increasing the buffer salinity. Because of the adaptability of investigating four fluorophores simultaneously with a cost-effective method, this technique will have wide application for examining complex homo- and heterooligomerization in model and living systems.
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The precise spatiotemporal control of nanoscale membrane shape and composition is the result of a complex interplay of individual and collective molecular behaviors. Here, we employed single-molecule localization microscopy and computational simulations to observe single-lipid diffusion and sorting in model membranes with varying compositions, phases, temperatures, and curvatures. Supported lipid bilayers were created over 50-nm-radius nanoparticles to mimic the size of naturally occurring membrane buds, such as endocytic pits and the formation of viral envelopes. The curved membranes recruited liquid-disordered lipid phases while altering the diffusion and sorting of tracer lipids. Disorder-preferring fluorescent lipids sorted to and experienced faster diffusion on the nanoscale curvature only when embedded in a membrane capable of sustaining lipid phase separation at low temperatures. The curvature-induced sorting and faster diffusion even occurred when the sample temperature was above the miscibility temperature of the planar membrane, implying that the nanoscale curvature could induce phase separation in otherwise homogeneous membranes. Further confirmation and understanding of these results are provided by continuum and coarse-grained molecular dynamics simulations with explicit and spontaneous curvature-phase coupling, respectively. The curvature-induced membrane compositional heterogeneity and altered dynamics were achieved only with a coupling of the curvature with a lipid phase separation. These cross-validating results demonstrate the complex interplay of lipid phases, molecular diffusion, and nanoscale membrane curvature that are critical for membrane functionality.
Assuntos
Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Temperatura , Difusão , Transporte Proteico , Membrana CelularRESUMO
Alpha/beta hydrolase domain-containing 5 (ABHD5), also termed CGI-58, is the key upstream activator of adipose triglyceride lipase (ATGL), which plays an essential role in lipid metabolism and energy storage. Mutations in ABHD5 disrupt lipolysis and are known to cause the Chanarin-Dorfman syndrome. Despite its importance, the structure of ABHD5 remains unknown. In this work, we combine computational and experimental methods to build a 3D structure of ABHD5. Multiple comparative and machine learning-based homology modeling methods are used to obtain possible models of ABHD5. The results from Gaussian accelerated molecular dynamics and experimental data of the apo models and their mutants are used to select the most likely model. Moreover, ensemble docking is performed on representative conformations of ABHD5 to reveal the binding mechanism of ABHD5 and a series of synthetic ligands. Our study suggests that the ABHD5 models created by deep learning-based methods are the best candidate structures for the ABHD5 protein. The mutations of E41, R116, and G328 disturb the hydrogen bonding network with nearby residues and suppress membrane targeting or ATGL activation. The simulations also reveal that the hydrophobic interactions are responsible for binding sulfonyl piperazine ligands to ABHD5. Our work provides fundamental insight into the structure of ABHD5 and its ligand-binding mode, which can be further applied to develop ABHD5 as a therapeutic target for metabolic disease and cancer.
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Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.
Assuntos
Toxina da Cólera/metabolismo , Receptores de Superfície Celular/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , EndocitoseRESUMO
Single-particle tracking (SPT) experiments of lipids and membrane proteins provide a wealth of information about the properties of biomembranes. Careful analysis of SPT trajectories can reveal deviations from ideal Brownian behavior. Among others, this includes confinement effects and anomalous diffusion, which are manifestations of both the nanoscale structure of the underlying membrane and the structure of the diffuser. With the rapid increase in temporal and spatial resolution of experimental methods, a new aspect of the motion of the particle, namely, anisotropic diffusion, might become relevant. This aspect that so far received only little attention is the anisotropy of the diffusive motion and may soon provide an additional proxy to the structure and topology of biomembranes. Unfortunately, the theoretical framework for detecting and interpreting anisotropy effects is currently scattered and incomplete. Here, we provide a computational method to evaluate the degree of anisotropy directly from molecular dynamics simulations and also point out a way to compare the obtained results with those available from SPT experiments. In order to probe the effects of anisotropic diffusion, we performed coarse-grained molecular dynamics simulations of peripheral and integral membrane proteins in flat and curved bilayers. In agreement with the theoretical basis, our computational results indicate that anisotropy can persist up to the rotational relaxation time [τ=(2Dr)-1], after which isotropic diffusion is observed. Moreover, the underlying topology of the membrane bilayer can couple with the geometry of the particle, thus extending the spatiotemporal domain over which this type of motion can be detected.
Assuntos
Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Anisotropia , DifusãoRESUMO
Phase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. Traditionally, SPT is sensitive to diffuser aggregation, whereas the diffusion rates reported by FCS are unaffected by the presence of immobile aggregates. Within this manuscript, we report (1) improved single-particle tracking analysis of lipid diffusion, (2) comparison and consistency between diffusion measurement methods for non-Brownian diffusers, and (3) the application of these methods to measure the phase, temperature, and composition dependencies in lipid diffusion. We demonstrate improved SPT analysis methods that yield consistent FCS and SPT diffusion results even when most fluorescent lipids are frequently confined within aggregates within the membrane. With varying membrane composition and temperature, we demonstrate differences in diffusion between the Ld and Lo phases of SLBs.
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Bicamadas Lipídicas/química , Lipídeos/química , Estrutura Molecular , Espectrometria de Fluorescência , TemperaturaRESUMO
AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.
Assuntos
Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Toxina da Cólera/farmacologia , Receptores de Superfície Celular/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Receptores de Superfície Celular/genéticaRESUMO
Nanoscale membrane curvature in cells is critical for endocytosis/exocytosis and membrane trafficking. However, the biophysical ramifications of nanoscale membrane curvature on the behavior of lipids remain poorly understood. Here, we created an experimental model system of membrane curvature at a physiologically-relevant scale and obtained nanoscopic information on single-lipid distributions and dynamics. Supported lipid bilayers were created over 50 and 70â¯nm radius nanoparticles to create membrane buds. Single-molecule localization microscopy was performed with diverse mixtures of fluorescent and non-fluorescent lipids. Variations in lipid acyl tales length, saturation, head-group, and fluorescent labeling strategy were tested while maintaining a single fluid lipid phase throughout the membrane. Monte Carlo simulations were used to fit our experimental results and quantify the effects of curvature on the lipid diffusion and sorting. Whereas varying the composition of the non-fluorescent lipids yielded minimal changes to the curvature effects, the labeling strategy of the fluorescent lipids yielded highly varying effects of curvature. Most conditions yield single-population Brownian diffusion throughout the membrane; however, curvature-induced lipid sorting, slowing, and aggregation were observed in some conditions. Head-group labeled lipids such as DPPE-Texas Red and POPE-Rhodamine diffused >2.4× slower on the curved vs. the planar membranes; tail-labeled lipids such as NBD-PPC, TopFluor-PPC, and TopFluor-PIP2, as well as DiIC12 and DiIC18 displayed no significant changes in diffusion due to the membrane curvature. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.
Assuntos
Membrana Celular/fisiologia , Bicamadas Lipídicas/química , Lipídeos/química , Difusão , Método de Monte Carlo , Nanopartículas/química , Transporte Proteico/fisiologia , Imagem Individual de Molécula/métodosRESUMO
Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.
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For endocytosis and exocytosis, membranes transition among planar, budding, and vesicular topographies through nanoscale reorganization of lipids, proteins, and carbohydrates. However, prior attempts to understand the initial stages of nanoscale bending have been limited by experimental resolution. Through the implementation of polarized localization microscopy, this article reports the inherent membrane bending capability of cholera toxin subunit B (CTxB) in quasi-one-component-supported lipid bilayers. Membrane buds were first detected with <50 nm radius, grew to >200 nm radius, and extended into longer tubules with dependence on the membrane tension and CTxB concentration. Compared to the concentration of the planar-supported lipid bilayers, CTxB was (12 ± 4)× more concentrated on the positive curvature top and (26 ± 11)× more concentrated on the negative Gaussian curvature neck of the nanoscale membrane buds. CTxB is frequently used as a marker for liquid-ordered lipid phases; however, the coupling between CTxB and membrane bending provides an alternate understanding of CTxB-induced membrane reorganization. These findings allow for the reinterpretation of prior observations by correlating CTxB clustering and diffusion to CTxB-induced membrane bending. Single-particle tracking was performed on single lipids and CTxB to reveal the correlations among single-molecule diffusion, CTxB accumulation, and membrane topography. Slowed lipid and CTxB diffusion was observed at the nanoscale bud locations, suggesting a local increase in the effective membrane viscosity or molecular crowding upon membrane bending. These results suggest inherent CTxB-induced membrane bending as a mechanism for initiating CTxB internalization in cells that could be independent of clathrin, caveolin, actin, and lipid phase separation.
Assuntos
Toxina da Cólera/farmacologia , Bicamadas Lipídicas/química , Toxina da Cólera/química , Toxina da Cólera/metabolismo , Difusão , Endocitose , Microscopia de Fluorescência , Microscopia de Polarização , Nanopartículas , Imagem Individual de MoléculaRESUMO
The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initiation of membrane bending occurs at a length scale that is irresolvable by most superresolution optical microscopy methods. Here, we report the development of polarized localization microscopy (PLM), a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting. PLM combines polarized total internal reflection fluorescence microscopy and single-molecule localization microscopy to reveal membrane orientation with subdiffraction-limited resolution without reducing localization precision by point spread function manipulation. Membrane curvature detection with PLM requires fewer localization events to detect curvature than three-dimensional single-molecule localization microscopy (e.g., photoactivated localization microscopy or stochastic optical reconstruction microscopy), which enables curvature detection 10× faster via PLM. With rotationally confined lipophilic fluorophores and the polarized incident fluorescence excitation, membrane-bending events are revealed with superresolution. Engineered hemispherical membrane curvature with a radius ≥24 nm was detected with PLM, and individual fluorophore localization precision was 13 ± 5 nm. Further, deciphering molecular mobility as a function of membrane topology was enabled. The diffusion coefficient of individual DiI molecules was 25 ± 5× higher in planar supported lipid bilayers than within nanoscale membrane curvature. Through the theoretical foundation and experimental demonstration provided here, PLM is poised to become a powerful technique for revealing the underlying biophysical mechanisms of membrane bending at physiological length scales.
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Bicamadas Lipídicas/química , Microscopia de Fluorescência , Microscopia de Polarização , Imagem Individual de Molécula , Nanopartículas , Lipossomas Unilamelares/químicaRESUMO
The addition of trace molecules into membranes can significantly alter the morphology of the co-existing liquid phases and lipid phase transition temperature. Membrane additives may affect lipid phase dynamics through preferentially partitioning to the boundary between lipid phases or preferentially mixing into one lipid phase. The characteristic differences between these mechanisms are demonstrated here in a minimalistic nearest neighbor model to provide a framework for how slight changes to membrane composition may affect lipid-phase-dependent processes, such as lipid-raft formation, immunological signaling, and molecular sorting preceding endocytosis with coexisting liquid phases. Within the low mole fractions explored here (≤3 mol%), increasing the additive concentration linearly changed the phase miscibility temperature. Rotationally asymmetric Janus particles reduced the miscibility transition temperature for all fractions and degree of phase polarization. Rotationally symmetric additives, however, either increased or decreased the phase miscibility temperature depending on the phase preference of the additive. While most experimental molecules may contain aspects of both of these idealized additives, this model provides a broad framework to quantify the effects of membrane additives in regard to lipid phase preference, lipid-raft association, and contribution to lipid phase-dependent molecular sorting.
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Bicamadas Lipídicas/química , Lipídeos/química , Modelos Químicos , Transição de Fase , Temperatura de TransiçãoRESUMO
Porosomes are secretory portals located at the cell plasma membrane involved in the regulated release of intravesicular contents from cells. Porosomes have been immunoisolated from a number of cells including the exocrine pancreas and neurons, biochemically characterized, and functionally reconstituted into an artificial lipid membrane. In the current study, the proteome of the porosome complex in mouse insulinoma Min6 cells was determined, demonstrating among other proteins, the presence of 30 core proteins including the heat shock protein Hsp90. Half maximal inhibition of Hsp90 using the specific inhibitor 17-demethoxy-17-(2-prophenylamino) geldanamycin, results in the loss of proteins, including the calcium-transporting ATPase type 2C and the potassium channel subfamily K member 2 from the Min6 porosome. This loss of porosome proteins is reflected in the observed inhibition of glucose stimulated insulin release from Min6 cells exposed to the Hsp90 specific inhibitor. Results from the study implicate Hsp90 in the assembly and function of the porosome complex. BIOLOGICAL SIGNIFICANCE: In the present study, the porosome proteome in the insulin-secreting mouse ß-cell line Min6 has been determined. Nearly 30 core proteins including the heat shock protein Hsp90 are found to compose the Min6 porosome complex. Results from the study implicate Hsp90 in the assembly of the Min6 porosome. These new findings will facilitate understanding of the porosome assembly and its function in insulin secretion.
Assuntos
Proteínas de Choque Térmico HSP90/fisiologia , Células Secretoras de Insulina/metabolismo , Proteoma/metabolismo , Vesículas Secretórias/metabolismo , Animais , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/química , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/ultraestrutura , Espectrometria de Massas , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Proteoma/análise , Proteoma/efeitos dos fármacos , Vesículas Secretórias/química , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/fisiologiaRESUMO
The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (FcεRI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods.
Assuntos
Membrana Celular/química , Espectrometria de Fluorescência/métodos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Análise de Elementos Finitos , Bicamadas Lipídicas/química , Mastócitos/citologia , RatosRESUMO
We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin-a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 10(8) with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.