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Lipoprotein particles (LPs) are excellent transporters and have been intensively studied in cardiovascular diseases, especially regarding parameters such as their class distribution and accumulation, site-specific delivery, cellular internalization, and escape from endo/lysosomal compartments. The aim of the present work is the hydrophilic cargo loading of LPs. As an exemplary proof-of-principle showcase, the glucose metabolism-regulating hormone, insulin, was successfully incorporated into high-density lipoprotein (HDL) particles. The incorporation was studied and verified to be successful using Atomic Force Microscopy (AFM) and Fluorescence Microscopy (FM). Single-molecule-sensitive FM together with confocal imaging visualized the membrane interaction of single, insulin-loaded HDL particles and the subsequent cellular translocation of glucose transporter type 4 (Glut4).
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Cholesterol is one of the main constituents of plasma membranes; thus, its supply is of utmost importance. This review covers the known mechanisms of cholesterol transfer from circulating lipoprotein particles to the plasma membrane, and vice versa. To achieve homeostasis, the human body utilizes cellular de novo synthesis and extracellular transport particles for supply of cholesterol and other lipids via the blood stream. These lipoprotein particles can be classified according to their density: chylomicrons, very low, low, and high-density lipoprotein (VLDL, LDL, and HDL, respectively). They deliver and receive their lipid loads, most importantly cholesterol, to and from cells by several redundant routes. Defects in one of these pathways (e.g., due to mutations in receptors) usually are not immediately fatal. Several redundant pathways, at least temporarily, compensate for the loss of one or more of them, but the defects trigger systemic diseases, such as atherosclerosis later on. Recently, intracellular membrane-membrane contact sites were shown to be involved in intracellular cholesterol transfer and the plasma membrane itself has been proposed to act as a binding site for lipoprotein-mediated cargo unloading.
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High-resolution imaging is essential for analysis of the steps and way stations of cargo transport in in vitro models of the endothelium. In this study, we demonstrate a microfluidic system consisting of two channels horizontally separated by a cell-growth-promoting membrane. Its design allows for high-resolution (down to single-molecule level) imaging using a high numerical aperture objective with a short working distance. To reduce optical aberrations and enable single-molecule-sensitive imaging, an observation window was constructed in the membrane via laser cutting with subsequent structuring using 3D multiphoton lithography for improved cell growth. The upper channel was loaded with endothelial cells under flow conditions, which showed polarization and junction formation. A coculture of human vascular endothelial cells with pericytes was developed that mimics the blood-brain barrier. Finally, this dual channel microfluidics system enabled 3D localization microscopy of the cytoskeleton and 3D single-molecule-sensitive tracing of lipoprotein particles.
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Barrera Hematoencefálica , Microfluídica , Técnicas de Cocultivo , Células Endoteliales , Humanos , PericitosRESUMEN
Lipid transfer from lipoprotein particles to cells is essential for lipid homeostasis. High-density lipoprotein (HDL) particles are mainly captured by cell membrane-associated scavenger receptor class B type 1 (SR-B1) from the bloodstream, while low-density and very-low-density lipoprotein (LDL and VLDL, respectively) particles are mostly taken up by receptor-mediated endocytosis. However, the role of the target lipid membrane itself in the transfer process has been largely neglected so far. Here, we study how lipoprotein particles (HDL, LDL, and VLDL) interact with synthetic lipid bilayers and cell-derived membranes and transfer their cargo subsequently. Employing cryo-electron microscopy, spectral imaging, and fluorescence (cross) correlation spectroscopy allowed us to observe integration of all major types of lipoprotein particles into the membrane and delivery of their cargo in a receptor-independent manner. Importantly, the biophysical properties of the target cell membranes change upon delivery of cargo. The concept of receptor-independent interaction of lipoprotein particles with membranes helps us to better understand lipoprotein particle biology and can be exploited for novel treatments of dyslipidemia diseases.
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Membrana Celular/metabolismo , Lipoproteínas/metabolismo , Transporte Biológico , Membrana Dobles de Lípidos/metabolismo , Microscopía de Fuerza AtómicaRESUMEN
We present the software platform 2CALM that allows for a comparative analysis of 3D localisation microscopy data representing protein distributions in two biological samples. The in-depth statistical analysis reveals differences between samples at the nanoscopic level using parameters such as cluster-density and -curvature. An automatic classification system combines multiplex and multi-level statistical approaches into one comprehensive parameter for similarity testing of the compared samples. We demonstrated the biological importance of 2CALM, comparing the protein distributions of CD41 and CD62p on activated platelets in a 3D artificial clot. Additionally, using 2CALM, we quantified the impact of the inflammatory cytokine interleukin-1ß on platelet activation in clots. The platform is applicable to any other cell type and biological system and can provide new insights into biological and medical applications.
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Plaquetas/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía/métodos , Modelos Biológicos , Trombosis/metabolismo , Humanos , Aprendizaje Automático , Selectina-P/metabolismo , Prueba de Estudio ConceptualRESUMEN
Cholesterol homeostasis is of central importance for life. Therefore, cells have developed a divergent set of pathways to meet their cholesterol needs. In this review, we focus on the direct transfer of cholesterol from lipoprotein particles to the cell membrane. More molecular details on the transfer of lipoprotein-derived lipids were gained by recent studies using phospholipid bilayers. While amphiphilic lipids are transferred right after contact of the lipoprotein particle with the membrane, the transfer of core lipids is restricted. Amphiphilic lipid transfer gains special importance in genetic diseases impairing lipoprotein metabolism like familial hypercholesterolemia. Taken together, these data indicate that there is a constant exchange of amphiphilic lipids between lipoprotein particles and the cell membrane.
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Membrana Celular/metabolismo , Colesterol/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Apolipoproteína B-100/metabolismo , Transporte Biológico , Colesterol/sangre , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Endocitosis , Humanos , Receptores de LDL/metabolismo , Receptores Depuradores de Clase B/metabolismoRESUMEN
Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles-exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles-neither with native miRNA nor artificially loaded with it-did not alter the cellular miRNA level.
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Lipoproteínas/metabolismo , MicroARNs/metabolismo , Transporte Biológico , Colesterol/metabolismo , Humanos , Lipoproteínas/aislamiento & purificación , MicroARNs/genética , Microfluídica , Control de Calidad , Transcripción Reversa/genéticaRESUMEN
The fundamental task of lipoprotein particles is extracellular transport of cholesterol, lipids, and fatty acids. Besides, cholesterol-rich apoB-containing lipoprotein particles (i.e., low density lipoprotein LDL) are key players in progression of atherosclerotic cardiovascular disease and are associated with familial hypercholesterolemia (FH). So far, lipoprotein particle binding to the cell membrane and subsequent cargo transfer is directly linked to the lipoprotein receptors on the target cell surface. However, our observations showed that lipoprotein particle cargo transport takes place even in the absence of the receptor. This finding suggests that an alternative mechanism for lipoprotein-particle/membrane interaction, besides the receptor-mediated one, exists. Here, we combined several complementary biophysical techniques to obtain a comprehensive view on the nonreceptor mediated LDL-particle/membrane. We applied a combination of atomic force and single-molecule-sensitive fluorescence microscopy (AFM and SMFM) to investigate the LDL particle interaction with membranes of increasing complexity. We observed direct transfer of fluorescently labeled amphiphilic lipid molecules from LDL particles into the pure lipid bilayer. We further confirmed cargo transfer by fluorescence cross-correlation spectroscopy (FCCS) and spectral imaging of environment-sensitive probes. Moreover, the integration of the LDL particle into the membranes was directly visualized by high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-EM). Overall, our data show that lipoprotein particles are able to incorporate into lipid membranes upon contact to transfer their cargo in the absence of specific receptors.
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Membrana Celular/ultraestructura , Enfermedad de la Arteria Coronaria/patología , Hiperlipoproteinemia Tipo II/metabolismo , Lipoproteínas LDL/química , Apolipoproteínas B/química , Fenómenos Biofísicos , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Enfermedad de la Arteria Coronaria/metabolismo , Microscopía por Crioelectrón , Progresión de la Enfermedad , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Humanos , Hiperlipoproteinemia Tipo II/patología , Membrana Dobles de Lípidos/química , Lipoproteínas LDL/farmacología , Lipoproteínas LDL/ultraestructura , Microscopía de Fuerza AtómicaRESUMEN
microRNAs (miRNAs) are post-transcriptional regulators of messenger RNA (mRNA), and transported through the whole organism by-but not limited to-lipoprotein particles. Here, we address the miRNA profile in serum and lipoprotein particles of healthy individuals in comparison with patients with uremia. Moreover, we quantitatively determined the cellular lipoprotein-particle-uptake dependence on the density of lipoprotein particle receptors and present a method for enhancement of the transfer efficiency. We observed a significant increase of the cellular miRNA level using reconstituted high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yielded no measurable effect. Thus, we conclude that no relevant effect of lipoprotein-particle-mediated miRNA-transfer exists under in vivo conditions though the miRNA profile of lipoprotein particles can be used as a diagnostic marker.
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Mobility of proteins and lipids plays a major role in physiological processes. Platforms which were developed to study protein interaction between immobilized and mobile proteins suffer from shortcomings such as fluorescence quenching or complicated fabrication methods. Here we report a versatile platform comprising immobilized histidine-tagged proteins and biotinylated proteins in a mobile phase. Importantly, multiphoton photolithography was used for easy and fast fabrication of the platform and allows, in principle, extension of its application to three dimensions. The platform, which is made up of functionalized polymer structures embedded in a mobile lipid bilayer, shows low background fluorescence and allows for mobility of arbitrary proteins.
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Acrilatos/química , Membrana Dobles de Lípidos/química , Polímeros/química , Proteínas/química , Difusión , Fluorescencia , Procesos FotoquímicosRESUMEN
BACKGROUND AND AIMS: Exchange of cholesterol between high-density lipoprotein (HDL) particles and cells is a key process for maintaining cellular cholesterol homeostasis. Recently, we have shown that amphiphilic cargo derived from HDL can be transferred directly to lipid bilayers. Here we pursued this work using a fluorescence-based method to directly follow cargo transfer from HDL particles to the cell membrane. METHODS: HDL was either immobilized on surfaces or added directly to cells, while transfer of fluorescent cargo was visualized via fluorescence imaging. RESULTS: In Chinese hamster ovary (CHO) cells expressing the scavenger receptor class B type 1 (SR-B1), transfer of amphiphilic cargo from HDL particles to the plasma membrane was observed immediately after contact, whereas hydrophobic cargo remained associated with the particles; about 60% of the amphiphilic cargo of surface-bound HDL was transferred to the plasma membrane. Essentially no cargo transfer was observed in cells with low endogenous SR-B1 expression. Interestingly, transfer of fluorescently-labeled cholesterol was also facilitated by using an artificial linker to bind HDL to the cell surface. CONCLUSIONS: Our data hence indicate that the tethering function of SR-B1 is sufficient for efficient transfer of free cholesterol to the plasma membrane.
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Antígenos CD36/metabolismo , Membrana Celular/metabolismo , HDL-Colesterol/sangre , Microscopía Fluorescente , Imagen Individual de Molécula/métodos , Animales , Células CHO , Cricetulus , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Transporte de Proteínas , Propiedades de Superficie , Factores de TiempoRESUMEN
T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.
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Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno/inmunología , Complejo CD3/química , Complejo CD3/inmunología , Ratones , Ratones TransgénicosRESUMEN
Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm.
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Actinas/metabolismo , Plaquetas/ultraestructura , Actinas/ultraestructura , Plaquetas/metabolismo , Células Cultivadas , Colorantes Fluorescentes/química , Humanos , Microscopía Fluorescente/métodos , Sensibilidad y EspecificidadRESUMEN
Multiphoton polymerization (MPP) enables 3D fabrication of micro- and nanoscale devices with complex geometries. Using MPP, we create a 3D platform for protein assays. Elevating the protein-binding sites above the substrate surface allows an optically sectioned readout, minimizing the inevitable background signal from nonspecific protein adsorption at the substrate surface. Two fluorescence-linked immunosorbent assays are demonstrated, the first one relying on streptavidin-biotin recognition and the second one on antibody recognition of apolipoprotein A1, a major constituent of high-density lipoprotein particles. Signal-to-noise ratios exceeding 1000 were achieved. The platform has high potential for 3D multiplexed recognition assays with an increased binding surface for on-chip flow cells.
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Proteínas/análisis , Adsorción , Anticuerpos , Biotina , Polimerizacion , EstreptavidinaRESUMEN
T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (µM range) to antigen when binding is measured in vitro(1). In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) (2). Measurements are based on Förster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.
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Antígenos/inmunología , Transferencia Resonante de Energía de Fluorescencia/métodos , Complejo Mayor de Histocompatibilidad/inmunología , Microscopía Fluorescente/métodos , Receptores de Antígenos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Células Presentadoras de Antígenos/inmunología , Humanos , Membrana Dobles de Lípidos , Datos de Secuencia Molecular , Unión Proteica , Linfocitos T/inmunologíaRESUMEN
In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues (1). This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light (2). Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode (3,4). They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.
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Membrana Dobles de Lípidos/química , Microscopía Fluorescente/métodos , Proteínas/química , Membrana Celular/química , Exocitosis , Vidrio/química , Imagen Molecular/métodos , Proteínas/análisisRESUMEN
Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in <1 min. Strikingly, active Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.
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Proteína X Asociada a bcl-2/metabolismo , Membrana Dobles de Lípidos , Microscopía/métodos , Unión Proteica , Espectrometría de Fluorescencia/métodos , Proteína X Asociada a bcl-2/químicaRESUMEN
Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90-140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per µm(2). We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.
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Células Presentadoras de Antígenos/inmunología , Activación de Linfocitos/inmunología , Nanopartículas/química , Linfocitos T/inmunología , Células Presentadoras de Antígenos/química , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Proteínas de la Membrana/inmunología , Sinapsis/inmunología , Linfocitos T/químicaRESUMEN
The binding of peptide-loaded major histocompatibility complex (pMHC) to the T cell receptor (TCR) represents the central step in T cell antigen recognition. It proceeds in the cell contact area between a T cell and an antigen-presenting cell termed the immunological synapse. An important and unresolved issue is how T cells discriminate between potentially harmful and harmless antigens. One limitation has been the difficulty to measure interaction parameters directly, that is, as they occur in the immunological synapse. Here we present a single-molecule approach to determine pMHC-TCR interaction kinetics in situ based on diffusion analysis of dye-labeled pMHC. We find synaptic off-rates >10-fold accelerated when compared to the dissociation of purified proteins measured in vitro.
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Biofisica/métodos , Antígenos de Histocompatibilidad Clase II/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Difusión , Humanos , Cinética , Modelos Inmunológicos , Unión Proteica/inmunología , Factores de TiempoRESUMEN
Diffusing membrane constituents are constantly exposed to a variety of forces that influence their stochastic path. Single molecule experiments allow for resolving trajectories at extremely high spatial and temporal accuracy, thereby offering insights into en route interactions of the tracer. In this review we discuss approaches to derive information about the underlying processes, based on single molecule tracking experiments. In particular, we focus on a new versatile way to analyze single molecule diffusion in the absence of a full analytical treatment. The method is based on comprehensive comparison of an experimental data set against the hypothetical outcome of multiple experiments performed on the computer. Since Monte Carlo simulations can be easily and rapidly performed even on state-of-the-art PCs, our method provides a simple way for testing various - even complicated - diffusion models. We describe the new method in detail, and show the applicability on two specific examples: firstly, kinetic rate constants can be derived for the transient interaction of mobile membrane proteins; secondly, residence time and corral size can be extracted for confined diffusion.