Fluorescence Fluctuation Spectroscopy Techniques Research Articles

Simulations of fluorescence fluctuation spectroscopy measurements of molecular diffusion in biomembranes

Studying the diffusion of individual molecules can yield important insights into the complex organization and function of the cell plasma membrane. Fluorescence fluctuation spectroscopy techniques allow the study of diffusion with single moleculesensitivity. Here, we present a novel, versatile and open accessible program based on Python for simulating molecular membrane diffusion. There have been different approaches to describe the diffusion laws governing the experimentally measured complex trajectories at the plasma membrane of live cells.

Dengue Virus Capsid-Protein Dynamics in Live Infected Cells Studied by Pair Correlation Analysis.

It has become increasingly evident that unveiling the mechanisms of virus entry, assembly, and virion release is fundamental for identifying means for preventing viral spread and controlling viral disease. Due to virus mobility and structural and/or functional heterogeneity among viral particles, high spatiotemporal resolution single-virus/single-particle techniques are required to capture the behavior of viral particles inside infected cells.In this chapter, we present fluorescence imaging analysis methods for studying the mobility of fluorescently labeled dengue virus (DENV) proteins in live infected cells. Some of the most recent Fluorescence Fluctuation Spectroscopy (FFS) methods will be presented and, in particular, the pair Correlation Functions (pCF) approach will be discussed. The pCF method does not require individual molecule isolation, as in a particle-tracking experiment, to capture single viral protein behavior. In this regard, image acquisition is followed by the spatiotemporal cross-correlation function at increasing time delays, yielding a quantitative view of single-particle mobility in intact live infected cells.We provide a general overview and a practical guidance for the implementation of advanced FFS techniques, and the pair Correlation Functions analysis, as quantitative tools to reveal insights into previously unreported DENV mechanisms. We expect this protocol report will serve as an incentive for further applying correlation imaging studies in virology research.

Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images—of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

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.

Challenges and Opportunities for Characterizing the Assembly of Nuclear Envelope Proteins by Fluorescence Fluctuation Spectroscopy

The nuclear envelope (NE), consisting of two membranes separated by a thin perinuclear space, has proven to be a challenging environment for measuring protein dynamics in living cells. First, exogenously expressed, fluorescently tagged proteins must be properly localized to the NE. Furthermore, undulations of the nuclear membranes complicate the application of fluorescence fluctuation spectroscopy (FFS) to proteins located within the perinuclear space by adding a slowly fluctuating signal. While this signal is absent for proteins associated with the nuclear membranes, distinguishing soluble from membrane-associated protein complexes by FFS presents a significant challenge since the diffusion time depends strongly on molecular size within the perinuclear space. Some proteins, such as the AAA+ ATPase TorsinA, may exist in both a membrane-associated and soluble form depending on the conditions in the cell. Determining the extent to which a protein associates with the membrane can provide valuable insight into its function within the cell. While diffusion time alone is insufficient to differentiate these populations, we explore whether the dependence of diffusion time on temperature can provide insight into a protein's localization within the NE. In addition, we explore how spatial correlations can be used to study the dynamics of proteins within the NE and the membrane undulations we previously reported. Finally, z-scan FFS provides a means to quantify the localization of proteins to the NE. These examples highlight the ways in which FFS techniques can be used to overcome the challenges of quantifying protein behavior in the NE of living cells. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).

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.

Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy.

Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques.

Imaging Fluorescence Fluctuation Spectroscopy: New Tools for Quantitative Bioimaging

Fluorescence fluctuation spectroscopy (FFS) techniques provide information at the single-molecule level with excellent time resolution. Usually applied at a single spot in a sample, they have been recently extended into imaging formats, referred to as imaging FFS. They provide spatial information at the optical diffraction limit and temporal information in the microsecond to millisecond range. This review provides an overview of the different modalities in which imaging FFS techniques have been implemented and discusses present imaging FFS capabilities and limitations. A combination of imaging FFS and nanoscopy would allow one to record information with the detailed spatial information of nanoscopy, which is ∼20 nm and limited only by fluorophore size and labeling density, and the time resolution of imaging FFS, limited by the fluorescence lifetime. This combination would provide new insights into biological events by providing spatiotemporal resolution at unprecedented levels.

Electrostatics Control Actin Filament Nucleation and Elongation Kinetics

The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment.

Time-Integrated Fluorescence Cumulant Analysis (TIFCA) Enables Model Selection in Ligand Binding Studies

TIFCA is a fluorescence fluctuation spectroscopy technique that makes use of both the molecular brightness and diffusion time of freely diffusing fluorescent species. TIFCA was introduced to calculate statistically significant higher order cumulants to accurately describe protein interactions using rigorous fitting procedures. Previously, we have applied TIFCA to perform a titration, confirm antibody stoichiometry and measure the binding affinity by first assuming a bivalent antibody model. The cumulants were calculated up to forth order and globally fit to resolve the fraction of bound and unbound ligand. The global fitting routine uses all points in the titration simultaneously and returns a global reduced chi2 for the entire titration series. It was found that analysis of the global reduced chi2 alone is not sensitive to the chosen binding model. Here we show that TIFCA has the ability to determine proper binding models by evaluating fits to individual cumulants within the titration series. Using a small peptide and antibody, we show that local reduced chi2 values are rather high for expressions describing a monovalent antibody. Only the correct number of species describing the bivalent nature of antibodies results in acceptable local reduced chi2 values for all orders of cumulants. More specifically, it is found that the second order cumulant is highly dependent upon the number of binding sites in an antibody molecule leading to the correct binding model.J.D.M. acknowledges support from the National Institute of Health (GM64589).

Probing Nucleocytoplasmic Transport with Fluorescence Fluctuation Spectroscopy and Two-photon Activation of Photoactivable GFP

Large proteins and macromolecular complexes have to enter and leave the nucleus in an efficient and selective manner. Macromolecules that are greater than 40 kD are transported actively across the nuclear envelope through nuclear pore complexes using soluble transport factors or carrier molecules that cycle between the cytoplasm and nucleus. The carrier proteins themselves interact with each other in order to transport cargo proteins across the nuclear pore complexes. In this work, we apply dual-color time-integrated fluorescence cumulant analysis (TIFCA), a fluorescence fluctuation spectroscopy technique, to investigate the protein interactions of the carrier proteins directly in cells. In addition, we apply two-photon activation to directly examine the nucleocytoplasmic transport of photoactivable GFP tagged carrier proteins. With these two approaches, we are able to probe the nucleocytoplasmic transport process of NTF2 directly inside cells and under equilibrium conditions. We investigate the oligomerization of NTF2 in cells and its transport properties when crossing the nuclear pore complexes.The experiments identify the presence of NTF2 dimers in the cytoplasm and nucleus of cells, while the measured transport properties across the nuclear pore complex are not consistent with dimeric NTF2. We will discuss the implications of these results for functions of nuclear envelope and models of nucleocytoplasmic transport. This work is supported by the National Science Foundation (PHY-0346782) and NIH grant R01GM064589.