Live Cell FRET Imaging Reveals Amyloid ß-Peptide Oligomerization in Hippocampal Neurons.

Amyloid ß-peptide (Aß) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of Aß oligomerization in neurons still need to be revealed. Förster resonance energy transfer (FRET) is a simple but effective way to study molecular interactions. Here, we used a confocal microscope with a sensitive Airyscan detector for FRET detection. By live cell FRET imaging, we detected Aß42 oligomerization in primary neurons. The neurons were incubated with fluorescently labeled Aß42 in the cell culture medium for 24 h. Aß42 were internalized and oligomerized in the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of Aß42 were significantly higher than for Aß40. These findings provide a better understanding of Aß42 oligomerization in neurons.

Interactions of the Lysosomotropic Detergent O-Methyl-Serine Dodecylamide Hydrochloride (MSDH) with Lipid Bilayer Membranes-Implications for Cell Toxicity.

O-methyl-serine dodecylamine hydrochloride (MSDH) is a detergent that accumulates selectively in lysosomes, a so-called lysosomotropic detergent, with unexpected chemical properties. At physiological pH, it spontaneously forms vesicles, which disassemble into small aggregates (probably micelles) below pH 6.4. In this study, we characterize the interaction between MSDH and liposomes at different pH and correlate the findings to toxicity in human fibroblasts. We find that the effect of MSDH on lipid membranes is highly pH-dependent. At neutral pH, the partitioning of MSDH into the liposome membrane is immediate and causes the leakage of small fluorophores, unless the ratio between MSDH and lipids is kept low. At pH 5, the partitioning of MSDH into the membrane is kinetically impeded since MSDH is charged and a high ratio between MSDH and the lipids is required to permeabilize the membrane. When transferred to cell culture conditions, the ratio between MSDH and plasma membrane lipids must therefore be low, at physiological pH, to maintain plasma membrane integrity. Transmission electron microscopy suggests that MSDH vesicles are taken up by endocytosis. As the pH of the endosomal compartment progressively drops, MSDH vesicles disassemble, leading to a high concentration of increasingly charged MSDH in small aggregates inside the lysosomes. At sufficiently high MSDH concentrations, the lysosome is permeabilized, the proteolytic content released to the cytosol and apoptotic cell death is induced.

Potentials and pitfalls of inverse fluorescence correlation spectroscopy.

Inverse Fluorescence Correlation Spectroscopy (iFCS) is a variant of FCS where unlabeled particles in solution, or domains in membranes, displace their surrounding, signal-generating molecules and thereby generate fluctuations. iFCS has to date been applied to unlabeled as well as labeled particles and protein molecules, using fluorescence as well as Raman scattering as a signal source, in diffraction-limited detection volumes as well as in nano-wells, and on fixed surfaces as well as in lipid bilayers. This review describes these applications and discusses the potentials and pitfalls when using iFCS.

A Protein-Based Encapsulation System with Calcium-Controlled Cargo Loading and Detachment.

Protein-based encapsulation systems have a wide spectrum of applications in targeted delivery of cargo molecules and for chemical transformations in confined spaces. By engineering affinity between cargo and container proteins it has been possible to enable the efficient and specific encapsulation of target molecules. Missing in current approaches is the ability to turn off the interaction after encapsulation to enable the cargo to freely diffuse in the lumen of the container. Separation between cargo and container is desirable in drug delivery applications and in the use of capsids as catalytic nanoparticles. We describe an encapsulation system based on the hepatitis B virus capsid in which an engineered high-affinity interaction between cargo and capsid proteins can be modulated by Ca2+ . Cargo proteins are loaded into capsids in the presence of Ca2+ , while ligand removal triggers unbinding inside the container. We observe that confinement leads to hindered rotation of cargo inside the capsid. Application of the designed container for catalysis was also demonstrated by encapsulation of an enzyme with ß-glucosidase activity.

Spontaneous Vesiculation and pH-Induced Disassembly of a Lysosomotropic Detergent: Impacts on Lysosomotropism and Lysosomal Delivery.

Lysosomotropic detergents (LDs) selectively rupture lysosomal membranes through mechanisms that have yet to be characterized. A consensus view, currently, holds that LDs, which are weakly basic, diffuse across cellular membranes as monomers in an uncharged state, and via protonation in the acidic lysosomal compartment, they become trapped, accumulate, and subsequently solubilize the membrane and induce lysosomal membrane permeabilization. Here we demonstrate that the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) spontaneously assembles into vesicles at, and above, cytosolic pH, and that the vesicles disassemble as the pH reaches 6.4 or lower. The aggregation commences at concentrations below the range of those used in cell studies. Assembly and disassembly of the vesicles was studied via dynamic light scattering, zeta potential measurements, cryo-TEM, and fluorescence correlation spectroscopy and was found to be reversible via control of the pH. Aggregation of MSDH into closed vesicles under cytosolic conditions is at variance with the commonly held view of LD behavior, and we propose that endocytotic pathways should be considered as possible routes of LD entry into lysosomes. We further demonstrate that MSDH vesicles can be loaded with fluorophores via a solution transition from low to high pH, for subsequent release when the pH is lowered again. The ability to encapsulate molecular cargo into MSDH vesicles together with its ability to disaggregate at low pH and to permeabilize the lysosomal membrane presents an intriguing possibility to use MSDH as a delivery system.

Luminescent Nanocellulose Platform: From Controlled Graft Block Copolymerization to Biomarker Sensing.

A strategy is devised for the conversion of cellulose nanofibrils (CNF) into fluorescently labeled probes involving the synthesis of CNF-based macroinitiators that initiate radical polymerization of methyl acrylate and acrylic acid N-hydroxysuccinimide ester producing a graft block copolymer modified CNF. Finally, a luminescent probe (Lucifer yellow derivative) was labeled onto the modified CNF through an amidation reaction. The surface modification steps were verified with solid-state (13)C nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy. Fluorescence correlation spectroscopy (FCS) confirmed the successful labeling of the CNF; the CNF have a hydrodynamic radius of about 700 nm with an average number of dye molecules per fibril of at least 6600. The modified CNF was also imaged with confocal laser scanning microscopy. Luminescent CNF proved to be viable biomarkers and allow for fluorescence-based optical detection of CNF uptake and distribution in organisms such as crustaceans. The luminescent CNF were exposed to live juvenile daphnids and microscopy analysis revealed the presence of the luminescent CNF all over D. magna's alimentary canal tissues without any toxicity effect leading to the death of the specimen.

Highly Sensitive FRET-FCS Detects Amyloid ß-Peptide Oligomers in Solution at Physiological Concentrations.

Oligomers formed by the amyloid ß-peptide (Aß) are pathogens in Alzheimer's disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, Aß oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of Förster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of Aß, FRET-FCS detected oligomers consisting of less than 10 Aß molecules, which coexisted with the monomers at fractions as low as 2 ± 2%. Thus, we demonstrate for the first time direct detection of small fractions of Aß oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

Scanning inverse fluorescence correlation spectroscopy.

Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross- and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 ± 12 nm diameter, and 40 nm domains were estimated to 65 ± 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.

Removal of total organic carbon from sewage wastewater using poly(ethylenimine)-functionalized magnetic nanoparticles.

The increased levels of organic carbon in sewage wastewater during recent years impose a great challenge to the existing wastewater treatment process (WWTP). Technological innovations are therefore sought that can reduce the release of organic carbon into lakes and seas. In the present study, magnetic nanoparticles (NPs) were synthesized, functionalized with poly(ethylenimine) (PEI), and characterized using TEM (transmission electron microscopy), X-ray diffraction (XRD), FTIR (Fourier transform infrared spectroscopy), CCS (confocal correlation spectroscopy), SICS (scattering interference correlation spectroscopy), magnetism studies, and thermogravimetric analysis (TGA). The removal of total organic carbon (TOC) and other contaminants using PEI-coated magnetic nanoparticles (PEI-NPs) was tested in wastewater obtained from the Hammarby Sjöstadsverk sewage plant, Sweden. The synthesized NPs were about 12 nm in diameter and showed a homogeneous particle size distribution in dispersion by TEM and CCS analyses, respectively. The magnetization curve reveals superparamagnetic behavior, and the NPs do not reach saturation because of surface anisotropy effects. A 50% reduction in TOC was obtained in 60 min when using 20 mg/L PEI-NPs in 0.5 L of wastewater. Along with TOC, other contaminants such as turbidity (89%), color (86%), total nitrogen (24%), and microbial content (90%) were also removed without significant changes in the mineral ion composition of wastewater. We conclude that the application of PEI-NPs has the potential to reduce the processing time, complexity, sludge production, and use of additional chemicals in the WWTP.

Detection and quantification of Na,K-ATPase dimers in the plasma membrane of living cells by FRET-FCS.

The sodium potassium pump, Na,K-ATPase (NKA), is an integral plasma membrane protein, expressed in all eukaryotic cells. It is responsible for maintaining the transmembrane Na+ gradient and is the major determinant of the membrane potential. Self-interaction and oligomerization of NKA in cell membranes has been proposed and discussed but is still an open question. Here, we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, to analyze NKA in the plasma membrane of living cells. Click chemistry was used to conjugate the fluorescent labels Alexa 488 and Alexa 647 to non-canonical amino acids introduced in the NKA α1 and ß1 subunits. We demonstrate that FRET-FCS can detect an order of magnitude lower concentration of green-red labeled protein pairs in a single-labeled red and green background than what is possible with cross-correlation (FCCS). We show that a significant fraction of NKA is expressed as a dimer in the plasma membrane. We also introduce a method to estimate not only the number of single and double labeled NKA, but the number of unlabeled, endogenous NKA and estimate the density of endogenous NKA at the plasma membrane to 1400 ± 800 enzymes/µm2.

Aß Oligomer Dissociation Is Catalyzed by Fibril Surfaces.

Oligomeric assemblies consisting of only a few protein subunits are key species in the cytotoxicity of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their lifetime in solution and abundance, governed by the balance of their sources and sinks, are thus important determinants of disease. While significant advances have been made in elucidating the processes that govern oligomer production, the mechanisms behind their dissociation are still poorly understood. Here, we use chemical kinetic modeling to determine the fate of oligomers formed in vitro and discuss the implications for their abundance in vivo. We discover that oligomeric species formed predominantly on fibril surfaces, a broad class which includes the bulk of oligomers formed by the key Alzheimer's disease-associated Aß peptides, also dissociate overwhelmingly on fibril surfaces, not in solution as had previously been assumed. We monitor this "secondary nucleation in reverse" by measuring the dissociation of Aß42 oligomers in the presence and absence of fibrils via two distinct experimental methods. Our findings imply that drugs that bind fibril surfaces to inhibit oligomer formation may also inhibit their dissociation, with important implications for rational design of therapeutic strategies for Alzheimer's and other amyloid diseases.

A zeptoliter volume meter for analysis of single protein molecules.

A central goal in bioanalytics is to determine the concentration of and interactions between biomolecules. Nanotechnology allows performing such analyses in a highly parallel, low-cost, and miniaturized fashion. Here we report on label-free volume, concentration, and mobility analysis of single protein molecules and nanoparticles during their diffusion through a subattoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Nonfluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration, and mobility of the nonfluorescent analytes through fluctuation analysis in both time and amplitude.

Interferometry and fluorescence detection for simultaneous analysis of labeled and unlabeled nanoparticles in solution.

A novel fluctuation spectroscopy technique based on interferometry is described. The technique, termed scattering interference correlation spectroscopy (SICS), autocorrelates the signals from the forward-scattered and transmitted laser light from nanoparticles (NPs) in solution. SICS has two important features: First, for unlabeled NPs with known refractive index, it analyzes not only the diffusion coefficient but also the effective cross section and concentration in a single measurement. Second, it can be combined with fluorescence correlation spectroscopy (FCS) for simultaneous analysis of labeled and unlabeled NPs. SICS is here demonstrated on unlabeled M13 phages and on unlabeled NPs with diameters of 210 nm down to 26 nm. It is also shown how the combination of SICS and FCS can be used to determine the fraction of fluorescent NPs in a mixture and estimate K(d) from a single binding measurement.

α-Synuclein-induced deformation of small unilamellar vesicles.

α-Synuclein is a small neuronal protein that reversibly associates with lipid membranes. The membrane interactions are believed to be central to the healthy function of this protein involved in synaptic plasticity and neurotransmitter release. α-Synuclein has been speculated to induce vesicle fusion as well as fission, processes which are analogous to each other but proceed in different directions and involve different driving forces. In the current work, we analyse α-synuclein-induced small unilamellar vesicle deformation from a thermodynamics point of view. We show that the structures interpreted in the literature as fusion intermediates are in fact a stable deformed state and neither fusion nor vesicle clustering occurs. We speculate on the driving force for the observed deformation and put forward a hypothesis that α-synuclein self-assembly on the lipid membrane precedes and induces membrane remodelling.

The structure of cellulose nanofibril networks at low concentrations and their stabilizing action on colloidal particles.

The structure and dynamics of networks formed by rod-shaped particles can be indirectly investigated by measuring the diffusion of spherical tracer particles. This method was used to characterize cellulose nanofibril (CNF) networks in both dispersed and arrested states, the results of which were compared with coarse-grained Brownian dynamics simulations. At a CNF concentration of 0.2 wt% a transition was observed where, below this concentration tracer diffusion is governed by the increasing macroscopic viscosity of the dispersion. Above 0.2 wt%, the diffusion of small particles (20-40 nm) remains viscosity controlled, while particles (100-500 nm) become trapped in the CNF network. Sedimentation of silica microparticles (1-5 µm) in CNF dispersions was also determined, showing that sedimentation of larger particles is significantly affected by the presence of CNF. At concentrations of 0.2 wt%, the sedimentation velocity of 5 µm particles was reduced by 99 % compared to pure water.

Soluble TatA forms oligomers that interact with membranes: Structure and insertion studies of a versatile protein transporter.

The twin-arginine translocase (Tat) mediates the transport of already-folded proteins across membranes in bacteria, plants and archaea. TatA is a small, dynamic subunit of the Tat-system that is believed to be the active component during target protein translocation. TatA is foremost characterized as a bitopic membrane protein, but has also been found to partition into a soluble, oligomeric structure of yet unknown function. To elucidate the interplay between the membrane-bound and soluble forms we have investigated the oligomers formed by Arabidopsis thaliana TatA. We used several biophysical techniques to study the oligomeric structure in solution, the conversion that takes place upon interaction with membrane models of different compositions, and the effect on bilayer integrity upon insertion. Our results demonstrate that in solution TatA oligomerizes into large objects with a high degree of ordered structure. Upon interaction with lipids, conformational changes take place and TatA disintegrates into lower order oligomers. The insertion of TatA into lipid bilayers causes a temporary leakage of small molecules across the bilayer. The disruptive effect on the membrane is dependent on the liposome's negative surface charge density, with more leakage observed for purely zwitterionic bilayers. Overall, our findings indicate that A. thaliana TatA forms oligomers in solution that insert into bilayers, a process that involves reorganization of the protein oligomer.

Cooperativity of α-Synuclein Binding to Lipid Membranes.

Cooperative binding is a key feature of metabolic pathways, signaling, and transport processes. It provides tight regulation over a narrow concentration interval of a ligand, thus enabling switching to be triggered by small concentration variations. The data presented in this work reveal strong positive cooperativity of α-synuclein binding to phospholipid membranes. Fluorescence cross-correlation spectroscopy, confocal microscopy, and cryo-TEM results show that in excess of vesicles α-synuclein does not distribute randomly but binds only to a fraction of all available vesicles. Furthermore, α-synuclein binding to a supported lipid bilayer observed with total internal reflection fluorescence microscopy displays a much steeper dependence of bound protein on total protein concentration than expected for independent binding. The same phenomenon was observed in the case of α-synuclein binding to unilamellar vesicles of sizes in the nm and µm range as well as to flat supported lipid bilayers, ruling out that nonuniform binding of the protein is governed by differences in membrane curvature. Positive cooperativity of α-synuclein binding to lipid membranes means that the affinity of the protein to a membrane is higher where there is already protein bound compared to a bare membrane. The phenomenon described in this work may have implications for α-synuclein function in synaptic transmission and other membrane remodeling events.

Inverse-fluorescence cross-correlation spectroscopy.

Inverse-fluorescence correlation spectroscopy (iFCS) was recently introduced as an alternative version of FCS that does not require labeling of the analyzed particles or biomolecules. In iFCS, the signal from a medium surrounding the particles is analyzed, as opposed to a signal from the studied particles themselves. As unlabeled particles diffuse through the detection volume, they displace a fraction of the fluorescent medium, causing transient dips in the detected signal which give information about the mobility and concentration of the analyzed particles. Here inverse-fluorescence cross-correlation spectroscopy (iFCCS) is introduced as an extension of iFCS. In iFCCS, labeled particles/biomolecules are analyzed and their fluorescence signal is cross-correlated with the signal from the surrounding medium. When labeled particles are analyzed, a direct estimate of the volume of the particles is obtained or, alternatively, an estimate of the size of the detection volume. Another possibility is to analyze the interaction of small, labeled molecules with unlabeled particles, resulting in cross-correlation as an indication of binding, even though only one binding partner is labeled. This also enables accurate estimation of the degree of labeling, since the amounts of labeled and unlabeled particles are estimated independently.

Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) is frequently used to study diffusion in cell membranes, primarily the plasma membrane. The diffusion coefficients reported in the plasma membrane of the same cell type and even within single cells typically display a large spread. We have investigated whether this spread can be explained by variations in membrane topography throughout the cell surface, that changes the amount of membrane in the FCS focal volume at different locations. Using FCS, we found that diffusion of the membrane dye DiI in the apical plasma membrane was consistently faster above the nucleus than above the cytoplasm. Using live cell scanning ion conductance microscopy (SICM) to obtain a topography map of the cell surface, we demonstrate that cell surface roughness is unevenly distributed with the plasma membrane above the nucleus being the smoothest, suggesting that the difference in diffusion observed in FCS is related to membrane topography. FCS modeled on simulated diffusion in cell surfaces obtained by SICM was consistent with the FCS data from live cells and demonstrated that topography variations can cause the appearance of anomalous diffusion in FCS measurements. Furthermore, we found that variations in the amount of the membrane marker DiD, a proxy for the membrane, but not the transmembrane protein TCRζ or the lipid-anchored protein Lck, in the FCS focal volume were related to variations in diffusion times at different positions in the plasma membrane. This relationship was seen at different positions both at the apical cell and basal cell sides. We conclude that it is crucial to consider variations in topography in the interpretation of FCS results from membranes.

Inverse-fluorescence correlation spectroscopy.

An alternative version of fluorescence correlation spectroscopy is presented, where the signal from a medium surrounding the particles of interest is analyzed, as opposed to a signal from the particles themselves. This allows for analysis of unlabeled particles and potentially of biomolecules. Here, the concept together with principal experiments on polystyrene beads of 100, 200, 400, and 800 nm diameter in an aqueous solution of alexa 488-fluorophores are presented. The use of photo detectors allowing higher photon fluxes, or of reduced detection volumes, should enable analysis of significantly smaller particles or even biomolecules.