Scanning Fluorescence Correlation Spectroscopy Research Articles

Influenza A Virus Infection Alters Lipid Packing and Surface Electrostatic Potential of the Host Plasma Membrane.

The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Förster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid-lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken together, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.

Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells.

Protein-protein-interactions play an important role in many cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced "blinking" and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N&B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for protein-protein-interactions quantification via fluorescence fluctuation-based methods.

Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS).

Activation of immune cells and formation of immunological synapses (IS) rely critically on the reorganization of the plasma membrane. These highly orchestrated processes are driven by diffusion and oligomerization dynamics, as well as by single molecule interactions. While slow macro- and meso-scale changes in organization can be observed with conventional imaging, fast nano-scale dynamics are often missed with traditional approaches, but resolving them is, nonetheless, essential to understand the underlying biological mechanisms at play. Here, we describe the use of scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS) to study reorganization and changes in molecular diffusion dynamics and interactions during IS formation and in other biological settings. We focus on the practical aspects of the measurements including calibration and alignment of the optical setup, present a comprehensive protocol to perform the measurements, and provide data analysis pipelines and strategies. Finally, we show an exemplary application of the technology to studying Lck diffusion during T-cell signaling.

POPEYE intercellular localization mediates cell-specific iron deficiency responses.

Plants must tightly regulate iron (Fe) sensing, acquisition, transport, mobilization, and storage to ensure sufficient levels of this essential micronutrient. POPEYE (PYE) is an iron responsive transcription factor that positively regulates the iron deficiency response, while also repressing genes essential for maintaining iron homeostasis. However, little is known about how PYE plays such contradictory roles. Under iron-deficient conditions, pPYE:GFP accumulates in the root pericycle while pPYE:PYE-GFP is localized to the nucleus in all Arabidopsis (Arabidopsis thaliana) root cells, suggesting that PYE may have cell-specific dynamics and functions. Using scanning fluorescence correlation spectroscopy and cell-specific promoters, we found that PYE-GFP moves between different cells and that the tendency for movement corresponds with transcript abundance. While localization to the cortex, endodermis, and vasculature is required to manage changes in iron availability, vasculature and endodermis localization of PYE-GFP protein exacerbated pye-1 defects and elicited a host of transcriptional changes that are detrimental to iron mobilization. Our findings indicate that PYE acts as a positive regulator of iron deficiency response by regulating iron bioavailability differentially across cells, which may trigger iron uptake from the surrounding rhizosphere and impact root energy metabolism.

Quantifying Intercellular Movement and Protein Stoichiometry for Computational Modeling.

Analyzing protein movement dynamics and their regulation has shown to be important in the study of cell fate decisions. Such analyses can be performed with scanning fluorescence correlation spectroscopy (scanning FCS), a versatile imaging methodology that has been applied in the animal kingdom and recently adapted to the plant kingdom. Specifically, scanning FCS allows for qualitatively capturing protein movement across barriers, such as the active transport through plasmodesmata, the analysis of protein movement rates, and the quantification of the stoichiometry of protein complexes, composed of one or more different proteins. Importantly, the quantifiable data generated with scanning FCS can be used to inform computational models, enhancing model simulations of in vivo events, such as cell fate decisions, during plant development.

Spectral Detection Enables Multi-Color Fluorescence Fluctuation Spectroscopy Studies in Living Cells

Fluorescence fluctuation spectroscopy techniques provide a powerful toolbox to quantify transport dynamics and interactions of biomolecules in living cells. Cross-correlation analysis of spectrally well-separated fluctuations allows to investigate hetero-interactions between different biomolecules. This analysis is conventionally limited to two fluorescent species that are excited with one or two laser lines and detected in two non-overlapping spectral channels. However, signaling pathways in biological systems often involve interactions between multiple biomolecules, e.g. formation of ternary or quaternary protein complexes. Here, we implement a methodology to investigate such interactions at the plasma membrane (PM) of living cells, in the context of e.g. viral assembly or receptor-ligand interactions. To this aim, we introduce scanning spectral fluorescence correlation spectroscopy (SSFCS), a combination of scanning FCS with spectrally resolved detection and decomposition, inspired by fluorescence lifetime correlation spectroscopy. We first demonstrate that SSFCS allows cross-talk free cross-correlation analysis on cells expressing spectrally highly overlapping fluorescent proteins (FPs) such as mEGFP and mEYFP at the PM, with a single excitation line. We then verify the performance of SSFCS for the quantification of diffusion dynamics and protein oligomerization (based on molecular brightness analysis) of two protein species tagged with spectrally overlapping FPs. Adding a second laser line, we show the possibility of three- and four-species (cross-) correlation analysis using mApple and mCherry2 as overlapping FP tags in the red spectral region. Using this set of fluorophores, we furthermore extend the recently presented raster spectral image correlation spectroscopy (RSICS) approach to four species, and successfully demonstrate multiplexed RSICS measurements of protein interactions in cellular cytoplasm. Finally, we apply SSFCS and RSICS to investigate the assembly of the Influenza A virus matrix proteins and ternary polymerase complex at the PM and in the nucleus of living cells, respectively.

Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models.

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid–lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.

Scanning FCS and Superresolution Microscopy on 2D Lipid Membranes

Fluorescence Correlation Spectroscopy (FCS) is an established tool for understanding the dynamics of complex cellular processes. By applying the approach scanning FCS (sFCS), significant improvements can be obtained when studying slowly diffusing species, as is often the case in cell membranes. In sFCS, the excitation volume is scanned rapidly through the sample allowing for the simultaneous measurement of the diffusion of species at multiple locations within the sample. This significantly increases the statistical accuracy. In addition, shorter residence times of the fluorophores lead to lower photon doses experienced by each detected molecule, reducing the risk of photobleaching. This is especially important for sensitive fluorophores or when performing combined Stimulated Emission Depletion (STED) and FCS measurements. An added advantage of the scanning process is the ability to determine the observation volume without prior calibration. Here, we show results obtained with a confocal time-resolved fluorescence microscope (MicroTime 200 STED equipped with a FLIMbee galvo scanner, PicoQuant). We performed sFCS measurements on supported lipid bilayers (SLBs) which are commonly used as a simple model system for biological membranes. As the thickness of a bilayer is just a few nanometers, the diffusion properties determined with FCS or sFCS usually correspond to an average over both leaflets. We utilize the fluorescence lifetime information in order to achieve an axial nanometric localization based on Metal Induced Energy Transfer (MIET) [Karedla et al. (2014).ChemPhysChem 15(4):705-711]. The measured lifetime values enable us to separate the different molecular diffusion properties within the upper and the lower leaflet of the SLB measured on graphene.

The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.

Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.

Influenza A matrix protein M1 induces lipid membrane deformation via protein multimerization.

The matrix protein M1 of the Influenza A virus (IAV) is supposed to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle toward the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In the present study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed able to cause membrane curvature in lipid bilayers containing negatively charged lipids, in the absence of other viral components. Furthermore, we prove that protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1–M1 interactions and multimer formation are required in order to alter the bilayer three-dimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context, M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains).

Measuring Hindered Diffusion Dynamics in Live Cell Plasma Membranes with Confocal and Super-Resolution Imaging

The cellular plasma membrane is a highly heterogeneous structure organised on nano-scales and displays a crucial interaction platform for proteins, lipids and soluble ligands. Investigating the molecular membrane organisation by measuring diffusion dynamics offers a better understanding of its biological function. Fluorescence correlation spectroscopy (FCS) is one of the prominent tools to elucidate these dynamics in living cells but can only report on the dynamics at one given spatial position at a time. Using scanning fluorescence correlation spectroscopy (sFCS), we obtain a multitude of FCS measurements at different spatial locations. Here, we present a statistical analysis pipeline for sFCS data which allows for the accurate determination of the diffusion dynamics and the differentiation of free (Brownian) from hindered (non-Brownian) diffusion modes. We show free diffusion for phospholipids in model membranes and cells but reveal hindered diffusion of sphingolipids and GPI-anchored proteins in cells. Notably, these measurements can be performed using standard fluorescent dyes or proteins on a conventional confocal laser scanning microscope. To further investigate the dynamics on single cell level, we combine sFCS with stimulated emission depletion (STED) microscopy and by alternating conventional and super-resolved excitation we introduce line interleaved excitation scanning STED-FCS (LIESS-FCS). With LIESS-FCS the diffusion modes can be directly determined at multiple spots within the cellular plasma membrane providing detailed insights into organisation and function. Overall we are presenting a novel toolkit to investigate nano-scale molecular diffusion dynamics for shedding a new light on membrane organisation and heterogeneity.

Quantification of the Interactions Between BCL-2 Proteins by Fluorescence Correlation Spectroscopy.

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. Quantification 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 fluorescently labeled biomolecules that has provided useful insight into the binding affinity of the Bcl-2 interactome. FCS is based on the detection of fluorescence fluctuations 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.

Scanning Fluorescence Correlation Spectroscopy for Quantification of the Dynamics and Interactions in Tube Organelles of Living Cells.

Single-molecule spectroscopic quantification of protein-protein interactions directly in the organelles of living cells is highly desirable but remains challenging. Bulk methods, such as Förster resonance energy transfer (FRET), currently only give a relative quantification of the strength of protein-protein interactions. Here, we introduce tube scanning fluorescence cross-correlation spectroscopy (tubeSFCCS) for the absolute quantification of diffusion and complex formation of fluorescently labeled molecules in the mitochondrial compartments. We determined the extent of association between the apoptosis regulators Bcl-xL and tBid at the mitochondrial outer membrane of living cells and discovered that practically all mitochondria-bound Bcl-xL and tBid are associated with each other, in contrast to undetectable association in the cytosol. Furthermore, we show further applicability of our method to other mitochondrial proteins, as well as to proteins in the endoplasmic reticulum (ER) membrane.

Statistical Analysis of Scanning Fluorescence Correlation Spectroscopy Data Differentiates Free from Hindered Diffusion.

Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly applicable statistical analysis pipeline for large scanning fluorescence correlation spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.

Scanning fluorescence correlation spectroscopy comes full circle.

In this article, we review the application of fluorescence correlation spectroscopy (FCS) methods to studies on live cells. We begin with a brief overview of the theory underlying FCS, highlighting the type of information obtainable. We then focus on circular scanning FCS. Specifically, we discuss instrumentation and data analysis and offer some considerations regarding sample preparation. Two examples from the literature are discussed in detail. First, we show how this method, coupled with the photon counting histogram analysis, can provide information on yeast ribosomal structures in live cells. The combination of scanning FCS with dual channel detection in the study of lipid domains in live cells is also illustrated.

Circle scanning STED fluorescence correlation spectroscopy to quantify membrane dynamics and compartmentalization.

Quantifying molecular dynamics of cell membrane constituents is required to understand organization and function of biological membranes. Because of its complex structure unambiguous interpretation of molecular membrane dynamics requires high spatial and temporal resolution measurements. In this paper, we provide a comprehensive description of circle scanning fluorescence correlation spectroscopy and its combination with stimulated emission depletion microscopy (CS-STED-FCS). This method allows quantification of sub-diffusion processes and direct mapping of heterogeneities in membranes with high spatiotemporal resolution. We show how to use model membranes to calibrate and test the technique and how to apply it in the context of living cells to quantify membrane dynamics with high spatiotemporal resolution and good statistics.

Direct evidence of amyloid precursor–like protein 1 trans interactions in cell–cell adhesion platforms investigated via fluorescence fluctuation spectroscopy

The amyloid precursor-like protein 1 (APLP1) is a type I transmembrane protein that plays a role in synaptic adhesion and synaptogenesis. Past investigations indicated that APLP1 is involved in the formation of protein-protein complexes that bridge the junctions between neighboring cells. Nevertheless, APLP1-APLP1 trans interactions have never been directly observed in higher eukaryotic cells. Here, we investigated APLP1 interactions and dynamics directly in living human embryonic kidney cells using fluorescence fluctuation spectroscopy techniques, namely cross-correlation scanning fluorescence correlation spectroscopy and number and brightness analysis. Our results show that APLP1 forms homotypic trans complexes at cell-cell contacts. In the presence of zinc ions, the protein forms macroscopic clusters, exhibiting an even higher degree of trans binding and strongly reduced dynamics. Further evidence from giant plasma membrane vesicles suggests that the presence of an intact cortical cytoskeleton is required for zinc-induced cis multimerization. Subsequently, large adhesion platforms bridging interacting cells are formed through APLP1-APLP1 trans interactions. Taken together, our results provide direct evidence that APLP1 functions as a neuronal zinc-dependent adhesion protein and allow a more detailed understanding of the molecular mechanisms driving the formation of APLP1 adhesion platforms.

Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data.

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

Envelope glycoprotein mobility on HIV-1 particles depends on the virus maturation state

Human immunodeficiency virus type 1 (HIV-1) assembles as immature particles, which require the proteolytic cleavage of structural polyprotein Gag and the clustering of envelope glycoprotein Env for infectivity. The details of mechanisms underlying Env clustering remain unknown. Here, we determine molecular dynamics of Env on the surface of individual HIV-1 particles using scanning fluorescence correlation spectroscopy on a super-resolution STED microscope. We find that Env undergoes a maturation-induced increase in mobility, highlighting diffusion as one cause for Env clustering. This mobility increase is dependent on Gag-interacting Env tail but not on changes in viral envelope lipid order. Diffusion of Env and other envelope incorporated proteins in mature HIV-1 is two orders of magnitude slower than in the plasma membrane, indicating that HIV-1 envelope is intrinsically a low mobility environment, mainly due to its general high lipid order. Our results provide insights into dynamic properties of proteins on the surface of individual virus particles.