Cross-correlation Spectroscopy Research Articles

Decoding the Role of Receptor Dimerization in Plexin-Semaphorin Signaling

Plexin-semaphorin signaling plays an important role in the formation of neural networks by acting as the guidance cue for axon growth or retraction. The plexin receptor is a single-pass transmembrane protein with a sema domain at the extracellular N-terminus and a GTP-ase activating protein domain on the cytoplasmic C-terminus. Binding of semaphorin is known to activate the plexin receptor, but the mechanism of how that binding event leads to activation is not understood. We will present our recent work using pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to measure plexin protein-protein interactions in live cells. PIE-FCCS transforms fluctuations in fluorescence intensity (arising mainly from diffusion) into information about a protein's mobility and concentration. By cross-correlating the fluorescence fluctuations in two color channels, PIE-FCCS also quantifies co-localization and co-diffusion with high accuracy. This method has been used to resolve the mechanism of epidermal growth factor receptor (EGFR) activation and inhibition, as well as the dimerization constant of the opsin G protein-coupled receptor. In this work we measure the ligand-free dimerization of plexin receptors in live cell membranes and the response of the receptor to semaphorin-induced activation.

PIE-FCCS Study of the Effects of Polycationic Macromolecules on Phosphatidylserine and Phosphatidylinositol Phosphate Lipid Mobility

The interaction between proteins and anionic lipids in the plasma membrane is a common motif in cell communication at the plasma membrane. Such interactions can gate membrane protein function and have also been proposed to sequester membrane lipids during quiescent phases of signaling. To date, however, the molecular structure and dynamics of the lipid-protein interface is poorly understood. To isolate these interactions in a biophysical assay, we have investigated the behavior of a polycationic polymer, quaternized polyvinylpyridine (QPVP), on supported lipid bilayers doped with tail-labeled phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP) lipids. To measure the mobility and association of the lipid and adsorbed polymer, we use a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a dual-color fluorescence spectroscopy that translates fluctuations in fluorescence signal into a measurement of diffusion and co-localization. With PIE-FCCS we have investigated the polymer adsorption-dependent translational mobility of the lipids and systematically studied the influence of lipid head-group charge and solvent ionic strength. Our results indicate that alteration of anionic lipid lateral mobility is dependent on the net charge of the lipid head group and is significantly screened by the ionic strength of the solution. At physiological salt concentration we observe that the lipid lateral mobility is nearly unaffected by mobile, adsorbed polymers and that there is no evidence of stable lipid-polymer complexes.

Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508-3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842-2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833-10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543-556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.

Epidermal Growth Factor Receptor (EGFR) Membrane Organization and Dynamics Investigated by Sw-Fccs and Imaging Fcs

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in various cellular processes including cell differentiation, proliferation, and migration. Despite many years of intensive research its mode of activation is still not fully understood, partly because its dimerization and its membrane localization are still under discussion. Here we use Single Wavelength excitation Fluorescence Cross-Correlation Spectroscopy (SW-FCCS) and Imaging Fluorescence Correlation Spectroscopy (Imaging FCS), to investigate receptor dimerization and receptor localization in membranes.The fraction of EGFR preformed dimers, i.e. dimerization in the absence of ligands, has been determined under various conditions with very different results, ranging from exclusive monomeric states to distributions between monomers dimers and possibly higher oligomers. The reason for these discrepancies is unclear. We therefore measure the dimerization on different cell lines (CHO-K1, Cos7, HEK293) at different membrane locations (basal and apical membrane) and at room and physiological temperature to determine whether dimerization is sensitive to these different conditions. We observe a cell line specific sensitivity of dimerization to temperature and membrane localization that can change the detected dimer fraction by more than a factor of 2, hinting at an explanation why different experiments provide different answers to the question of preformed dimers. Furthermore, we use Imaging FCS to investigate the localization of the EGFR within the membrane and its interaction with the cytoskeleton. Our study shows that the EGFR is interacting with cholesterol dependent and independent domains and its motion is influenced by the cytoskeleton. Overall these measurements provide a complex picture of EGFR organization and dynamics, which might play essential roles in receptor function and signaling.

Dynamics and Interaction of Interleukin-4 Receptor Subunits in Living Cells

It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process. Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (Kd,2D) of 180 and 480 receptors per μm2 for the type-2 complexes IL-4:IL-4Rα/IL-13Rα1 and IL-13:IL-13Rα1/IL-4Rα, respectively. For the lower affinity type-1 complex IL-4:IL-4Rα/IL-2Rγ, we estimated a Kd,2D of ∼1000 receptors per μm2. The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.

Determination of the dissociation constant of the NFκB p50/p65 heterodimer in living cells using fluorescence cross-correlation spectroscopy.

Fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for observing and quantifying protein-protein interactions in vitro and in vivo. FCCS has emerged as a useful tool for obtaining parameters of the concentration of labeled particles, their molecular dynamics, as well as the size of their complexes. This chapter discusses aspects of preparing a biological system for FCCS experiments and suggests practical advice for performing FCCS in living cells. Moreover, we describe the method of FCCS to determine the dissociation constant of a transcription factor dimer in the living cell.

Coincidence Measurements in Dual-Color Confocal Microscopy: A Combined Single-Particle and Fluorescence Correlation Approach

In this paper we discuss how the coincident detection of mobile particles in dual-color confocal images can be improved. Optimal coincidence detection requires a careful choice of experimental conditions and image acquisition parameters in order to maximize the overlap between the two detection volumes. By measuring this overlap with fluorescence cross-correlation spectroscopy, we show in particular that a small confocal field of view is necessary in order to maintain good coincidence. Most importantly, coincidence detection also requires a dedicated image analysis strategy. Traditionally, two approaches have been adopted to assess coincidence of mobile particles: fluorescence fluctuation measurements, notably cross-correlation spectroscopy, and single particle detection. Here we propose to combine these two approaches by calculating a cross-correlation coefficient for each of the detected single particles. We show that this allows to remove accidental coincidence events from a data set, and thus to unambiguously identify particles that instead carry two different fluorophores. This strategy can help increase the available concentration range for confocal coincidence measurements and detect rare binding events. [Formula: see text]Special Issue Comments: This article about coincident detection of mobile particle in two-color confocal images is thematically related to several articles in this Special Issue, namely the review of FRET-based single-molecule fluorescence techniques by Ruedas-Rama et al.,1 the single particle detection work presented by de Keersmaecker et al.2 and the general considerations on the mathematical treatment of single molecule trajectories presented by Flomenbom3. Our study of single liposomes is also relevant to experiments involving proteins and liposomes, such as the enzyme experiments described in the review by Jørgensen and Hatzakis.4

Resolving particle size modality in bi-modal iron oxide nanoparticle suspensions

Abstract Particle size modality in bi-modal iron oxide suspensions was resolved by exploiting complex ac-susceptibility (ACS), small angle X-ray scattering (SAXS) and photon cross-correlation spectroscopy. To explain dynamic magnetic response of bi-modal suspensions, the Debye model was expanded to a linear superposition form allowing for the contribution of both particle fractions. This modified and adopted model is able to resolve the bi-modal particle size distributions. The SAXS curves of mono- and bi-modal suspensions were fitted well using a Monte Carlo simulation scheme, allowing the detection of bi-modal particle size distributions with high precision.

Non-Destructive Global and Localized 2D T1/T2 NMR Relaxometry to Resolve Microstructure in Apples Affected by Watercore

Apples can be considered as having a complex system formed by several structures at different organization levels: macroscale (>100 μm) and microscale (<100 μm). This work implements 2D T1/T2 global and localized relaxometry sequences on whole apples to be able to perform an intensive non-destructive and non-invasive microstructure study. The 2D T1/T2 cross-correlation spectroscopy allows the extraction of quantitative information about the water compartmentation in different subcellular organelles. A clear difference is found as sound apples show neat peaks for water in different subcellular compartments, such as vacuolar, cytoplasmatic and extracellular water, while in watercore-affected tissues such compartments appear merged. Localized relaxometry allows for the predefinition of slices in order to understand the microstructure of a particular region of the fruit, providing information that cannot be derived from global 2D T1/T2 relaxometry.

The Specific Monomer/Dimer Equilibrium of the Corticotropin-releasing Factor Receptor Type 1 Is Established in the Endoplasmic Reticulum

G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment.

Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells.

In living cells, most proteins diffuse over distances of micrometres within seconds. Protein translocation is constrained due to the cellular organization into subcompartments that impose diffusion barriers and guide enzymatic activities to their targets. Here, we introduce an approach to retrieve structural features from the scale-dependent mobility of green fluorescent protein monomer and multimers in human cells. We measure protein transport simultaneously between hundreds of positions by multi-scale fluorescence cross-correlation spectroscopy using a line-illuminating confocal microscope. From these data we derive a quantitative model of the intracellular architecture that resembles a random obstacle network for diffusing proteins. This topology partitions the cellular content and increases the dwell time of proteins in their local environment. The accessibility of obstacle surfaces depends on protein size. Our method links multi-scale mobility measurements with a quantitative description of intracellular structure that can be applied to evaluate how drug-induced perturbations affect protein transport and interactions.

Near-field fluorescence cross-correlation spectroscopy on planar membranes.

The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (FcεRI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods.

Altered spatio-temporal dynamics of RNase H2 complex assembly at replication and repair sites in Aicardi-Goutières syndrome.

Ribonuclease H2 plays an essential role for genome stability as it removes ribonucleotides misincorporated into genomic DNA by replicative polymerases and resolves RNA/DNA hybrids. Biallelic mutations in the genes encoding the three RNase H2 subunits cause Aicardi-Goutières syndrome (AGS), an early-onset inflammatory encephalopathy that phenotypically overlaps with the autoimmune disorder systemic lupus erythematosus. Here we studied the intracellular dynamics of RNase H2 in living cells during DNA replication and in response to DNA damage using confocal time-lapse imaging and fluorescence cross-correlation spectroscopy. We demonstrate that the RNase H2 complex is assembled in the cytosol and imported into the nucleus in an RNase H2B-dependent manner. RNase H2 is not only recruited to DNA replication foci, but also to sites of PCNA-dependent DNA repair. By fluorescence recovery after photobleaching, we demonstrate a high mobility and fast exchange of RNase H2 at sites of DNA repair and replication. We provide evidence that recruitment of RNase H2 is not only PCNA-dependent, mediated by an interaction of the B subunit with PCNA, but also PCNA-independent mediated via the catalytic domain of the A subunit. We found that AGS-associated mutations alter complex formation, recruitment efficiency and exchange kinetics at sites of DNA replication and repair suggesting that impaired ribonucleotide removal contributes to AGS pathogenesis.

Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos.

Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms-3 μm time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos' vessels.

Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.

Symmetric exchange of multi-protein building blocks between stationary focal adhesions and the cytosol

How can the integrin adhesome get self-assembled locally, rapidly, and correctly as diverse cell-matrix adhesion sites? Here, we investigate this question by exploring the cytosolic state of integrin-adhesome components and their dynamic exchange between adhesion sites and cytosol. Using fluorescence cross-correlation spectroscopy (FCCS) and fluorescence recovery after photobleaching (FRAP) we found that the integrin adhesome is extensively pre-assembled already in the cytosol as multi-protein building blocks for adhesion sites. Stationary focal adhesions release symmetrically the same types of protein complexes that they recruit, thereby keeping the cytosolic pool of building blocks spatiotemporally uniform. We conclude a model in which multi-protein building blocks enable rapid and modular self-assembly of adhesion sites and symmetric exchange of these building blocks preserves their specifications and thus the assembly logic of the system.DOI: http://dx.doi.org/10.7554/eLife.02257.001.

Time-resolved fluorescence spectroscopy measures clustering and mobility of a G protein-coupled receptor opsin in live cell membranes.

Determining membrane protein quaternary structure is extremely challenging, especially in live cell membranes. We measured the oligomerization of opsin, a prototypical G protein-coupled receptor with pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Individual cell measurements revealed that opsin is predominantly organized into dimeric clusters. At low concentrations, we observed that the population of oligomers increased linearly with the square of the individual monomer populations. This finding supports a monomer–dimer equilibrium and provides an experimental measurement of the equilibrium constant.

Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin

Lens epithelium-derived growth factor (LEDGF/p75) is a transcriptional co-activator involved in targeting human immunodeficiency virus (HIV) integration and the development of MLL fusion-mediated acute leukemia. A previous study revealed that LEDGF/p75 dynamically scans the chromatin, and upon interaction with HIV-1 integrase, their complex is locked on chromatin. At present, it is not known whether LEDGF/p75-mediated chromatin locking is typical for interacting proteins. Here, we employed continuous photobleaching and fluorescence correlation and cross-correlation spectroscopy to investigate in vivo chromatin binding of JPO2, a LEDGF/p75- and c-Myc-interacting protein involved in transcriptional regulation. In the absence of LEDGF/p75, JPO2 performs chromatin scanning inherent to transcription factors. However, whereas the dynamics of JPO2 chromatin binding are decelerated upon interaction with LEDGF/p75, very strong locking of their complex onto chromatin is absent. Similar results were obtained with the domesticated transposase PogZ, another cellular interaction partner of LEDGF/p75. We furthermore show that diffusive JPO2 can oligomerize; that JPO2 and LEDGF/p75 interact directly and specifically in vivo through the specific interaction domain of JPO2 and the C-terminal domain of LEDGF/p75, comprising the integrase-binding domain; and that modulation of JPO2 dynamics requires a functional PWWP domain in LEDGF/p75. Our results suggest that the dynamics of the LEDGF/p75-chromatin interaction depend on the specific partner and that strong chromatin locking is not a property of all LEDGF/p75-binding proteins.

Clathrin and Membrane Microdomains Cooperatively Regulate RbohD Dynamics and Activity in Arabidopsis.

Arabidopsis thaliana respiratory burst oxidase homolog D (RbohD) functions as an essential regulator of reactive oxygen species (ROS). However, our understanding of the regulation of RbohD remains limited. By variable-angle total internal reflection fluorescence microscopy, we demonstrate that green fluorescent protein (GFP)-RbohD organizes into dynamic spots at the plasma membrane. These RbohD spots have heterogeneous diffusion coefficients and oligomerization states, as measured by photobleaching techniques. Stimulation with ionomycin and calyculin A, which activate the ROS-producing enzymatic activity of RbohD, increases the diffusion and oligomerization of RbohD. Abscisic acid and flg22 treatments also increase the diffusion coefficient and clustering of GFP-RbohD. Single-particle analysis in clathrin heavy chain2 mutants and a Flotillin1 artificial microRNA line demonstrated that clathrin- and microdomain-dependent endocytic pathways cooperatively regulate RbohD dynamics. Under salt stress, GFP-RbohD assembles into clusters and then internalizes into the cytoplasm. Dual-color fluorescence cross-correlation spectroscopy analysis further showed that salt stress stimulates RbohD endocytosis via membrane microdomains. We demonstrate that microdomain-associated RbohD spots diffuse at the membrane with high heterogeneity, and these dynamics closely relate to RbohD activity. Our results provide insight into the regulation of RbohD activity by clustering and endocytosis, which facilitate the activation of redox signaling pathways.

Integrated Multichannel Electronics for High Performance Time Correlated Single Photon Counting

The analysis of extremely weak, ultrafast and periodical light signals is increasingly widespread in a large number of fields, from chemistry and biology to telemetry and communication, and in a great variety of applications, from quantum cryptography to fluorescence imaging.Speaking of fluorescent lifetime analysis, for example, the detection of very faint and fast optical signals is fundamental to investigate the complex interactions between molecules that play a key role in many crucial issues such as the origin and growth mechanisms of tumors. In this domain the Time-Correlated Single Photon Counting (TCSPC) technique is very effective to reconstruct the light waveform when both extremely high time resolution and linearity are required.TCSPC is based on the detection of single photons and on the record of their arrival times: if the probability of detecting a photon during one excitation cycle is far less than one, the histogram of photon arrivals per time bin exactly corresponds to the temporal trend of the light signal. In order to properly reconstruct the light waveform a time measurement block featuring very high performance is necessary: this circuit has to be able to measure time intervals of few hundreds of nanoseconds with picosecond time resolution and a differential non-linearity much lower than few percents of the time bin width.Moreover a growing number of techniques do now require to collect data from multiple channels at the same time. Förster Resonance Energy Transfer (FRET), Fluorescence Diffuse Optical Tomography (FDOT) and single-molecule spectroscopy, for example, can benefit a lot of a system capable of providing simultaneous analysis in temporal or spectral range. Indeed, concurrency not only reduces the measurement time, revealing dynamic phenomena even on very short timescales but it also opens the way to spectrally resolved measurements that analyze different events occurring simultaneously, like Fluorescence Cross-Correlation Spectroscopy (FCCS).Nevertheless the development of a truly parallel acquisition system featuring both high performance and a high number of independent channels is still an open challenge: to achieve this goal the design of a time measurement circuit capable of acquiring data from several channels in parallel while providing high performance on each channel is of the utmost importance. In this work a fully integrated array of 16 time to amplitude converters (TAC) designed in 0.35µm SiGe technology is presented.The first simulations show very high performance on each channel with a FWHM time resolution lower than 19ps and a differential non linearity lower than 0.4% peak-to-peak of the LSB. In order to reduce the overall area of the chip, pairs of converter stages share all the conversion chain through a 2 input multiplexer. The simulated dead time on the entire full scale range is 191ns, corresponding to a maximum conversion frequency of 5.24MHz per channel. The obtained results on the designed 16 channel TAC array are extremely good and, if the performance are verified by experimental measurements, this array will be one of the fundamental blocks of a fully parallel high-performance TCSPC acquisition system. Based on high efficiency fully custom single photon detectors (SPAD) and on a front-end electronics specifically designed to properly work with these sensors, the final system will be the upgrade of a few channels high performance acquisition chain already developed in the same technology.