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This study investigates the dynamics of oleate hydratase (OhyA), a bacterial flavoenzyme from Staphylococcus aureus, and its interactions with lipid membranes, focusing on the factors influencing membrane binding and oligomerization. OhyA catalyzes the hydration of unsaturated fatty acids, playing a key role in bacterial pathogenesis by neutralizing host antimicrobial fatty acids. OhyA binds the membrane bilayer to access membrane-embedded substrates for catalysis, and structural studies have revealed that OhyA forms oligomers on membrane surfaces, stabilized by both protein-protein and protein-lipid interactions. Using fluorescence correlation spectroscopy (FCS), we examined the effects of membrane curvature and lipid availability on OhyA binding to phosphatidylglycerol unilamellar vesicles. Our results reveal that OhyA preferentially binds to vesicles with moderate curvature, while the presence of substrate fatty acids slightly enhanced the overall interaction despite reducing the binding affinity by 3- to 4-fold. Complementary phosphorus-31 (31P) NMR spectroscopy further demonstrated two distinct binding modes: a fast-exchange interaction at lower protein concentrations and a longer lasting interaction at higher protein concentrations, likely reflecting cooperative oligomerization. These findings highlight the reversible, non-stoichiometric nature of OhyA•membrane interactions, with dynamic binding behaviors influenced by protein concentration and lipid environment. This research provides new insights into the dynamic behavior of OhyA on bacterial membranes, highlighting that initial interactions are driven by lipid-mediated protein binding, while sustained interactions are primarily governed by the protein:lipid molar ratio rather than the formation of new, specific lipid-protein interactions. These findings advance our understanding of the biophysical principles underlying OhyA’s role in bacterial membrane function and virulence.

Abstract Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.

Wnt, a family of secreted signaling proteins, serves diverse functions in embryogenesis, organogenesis, cancer, and stem cell functions. In the context of development, Wnt has been considered a representative morphogen, forming concentration gradients to give positional information to cells or tissues. However, although gradients are often illustrated in schemata, the reality of concentration gradients, or in other words, actual spatial distribution of Wnt ligands, and their behaviors in the extracellular space still remain poorly known. To understand extracellular behavior of Wnt ligands, quantitative analyses such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are highly informative because Wnt dispersal involves physical and biochemical processes, such as diffusion and binding to or dissociation from cell surface molecules, including heparan sulfate proteoglycans (HSPGs). Here, I briefly discuss representative methods to quantify morphogen dynamics. In addition, I discuss molecular manipulations of morphogens, mainly focusing on use of protein binders, and synthetic biology of morphogens as indicators of current and future directions in this field. [ABSTRACT FROM AUTHOR]

Simple Summary: Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of chemotherapy-resistant lymphomas, but CAR T cell therapy for solid tumors has been disappointing. This study examined the molecular structure, membrane organization, and mobility of HER2-specific CARs containing different costimulatory domains. Clustering behavior and diffusion kinetics of CARs were influenced by the costimulatory domains, proving to be significant predictors of immune synapse formation and early activation. Our results suggest that CAR T cell therapy for solid tumors needs to consider the molecular structure, membrane organization, and mobility of the chimeric antigen receptors alongside the long-term costimulation-based expansion capacity and anti-tumor activity for optimal therapeutic effect. Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While.z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile.z CAR induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the.z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect. [ABSTRACT FROM AUTHOR]

Seasonal periodic pandemics and epidemics caused by Influenza A viruses (IAVs) are associated with high morbidity and mortality worldwide. They are frequent and unpredictable in severity so there is a need for biophysical platforms that can be used to provide both mechanistic insights into influenza virulence and its potential treatment by anti-IAV agents. Host membrane viral association through the glycoprotein hemagglutinin (HA) of IAVs is one of the primary steps in infection. HA is thus a potential target for drug discovery and development against influenza. Deconvolution of the multivalent interactions of HA at the interfaces of the host cell membrane can help unravel therapeutic targets. In this contribution, we reported the effect of a multivalent HA glycoprotein association on various glycosphingolipid receptors (GD1a, GM3, GM1) doped asymmetrically into an artificial host membrane spanned across an aqueous filled microcavity array. The extent of HA association and its impact on membrane resistance, capacitance, and diffusivity was measured using highly sensitive electrochemical impedance spectroscopy (EIS) and fluorescence lifetime correlation spectroscopy (FLCS). Furthermore, we investigated the inhibition of the influenza HA glycoprotein association with the host mimetic surface by natural and synthetic sialic acid-based inhibitors (sialic acid, Siaα2,3-GalOMe, FB127, 3-sialyl lactose) using electrochemical impedance spectroscopy and observe that while all inhibit, they do not prevent host binding. Overall, the work demonstrates the platform provides a label-free screening platform for the biophysical evaluation of new inhibitors in the development of potential therapeutics for IAV infection prevention and treatment.

Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N- ethylmaleimide (NEM) or dl-dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.

Extracellular plaque deposits of ß-amyloid peptide (Aß) are one of the main pathological features of Alzheimer's disease (AD). The aggregation of Aß42 species, especially Aß42 oligomers, is still an active research field in AD pathogenesis. Secretory clusterin protein (sCLU), an extracellular chaperone, plays an important role in AD pathogenesis. Although sCLU interacts directly with Aß42 in vitro and in vivo, the mechanism is not clear. In this paper, His-tagged sCLU (sCLU-His) was cloned, expressed and purified, and we applied florescence resonance energy transfer-fluorescence correlation spectroscopy (FRET-FCS) to investigate the direct interaction of sCLU-His and Aß42 at the single-molecule fluorescence level in vitro. Here, we chose four different fluorescently labeled Aß42 oligomers to form two different groups of aggregation models, easy or difficult to aggregate. The results showed that sCLU-His could form complexes with both aggregation models, and sCLU-His inhibited the aggregation of Aß42/RB ~ Aß42/Atto647 (easy to aggregate model). The complexes were produced as the Aß42/Label adhered to the sCLU-His, which is similar to a "strawberry model," as strawberry seeds are dotted on the outer surface of strawberries. This work provided additional insight into the interaction mechanism of sCLU and Aß42. [ABSTRACT FROM AUTHOR]

Extracellular plaque deposits of β‐amyloid peptide (Aβ) are one of the main pathological features of Alzheimer's disease (AD). The aggregation of Aβ42 species, especially Aβ42 oligomers, is still an active research field in AD pathogenesis. Secretory clusterin protein (sCLU), an extracellular chaperone, plays an important role in AD pathogenesis. Although sCLU interacts directly with Aβ42 in vitro and in vivo, the mechanism is not clear. In this paper, His‐tagged sCLU (sCLU‐His) was cloned, expressed and purified, and we applied florescence resonance energy transfer‐fluorescence correlation spectroscopy (FRET‐FCS) to investigate the direct interaction of sCLU‐His and Aβ42 at the single‐molecule fluorescence level in vitro. Here, we chose four different fluorescently labeled Aβ42 oligomers to form two different groups of aggregation models, easy or difficult to aggregate. The results showed that sCLU‐His could form complexes with both aggregation models, and sCLU‐His inhibited the aggregation of Aβ42/RB ~ Aβ42/Atto647 (easy to aggregate model). The complexes were produced as the Aβ42/Label adhered to the sCLU‐His, which is similar to a "strawberry model," as strawberry seeds are dotted on the outer surface of strawberries. This work provided additional insight into the interaction mechanism of sCLU and Aβ42. [ABSTRACT FROM AUTHOR]

Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research. [ABSTRACT FROM AUTHOR]

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and selfpowered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and insolution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the singlemolecule level, in sharp contrast to the ~20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that pNPP-induced dye blinking at the ~10- ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models--including those of our own--in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale. [ABSTRACT FROM AUTHOR]

In the living cells, the majority of proteins does not work alone, but interact with other proteins or other biomolecules to maintain the cellular function, constituting a "protein community". Previous efforts on mass spectroscopy-based protein interaction networks, interactomes, have provided a picture on the protein community. However, these were static information after cells were disrupted. For a better understanding of the protein community in cells, it is important to know the properties of intracellular dynamics and interactions. Since hydrodynamic size and mobility of proteins are related into such properties, direct measurement of diffusional motion of proteins in single living cells will be helpful for uncovering the properties. Here we completed measurement of the diffusion and homo-oligomeric properties of 369 cytoplasmic GFP-fusion proteins in living yeast Saccharomyces cerevisiae cells using fluorescence correlation spectroscopy (FCS). The large-scale analysis showed that the motions of majority of proteins obeyed a two-component (i.e. slow and fast components) diffusion model. Remarkably, both of the two components diffused more slowly than expected monomeric states. In addition, further analysis suggested that more proteins existed as homo-oligomeric states in living cells than previously expected. Our study, which characterizes the dynamics of proteins in living cells on a large-scale, provided a global view on intracellular protein dynamics to understand the protein community. [ABSTRACT FROM AUTHOR]

• Biomacromolecule-incorporated liposomes can be important in drug stabilization. • Casein-mixed-lecithin-liposomes have reduced size compared to unmixed systems. • Microenvironment of the bi-layer of mixed system is hydrophobic and rigid. • Mixed system is much efficient to stabilize curcumin, β -carotene than pure systems. Formulation of new liposoma-based systems can always be in the spotlight for their unique utilization as carriers. Some changes in the composition of lipids may give rise to new mixed liposomes exhibiting modified size and physico-chemical characteristics. Consequently, these display different encapsulating properties toward various molecules. In this work, we have explored the variations in the model lecithin liposomes with casein additive from their size, shape, microenvironment and dynamics in solution. It is observed that with introduction of casein, the size of the liposome is substantially reduced due to incorporation of the additive in its bilayer. Strong interaction between the hydrophobic side chains of casein and lipid bilayer, and electrostatic repulsion of head groups of lipid are responsible to result small casein-mixed liposomes. Spectral properties of coumarin-153 disclose that the microenvironment of the bi-layer of mixed system is predominantly hydrophobic in nature and much rigid too. Fluorescence-lifetime-imaging-microscopy indicates that casein mixed systems exhibit wider lifetime distribution than pure liposomes with predominance of longer lifetimes, entirely arising from the outer bilayer. This indicates that casein gets incorporated in the bilayer of the vesicle to cater rigid and more hydrophobic microenvironment with an effective decrease in size. It is found that such mixed system is very efficient to stabilize the hydrophobic drugs (curcumin and β -carotene) for prolonged period than the pure liposome or casein-only. The findings are indeed important from the perspective of drug stabilization by liposomes, and is suggestive of possible utilization in oral drug delivery applications involving such size-reduced nano-encapsulates. [ABSTRACT FROM AUTHOR]

Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.

The inhibition of mitochondrial permeabilization by the anti-apoptotic protein Bcl-xL is crucial for cell survival and homeostasis. Its inhibitory role requires the partitioning of Bcl-xL to the mitochondrial outer membrane from an inactive state in the cytosol, leading to its extensive refolding. The molecular mechanisms behind these events and the resulting conformations in the bilayer are unclear, and different models have been proposed to explain them. In the most recently proposed non-canonical model, the active form of Bcl-xL employs its N-terminal BH4 helix to bind and block its pro-apoptotic target. Here, we used a combination of various spectroscopic techniques to study the release of the BH4 helix (α1) during the membrane insertion of Bcl-xL. This refolding was characterized by a gradual increase in helicity due to the lipid-dependent partitioning-coupled folding and formation of new helix αX (presumably in the originally disordered loop between helices α1 and α2). Notably, a comparison of various fluorescence and circular dichroism measurements suggested the presence of multiple Bcl-xL conformations in the bilayer. This conclusion was explicitly confirmed by single-molecule measurements of Fӧrster Resonance Energy Transfer from Alexa-Fluor-488-labeled Bcl-xL D189C to a mCherry fluorescent protein attached at the N-terminus. These measurements clearly indicated that the refolding of Bcl-xL in the bilayer is not a two-state transition and involves multiple membranous intermediates of variable compactness.

Proteins are broadly versatile biochemical materials, whose functionality is tightly related to their folding state. Native folding can be lost to yield misfolded conformations, often leading to formation of protein oligomers, aggregates, and biomolecular phase condensates. The fluorogenic hyaluronan HA-RB, a nonsulfonated glycosaminoglycan with a combination of polyanionic character and of hydrophobic spots due to rhodamine B dyes, binds to early aggregates of the model protein cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 1 from Arabidopsis thaliana (AtGAPC1) since the very onset of the oligomeric phase, making them brightly fluorescent. This initial step of aggregation has, until now, remained elusive with other fluorescence- or scattering-based techniques. The information gathered from nanotracking (via light-sheet fluorescence microscopy) and from FCS in a confocal microscope converges to highlight the ability of HA-RB to bind protein aggregates from the very early steps of aggregation and with high affinity. Altogether, this fluorescence-based approach allows one to monitor and track individual early AtGAPC1 aggregates in the size range from 10 to 100 nm with high time (∼10-2 s) and space (∼250 nm) resolution.

Advanced fluorescence techniques, commonly known as the F-techniques, measure the kinetics and the interactions of biomolecules with high sensitivity and spatiotemporal resolution. Applications of the F-techniques, which were initially limited to cells, were further extended to study in vivo protein organization and dynamics in whole organisms. The integration of F-techniques with multi-photon microscopy and light-sheet microscopy widened their applications in the field of developmental biology. It became possible to penetrate the thick tissues of living organisms and obtain good signal-to-noise ratio with reduced photo-induced toxicity. In this review, we discuss the principle and the applications of the three most commonly used F-techniques in developmental biology: Fluorescence Recovery After Photo-bleaching (FRAP), Förster Resonance Energy Transfer (FRET), and Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS). [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and Huntington's disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In the current review, we present an overviewof the principles and application of severalmethodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N&B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE. In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern (unobstructed diffusion) opposite to basal cholesterol conditions were most of Orai1 channels moves in a confined space, as assessed by Fluorescence Correlation Spectroscopy, Cav1 overexpression inhibited these alterations maintaining Orai1 into a confined and partially confined movement. These results not only highlight the complex effect of cholesterol regulation on SOCE, but also indicate a direct regulatory effect on Orai1 localization and compartmentalization by this lipid. [ABSTRACT FROM AUTHOR]

Objectives Alzheimer's disease (AD) is the most common cause of dementia in the world. As many AD biomarkers occur at rather low abundances in CSF or blood, techniques of very high sensitivity and accuracy are important as diagnostic tools in the clinic. Here, we aimed to provide proof of concept of the use of a single molecule detection technique, Fluorescence Correlation Spectroscopy (FCS) for detection of novel candidate biomarkers for AD. Design and methods FCS detects the diffusion times of the antigen-antibody complexes in highly diluted sample solutions, thus eliminating the need of large sample volumes and allows estimating the concentration of the target antigen. We developed a FCS set-up for contactin-2, a neuronal cell adhesion molecule and a ligand of beta-secretase 1 (BACE1) and amyloid precursor protein (APP), the latter proteins being important players in AD. With this method, we investigated whether contactin-2 concentrations are changed after delayed storage and in patients with Alzheimer's disease. Results The FCS set-up for measuring contactin-2 in CSF had a lower limit of quantification (LLOQ) of 0.2 ng/ml and intra- and inter-assay coefficients of variation (CVs) of 12.2% and 14.6% respectively. Contactin-2 levels were stable up to one week storage of CSF ( n = 3) at RT and 4 °C. Further, contactin-2 levels were similar in probable AD patients ( n = 34, p = 0.27) compared to patients with subjective cognitive decline (SCD) ( n = 11). Conclusions FCS is a sensitive tool, which can be used for detecting biomarkers in the clinical setting using very low sample volumes (10 μl) and can measure proteins in their native conformations in the body fluid. [ABSTRACT FROM AUTHOR]

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements. [ABSTRACT FROM AUTHOR]

Poly(ethylene glycol) (PEG) hydrogels are hydrophilic, high water content, polymeric networks that represent excellent candidates as engineering biomaterials for a broad range of applications. A key challenge for many biomedical applications is the control of transport properties within the resulting 3D crosslinked gels. The effects of the water content at synthesis and crosslinker molecular weights on gel chemical structure and equilibrium volumetric swelling ratio, Q, are studied for a series of PEG hydrogels. In addition, the translational diffusion coefficients of a model probe molecule, Rhodamine 110, are determined directly within the hydrogels by fluorescence correlation spectroscopy measurements. Increasing the water content at synthesis results in larger observed swelling behavior and faster particle transport within the formed PEG hydrogels for two different crosslinker molecular weights due to fewer physical crosslinks in the gels. Comparison of the particle translational diffusion coefficient to the swelling ratio shows a linear relationship for all crosslink densities examined. [ABSTRACT FROM AUTHOR]

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool for investigation of processes accompanied by changes in the mobility of molecules and complexes. In the present work, peak intensity analysis (PIA) in combination with the solution stirring using FCS setup was applied to explore the interaction between fluorescently labeled protein ligands and corresponding receptors located on membranes. In the system composed of biotinylated liposomes and fluorescently labeled streptavidin as a ligand, PIA allowed us to determine the optimum receptor concentration and demonstrate the essential dependence of the binding efficacy on the length of the linker between the biotin group and the polar head group of the lipid. The binding was dependent on the size of liposomes which was varied by lipid extrusion through filters of different pore diameters. The sensitivity of the method was higher with the liposomes of larger sizes. The PIA approach can be applied not only to liposomes but also to relatively large objects, e.g., erythrocytes or Sepharose beads derivatized with lactose as a receptor for the binding of viscumin and ricin. [ABSTRACT FROM AUTHOR]

Self-organization of cell-surface receptors in structurally distinct domains in the plasma membrane is of vital interest for correct cellular signaling. However, this dynamic process is difficult to study in cells with sufficiently high temporal and spatial resolution. We present here two quantitative high-resolution methods with single-molecule sensitivity, Fluorescence Correlation Spectroscopy (FCS) and pair-correlation Photoactivated Localization Microscopy (pcPALM), which enable nondestructive study of receptor diffusion and lateral organization at the nanoscale level. We introduce here the methods and review their application in studies of lateral organization of G Protein-Coupled Receptors (GPCRs). Examples from our own work on opioid receptor lateral organization are presented in order to illustrate the most recent advances in the field. [Projekat Ministarstva nauke Republike Srbije, br. 172015 i br. 45001]

[Display omitted] • Microscopic behaviours of ammonium-based hydroxyl ILs with varying number of hydroxyl groups have been investigated. • FLUPs technique coupled with TCSPC technique have been used to capture the complete solvation dynamics of relevant RTILs. • Rotational and translational diffusion of Perylene and MPTS have been studied using TRFA and FCS techniques. • Self-diffusion coefficients of the cations of the RTILS have been estimated using PFG-NMR techniques. The current study aims to understand the impact of variation of number of hydroxyl groups on the cationic head of ammonium-based room temperature ionic liquids (RTILs) towards inter/intra molecular hydrogen bonding interaction, local structural organization and dynamics of the solvent systems. For this purpose, three hydroxyl functionalized ammonium based RTILs (HFILs) bearing different numbers of hydroxyl groups on the cationic head as well as a non-hydroxyl ammonium based RTIL have been examined using both ensembled average and single-molecule spectroscopy techniques. Investigation of solvent relaxation dynamics of all the concerned RTILs by the combination of femto-second fluorescence up-conversion techniques and time corelated single photon counting techniques reveals a bimodal solvent relaxation behaviour having a very fast sub-picosecond and a relatively slower picosecond to nanosecond solvation time component for all the RTILs. Interestingly, analysis of the rotational and translational diffusion dynamics of a few particular solutes shows that the solvent–solvent interaction in the poly-hydroxyl-based HFILs is substantially stronger than the solute–solvent interaction. Additionally, analysis of the rotational diffusion data reveals that all RTILs exhibit significant dynamic heterogeneity, which increases with increase the number of hydroxyl groups on the cationic head of HFILs. Moreover, PFG-NMR study have shown that the HFILs had considerably greater hydrodynamic radii than the non-hydroxyl RTILs, which is likely due to the result of stronger hydrogen bonding interaction between the hydroxyl groups and the constituent of the HFILs. The outcomes of all these investigations have essentially demonstrated that subsequent addition of hydroxyl functionalities to the cationic head of the RTILs significantly alter the intra/inter molecular interaction, local structural organisation and heterogeneity of the medium. [ABSTRACT FROM AUTHOR]

Background: Genome organization into subchromosomal topologically associating domains (TADs) is linked to cell-type-specific gene expression programs. However, dynamic properties of such domains remain elusive, and it is unclear how domain plasticity modulates genomic accessibility for soluble factors. Results: Here, we combine and compare a high-resolution topology analysis of interacting chromatin loci with fluorescence correlation spectroscopy measurements of domain dynamics in single living cells. We identify topologically and dynamically independent chromatin domains of ~1 Mb in size that are best described by a loop-cluster polymer model. Hydrodynamic relaxation times and gyration radii of domains are larger for open (161 ± 15 ms, 297 ± 9 nm) than for dense chromatin (88 ± 7 ms, 243 ± 6 nm) and increase globally upon chromatin hyperacetylation or ATP depletion. Conclusions: Based on the domain structure and dynamics measurements, we propose a loop-cluster model for chromatin domains. It suggests that the regulation of chromatin accessibility for soluble factors displays a significantly stronger dependence on factor concentration than search processes within a static network. [ABSTRACT FROM AUTHOR]

Fluorescence correlation spectroscopy (FCS) has become an important technique for the characterization of molecular dynamics, especially at interfaces. Fluorescence correlation spectroscopy provides both temporal and spatial resolution for measuring fast processes at equilibrium through analysis of noise in fluorescence intensities from the statistical fluctuations in a small number of molecules. The small molecular populations produce very low-level fluorescence signals, where timeaveraging the fluorescence autocorrelation function is needed to generate reasonable signal-to-noise (S/N) ratios. Recently imaging cameras have been adapted to FCS measurements of molecular dynamics at interfaces (membranes and surfaces) through the use of electron-multiplying charge-coupled device (EM-CCD) detectors for acquisition of fluorescence from addressable areas on the detector. This approach provides a major advantage over traditional focused-spot FCS by allowing electronic control over the location and area of the acquired region on the sample surface. Imaging-FCS can also provide a spatial multiplexing advantage through its ability to measure intensity data from larger areas in parallel with no loss of time resolution. In this work, this multiplexing advantage is exploited to determine molecular diffusion rates from the simultaneous measurement of multiple areas on a surface, the autocorrelation traces from which are averaged to improve the S/N ratio. As proof of concept, the diffusion of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) on a C18-modified interface was measured using this multiplexed method and compared to autocorrelation data acquired from a single spot. Due to the slow thermal recovery of the EM-CCD that inhibits fast time-averaging, spatial multiplexing in imaging-FCS provides an eightyfold time savings to reach the same S/N ratio as multiple (time-averaged) measurements from a single spot. [ABSTRACT FROM AUTHOR]

The boundaries of brain regions are associated with the tissuespecific secretion of ligands from different signaling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. Using light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics, we demonstrated that Wnt3 regulates cerebellum development during embryogenesis using zebrafish wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP. The results suggest a state of dynamic equilibrium of Wnt3EGFP mobility in polarized neuroepithelial-like progenitors in the dorsal midline and cerebellar progenitors on the lateral side. Wnt3EGFP is secreted from the cerebellum as shown by measurements of its mobility in the ventricular cavity. The importance of Wnt secretion in brain patterning was validated with the Porcn inhibitor Wnt-C59 (C59), which, when applied early, reduced membrane-bound and secreted fractions of Wnt3EGFP and led to a malformed brain characterized by the absence of epithalamus, optic tectum and cerebellum. Likewise, interference with Wnt secretion later on during cerebellar development negatively impacted cerebellar growth and patterning. Our work, supported by quantitative analysis of protein dynamics in vivo, highlights the importance of membrane-localized and secreted Wnt3 during cerebellum development. [ABSTRACT FROM AUTHOR]

In this study, an aptamer-based single particle method was developed for the thrombin detection in human serum samples using fluorescence correlation spectroscopy (FCS). In this method, quantum dots (QDs) were used as the fluorescent probes and thrombin-binding aptamer (TBA) was used as molecular recognition unit. When two QDs probes labeled with TBA (QD-TBA1 and QD-TBA2) are mixed in a sample containing thrombin targets, the binding of targets will cause QDs to form dimers (or oligomers) with bigger sizes, which leads to the nearly double increase in the characteristic diffusion time of QDs in the detection volume of FCS. FCS method can detect the change in the characteristic diffusion time of QDs. Firstly, the diffusion and blinking behaviors of QD-TBA probes in the presence of thrombin were investigated by FCS and total internal reflection fluorescence microscopy (TIRFM) imaging system, and the experimental results documented that QD-TBAs were bound together with “one-by-one” structure when thrombin were added into the solution. And then, the assay conditions were optimized in order to improve the sensitivity and specificity of this method. Under the optimized conditions, the linear range of the method is from 5.0 nM to 500 nM of thrombin, and the limit of detection is about 2.6 nM. Finally, this method was applied to homogeneous determination of thrombin in human serum samples. [ABSTRACT FROM AUTHOR]

Highlights: [•] Fluorescence correlation spectroscopy used to monitor Aβ fibrillogenesis. [•] New analysis method using a 5 Gaussian distribution model. [•] Several different sized Aβ components in solution can be determined simultaneously. [•] Possible to measure in complex buffers or in biological fluids. [ABSTRACT FROM AUTHOR]

Abstract: Ceramides are involved in a variety of cellular processes and in disease. Their biological functions are thought to depend on ceramides’ unique biophysical properties, which promote strong alterations of cell membrane properties and consequent triggering of signaling events. Over the last decades, efforts were made to understand the impact of ceramide on membrane biophysical features. Several studies, performed in a multitude of membrane models, address ceramides’ specific interactions, the effect of their acyl chain structure and the influence of membrane lipid composition and properties on ceramide biophysical outcome. In this review, a rationale for the multiple and complex changes promoted by ceramide is provided, highlighting, on a comprehensive and critical manner, the interactions between ceramides and specific lipids and/or lipid phases. Focus is also given to the interplay between ceramide and cholesterol, particularly in lipid raft-mimicking mixtures, an issue of intense debate due to the urgent need to understand the biophysical impact of ceramide formation in models resembling the cell membrane. The implications of ceramide-induced biophysical changes on lipid–protein interactions and cell signaling are also discussed, together with the emerging evidence for the existence of ceramide-gel like domains in cellular membranes. [Copyright &y& Elsevier]

Abstract: The organization of the plasma membrane is regulated by the dynamic equilibrium between the liquid ordered (Lo) and liquid disordered (Ld) phases. The abundance of the Lo phase is assumed to be a consequence of the interaction between cholesterol and the other lipids, which are otherwise in either the Ld or gel (So) phase. The characteristic lipid packing in these phases results in significant differences in their respective lateral dynamics. In this study, imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is applied to monitor the diffusion within supported lipid bilayers (SLBs) as functions of temperature and composition. We show that the temperature dependence of membrane lateral diffusion, which is parameterized by the Arrhenius activation energy (E Arr), can resolve the sub-resolution phase behavior of lipid mixtures. The FCS diffusion law, a novel membrane heterogeneity ruler implemented in ITIR-FCS, is applied to show that the domains in the So–Ld phase are static and large while they are small and dynamic in the Lo–Ld phase. Diffusion measurements and the subsequent FCS diffusion law analyses at different temperatures show that the modulation in membrane dynamics at high temperature (313K) is a cumulative effect of domain melting and rigidity relaxation. Finally, we extend these studies to the plasma membranes of commonly used neuroblastoma, HeLa and fibroblast cells. The temperature dependence of membrane dynamics for neuroblastoma cells is significantly different from that of HeLa or fibroblast cells as the different cell types exhibit a high level of compositional heterogeneity. [Copyright &y& Elsevier]

[Display omitted] Globally, over 2 billion people suffer from vision impairment. Despite complex multifactorial etiology, advanced glycation end products are involved in the pathogenesis of many causative age- and diabetes-related eye diseases. Deglycating enzyme fructosamine-3-kinase (FN3K) was recently proposed as a potential therapeutic, but for further biopharmaceutical development, knowledge on its manufacturability and stability and mobility in the vitreous fluid of the eye is indispensable. We evaluated recombinant production of FN3K in two host systems, and its diffusion behavior in both bovine and human vitreous. Compared to Escherichia coli, intracellular production in Pichia pastoris yielded more and higher purity FN3K. The yeast-produced enzyme was used in a first attempt to use fluorescence correlation spectroscopy to study protein mobility in non-sonicated bovine vitreous, human vitreous, and intact bovine eyes. It was demonstrated that FN3K retained mobility upon intravitreal injection, although a certain delay in diffusion was observed. Alkylation of free cysteines was tolerated both in terms of enzymatic activity and vitreous diffusion. Ex vivo diffusion data gathered and the availability of yeast-produced high purity enzyme now clear the path for in vivo pharmacokinetics studies of FN3K. [ABSTRACT FROM AUTHOR]

Abstract: Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme–membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors. [Copyright &y& Elsevier]

Abstract: Proteoliposomes represent nanoscale assemblies of indispensable value for studying membrane proteins in general and membrane transporters in particular. Since no universal protocol exists, conditions for proteoliposome formation must be determined on a case-by-case basis. This process will be significantly expedited if the size and composition of the assemblies can be analyzed in a single step using only microliters of sample. Here we show that dual-color fluorescence cross-correlation spectroscopy (FCCS) is of great value for optimizing the reconstitution process, because it distinguishes micelles, liposomes and aggregates in heterogeneous mixtures and permits direct monitoring of the co-localization of proteins and lipids in the diffusing assemblies. As proof-of-principle, liposomes containing the functional multidrug resistance transporter NorA from Staphylococcus aureus were prepared, demonstrating that FCCS is an excellent tool to guide the development of reconstitution protocols. [Copyright &y& Elsevier]

Abstract: This mini-review reports on applications of particular multiphoton excitation microscopy-based methodologies employed in our laboratory to study skin. These approaches allow in-depth optical sectioning of the tissue, providing spatially resolved information on specific fluorescence probes’ parameters. Specifically, by applying these methods, spatially resolved maps of water dipolar relaxation (generalized polarization function using the 6-lauroyl-2-(N,N-dimethylamino)naphthale probe), activity of protons (fluorescence lifetime imaging using a proton sensitive fluorescence probe – 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) and diffusion coefficients of distinct fluorescence probes (raster imaging correlation spectroscopy) can be obtained from different regions of the tissue. Comparative studies of different tissue strata, but also between equivalent regions of normal and abnormal excised skin, including applications of fluctuation correlation spectroscopy on transdermal penetration of liposomes are presented and discussed. The data from the different studies reported reveal the intrinsic heterogeneity of skin and also prove these strategies to be powerful noninvasive tools to explore structural and dynamical aspects of the tissue. [Copyright &y& Elsevier]

Abstract: Membrane protein biogenesis in bacteria occurs via dedicated molecular systems SecYEG and YidC that function independently and in cooperation. YidC belongs to the universally conserved Oxa1/Alb3/YidC family of membrane insertases and is believed to associate with translating ribosomes at the membrane surface. Here, we have examined the architecture of the YidC:ribosome complex formed upon YidC-mediated membrane protein insertion. Fluorescence correlation spectroscopy was employed to investigate the complex assembly under physiological conditions. A slightly acidic environment stimulates binding of detergent-solubilized YidC to ribosomes due to electrostatic interactions, while YidC acquires specificity for translating ribosomes at pH-neutral conditions. The nanodisc reconstitution of the YidC to embed it into a native phospholipid membrane environment strongly enhances the YidC:ribosome complex formation. A single copy of YidC suffices for the binding of translating ribosome both in detergent and at the lipid membrane interface, thus being the minimal functional unit. Data reveal molecular details on the insertase functioning and interactions and suggest a new structural model for the YidC:ribosome complex. [Copyright &y& Elsevier]

Recent studies introduced a novel view that the SecYEG translocon functions as a monomer and interacts with the dimeric SecA ATPase, which fuels the preprotein translocation reaction. Here, we used nanodisc-reconstituted SecYEG to characterize the functional properties of single copies of the translocon. Using a method based on intermolecular Förster resonance energy transfer, we show for the first time that isolated nanodisc-reconstituted SecYEG monomers support preprotein translocation. When several copies of SecYEG were co-reconstituted within a nanodisc, no change in translocation kinetics was observed, suggesting that SecYEG oligomers do not facilitate enhanced translocation. In contrast, nanodisc-reconstituted monomers of the PrlA4 variant of SecYEG showed increased translocation rates. Experiments based on intramolecular Förster resonance energy transfer within the nanodisc-isolated monomeric SecYEG demonstrated a nucleotide-dependent opening of the channel upon interaction with SecA. In conclusion, the nanodisc-reconstituted SecYEG monomers are functional for preprotein translocation and provide a new prospect for single-molecule analysis of dynamic aspects of protein translocation. [ABSTRACT FROM AUTHOR]