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[Methods in Molecular Biology] BCL-2 Family Proteins Volume 1877 (Methods and Protocols) || Quantification of the Interactions Between BCL-2 Proteins by Fluorescence Correlation Spectroscopy
摘要： The proteins of the Bcl-2 family regulate apoptosis by forming a complex interaction network whose output determines whether mitochondrial outer membrane permeabilization is executed. Quanti?cation of complex formation between Bcl-2 proteins in solution and in membranes is therefore key to understand how the hierarchy of interactions controls cell death induction. Fluorescence correlation spectroscopy (FCS) is a noninvasive, nondestructive method to investigate the mobility and the association of ?uorescently labeled biomolecules that has provided useful insight into the binding af?nity of the Bcl-2 interactome. FCS is based on the detection of ?uorescence ?uctuations caused by the diffusion of individual molecules through a very tiny observation volume of the detection system. Scanning FCS (SFCS) solves some of the practical challenges of acquiring FCS in membranes and expands the application scope of the method. In this chapter, we explain the principle of FCS and describe protocols how it can be used to quantify interactions between Bcl-2 proteins in solution and in model membrane systems.
关键词： FCCS,Scanning FCCS,Bcl-xL,cBid,Bcl-2,Fluorescence correlation spectroscopy,FCS,Bax
更新于2025-11-21 11:08:18
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Diffusion Behavior of Differently Charged Molecules in Self-Assembled Organic Nanotubes Studied Using Imaging Fluorescence Correlation Spectroscopy
摘要： The diffusion behavior of fluorescent molecules within bolaamphiphile-based organic nanotubes (ONTs) was systematically investigated using imaging fluorescence correlation spectroscopy (imaging FCS). Anionic sulforhodamine B (SRB), zwitterionic/cationic rhodamine B (RB) or cationic rhodamine 123 (R123) was loaded into ONTs having cylindrical hollow structures (ca. 10 nm in inner diameter) with amine and glucose groups on the inner and outer surfaces, respectively. Wide-field fluorescence video microscopy was used to acquire imaging FCS data for dye-doped ONTs in aqueous solutions of different ionic strengths (1 – 500 mM) at different pH (3.4 – 8.4). The diffusion behavior of these dyes was discussed on the basis of their apparent diffusion coefficients (D) that were determined by autocorrelating the time transient of fluorescence intensity at each pixel on an ONT. Molecular diffusion in the ONTs was significantly slowed by molecule-nanotube interactions, as shown by the very small D (10-1 – 10-2 μm2/s). The pH-dependence of D revealed that dye diffusion was basically controlled by electrostatic interactions associated with the protonation of the amine groups on the ONT inner surface. The pH-dependent change in D was observed over a wide pH range, possibly due to electrostatically induced variations in the pKa of the densely packed ammonium ions on the ONT inner surface. On the other hand, the influence of ionic strength on D was relatively unclear, suggesting the involvement of non-coulombic interactions with the ONTs in molecular diffusion. Importantly, individual ONTs of different lengths (1 – 5 μm) afforded similar diffusion coefficients for each type of dye at each solution condition, implying that the properties of ONTs were uniform in terms of solute loading and release. These results highlight the characteristics of molecular diffusion behavior within the ONTs, and will help in the design of organic nanotubes better suited for use as drug vehicles and contaminant adsorbents.
关键词： Electrostatic Interactions,Diffusion Behavior,Ionic Strength,pH-dependence,Imaging Fluorescence Correlation Spectroscopy,Organic Nanotubes
更新于2025-11-19 16:56:42
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High-Performance Chromatographic Characterization of Surface Chemical Heterogeneities of Fluorescent Organic–Inorganic Hybrid Core–Shell Silica Nanoparticles
摘要： In contrast to small-molar-mass compounds, detailed structural investigations of inorganic core–organic ligand shell hybrid nanoparticles remain challenging. The assessment of batch-reaction-induced heterogeneities of surface chemical properties and their correlation with particle size has been a particularly long-standing issue. Applying a combination of high-performance liquid chromatography (HPLC) and gel permeation chromatography (GPC) to ultra-small (<10 nm diameter) poly(ethylene glycol)-coated (PEGylated) fluorescent core–shell silica nanoparticles, we elucidate here previously unknown surface heterogeneities resulting from varying dye conjugation to nanoparticle silica cores and surfaces. Heterogeneities are predominantly governed by dye charge, as corroborated by molecular dynamics simulations. We demonstrate that this insight enables the development of synthesis protocols to achieve PEGylated and targeting ligand-functionalized PEGylated silica nanoparticles with dramatically improved surface chemical homogeneity, as evidenced by single-peak HPLC chromatograms. Because surface chemical properties are key to all nanoparticle interactions, we expect these methods and fundamental insights to become relevant to a number of systems for applications, including bioimaging and nanomedicine.
关键词： surface chemistry heterogeneity,high-performance liquid chromatography,nanoparticle characterization,nanoparticle surface functionalization,fluorescence correlation spectroscopy,molecular dynamics,nanoparticle heterogeneity
更新于2025-09-23 15:23:52
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Monomeric cohesin state revealed by live‐cell single‐molecule spectroscopy
摘要： The cohesin complex plays an important role in the maintenance of genome stability. Cohesin is composed of four core subunits and a set of regulatory subunits that interact with the core subunits. Less is known about cohesin dynamics in live cells and on the contribution of individual subunits to the overall complex. Understanding the tethering mechanism of cohesin is still a challenge, especially because the proposed mechanisms are still not conclusive. Models proposed to describe tethering depend on either the monomeric cohesin ring or a cohesin dimer. Here, we investigate the role of cohesin dynamics and stoichiometry in live yeast cells at single-molecule resolution. We explore the effect of regulatory subunit deletion on cohesin mobility and found that depletion of different regulatory subunits has opposing effects. Finally, we show that cohesin exists mostly as a canonical monomer throughout the cell cycle, and its monomeric form is independent of its regulatory factors. Our results demonstrate that single-molecule tools have the potential to provide new insights into the cohesin mechanism of action in live cells.
关键词： cohesin,photon counting histogram,chromosome,fluorescence correlation spectroscopy,SMC complexes
更新于2025-09-16 10:30:52
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Controlling Surface Chemical Heterogeneities of Ultrasmall Fluorescent Core–Shell Silica Nanoparticles as Revealed by High-Performance Liquid Chromatography
摘要： Ultrasmall (diameter below 10 nm) fluorescent core?shell silica nanoparticles have garnered increasing attention in recent years as a result of their high brightness and favorable biodistribution properties important for applications including bioimaging and nanomedicine. Here, we present an in-depth study that provides new insights into the physical parameters that govern full covalent fluorescent dye encapsulation within the silica core of poly(ethylene glycol)-coated core?shell silica nanoparticles referred to as Cornell prime dots (C′ dots). We use a combination of high-performance liquid chromatography (HPLC), gel-permeation chromatography, and fluorescence correlation spectroscopy to monitor the result of ammonia concentration in the synthesis of C′ dots from negatively and positively charged versions of near-infrared dyes Cy5 and Cy5.5. HPLC, in particular, allows the distinction between cases of full versus partial dye encapsulation in the silica particle core leading to surface chemical heterogeneities in the form of hydrophobic surface patches, which, in turn, modulate biological response in ferroptotic cell death experiments. Our results demonstrate that there is a complex interplay between dye?dye and dye?silica cluster interactions originally formed in the sol?gel synthesis governing optimal dye encapsulation. We expect that the reduced surface chemical heterogeneities will make the resulting nanoparticles attractive for a number of applications in biology and medicine.
关键词： gel-permeation chromatography,ferroptotic cell death,high-performance liquid chromatography,fluorescent core?shell silica nanoparticles,fluorescence correlation spectroscopy
更新于2025-09-11 14:15:04
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Self‐assembly of lipid rafts revealed by fluorescence correlation spectroscopy in living breast cancer cells
摘要： In the study, imaging this polarisation Lipids and proteins in the plasma membrane are laterally heterogeneous and formalised as lipid rafts featuring unique biophysical properties. However, self-assembly mechanism of lipid raft cannot be revealed even its physical properties and components were determined in specific physiological two-photon processes. generalised and fluorescence correlation spectroscopy were used to study the fusion of lipid rafts through the membrane phase and the lateral diffusion of lipids in living breast cancer cells. A self-assembly model of lipid rafts and lipid associated with to phase was membrane demonstrate the lipid sorting ability of lipid rafts in the plasma membrane. The results showed that the increased proportion of slow subdiffusion of GM1-binding cholera toxin B-subunit (CT-B) is accompanied with an increased liquid-ordered domain during the β-estradiol-induced fusion of lipid rafts. And slow subdiffusion of CT-B was vanished with the depletion of lipid rafts. Whereas the dialkylindocarbocyanine (DiIC18) diffusion was not specifically regulated by lipid rafts. This study will open up a new insight for uncovering the self-assembly of lipid rafts in specific pathophysiological processes.
关键词： Lipid rafts,Self-assembly,Membrane phase,Fluorescence correlation spectroscopy,Anomalous diffusion
更新于2025-09-11 14:15:04
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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
摘要： A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
关键词： Issue 142,cell-cell adhesion,fluorescence correlation spectroscopy,cell-cell interactions,Protein-protein interactions,number and brightness,fluorescence fluctuation spectroscopy,Biochemistry,N&B
更新于2025-09-09 09:28:46
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High-speed velocimetry in microfluidic protein mixers using confocal fluorescence decay microscopy
摘要： A method to measure fluid speeds on the order of 10,000 mm/s in microchannels is presented. A microfluidic protein mixer is manufactured with a 170 μm microscope coverslip bottom that interfaces with a confocal florescence microscope using a water-immersed Olympus UPLSAPO 60XW objective to create a diffraction-limited confocal volume. A diode laser with a repetition rate of 1 MHz is used to study Poiseuille flows at average speeds of 5000, 6000 and 7000 mm/s by exciting tris(2,2′-bipyridine)ruthenium(II) hexafluorophosphate solution at 1 μmol/L concentration flowing through the micro-mixer in the confocal volume. Decays collected using a time-correlated single photon counting card at each grid point are characterized by the first moment of the decay and curve fitted with the theoretical Poiseuille flow solutions. It was found that curve fitting with higher average speeds results in lower errors. A fluorescence correlation study was then carried out at different depths in the micro-mixer to understand the raw data profiles observed using the diode laser. A mixing study was then carried out using a Ti-Sapphire laser with a repetition rate of 3.8 MHz. A Poiseuille flow at 7000 mm/s was measured using the Ti-Sapphire laser and then curve fitted to the theoretical Poiseuille flow solution. The curve fit was then applied to the complicated flow region to determine speed. Results of the experimental mixing study are also compared to direct numerical simulation results.
关键词： Microfluidic protein mixers,High-speed velocimetry,Poiseuille flows,Fluorescence correlation spectroscopy,Confocal fluorescence decay microscopy
更新于2025-09-09 09:28:46
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Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy
摘要： The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.
关键词： photo-induced DNA damage,surface-integrated fluorescence correlation spectroscopy (SI-FCS),reactive oxygen species,super-resolution microscopy,DNA-PAINT
更新于2025-09-09 09:28:46
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Nanoparticles at Biomimetic Interfaces: A Combined Experimental and Simulation Study on Charged Gold Nanoparticles/Lipid Bilayers Interfaces
摘要： The poor understanding of the interaction of nanomaterials with biologically relevant interfaces is recognized as one of the major issues currently limiting the development of nanomedicine. The central purpose of this study is to compare experimental (Confocal Microscopy, Fluorescence Correlation Spectroscopy, X-ray Reflectivity) and computational (Molecular Dynamics simulations) results to thoroughly describe the interaction of cationic gold nanoparticles (AuNP) with mixed zwitterionic/anionic lipid membranes. The adhesion of AuNPs to the lipid membrane is investigated on different length scales from a structural and dynamical point of view; with this approach, a series of complex phenomena, spanning from lipid extraction, localized membrane disruption, lateral phase separation and slaved diffusion, are characterized and interpreted from a molecular level to macroscopic observations.
关键词： Nanoparticles,Gold Nanoparticles,Molecular Dynamics,X-ray Reflectivity,Fluorescence Correlation Spectroscopy,Lipid Bilayers,Biomimetic Interfaces,Confocal Microscopy
更新于2025-09-04 15:30:14