K-space Image Correlation Spectroscopy Research Articles

Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs.

Some membrane proteins cluster in nanoscale cholesterol and sphingomyelin rich membrane domains, called lipid rafts, that transform into ceramide-rich rafts and coalesce into large ceramide-rich platforms during cell stress. However, the clustering mechanisms and their impact on membrane protein stability and function remain uncertain. We reported previously that the cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in the hyperinflammatory pulmonary disease cystic fibrosis (CF), forms clusters that are cholesterol-dependent and become incorporated into large and long-lived ceramide-rich platforms during hormonal stimulation. Using a combination of optimized fluorescence confocal imaging and quantitative analyses such as image correlation spectroscopy (ICS) and the k-space ICS (kICS), we report here that CFTR clustering at the plasma membrane of primary human bronchial epithelial cells does not involve known tethering interactions of CFTR with PDZ domain-containing proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are impaired by the CF disease-causing mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate that CF therapeutics that correct mutant protein folding restore both CFTR trafficking and normal lipid interactions in the plasma membrane which results in its stability at the plasma membrane and its functional restoration.

Using kICS to Reveal Changed Membrane Diffusion of AQP-9 Treated with Drugs.

The formation of nanodomains in the plasma membrane are thought to be part of membrane proteins regulation and signaling. Plasma membrane proteins are often investigated by analyzing the lateral mobility. k-space ICS (kICS) is a powerful image correlation spectroscopy (ICS) technique and a valuable supplement to fluorescence correlation spectroscopy (FCS). Here, we study the diffusion of aquaporin-9 (AQP9) in the plasma membrane, and the effect of different membrane and cytoskeleton affecting drugs, and therefore nanodomain perturbing, using kICS. We measured the diffusion coefficient of AQP9 after addition of these drugs using live cell Total Internal Reflection Fluorescence imaging on HEK-293 cells. The actin polymerization inhibitors Cytochalasin D and Latrunculin A do not affect the diffusion coefficient of AQP9. Methyl-β-Cyclodextrin decreases GFP-AQP9 diffusion coefficient in the plasma membrane. Human epidermal growth factor led to an increase in the diffusion coefficient of AQP9. These findings led to the conclusion that kICS can be used to measure diffusion AQP9, and suggests that the AQP9 is not part of nanodomains.

AQP2 Plasma Membrane Diffusion Is Altered by the Degree of AQP2-S256 Phosphorylation.

Fine tuning of urine concentration occurs in the renal collecting duct in response to circulating levels of arginine vasopressin (AVP). AVP stimulates intracellular cAMP production, which mediates exocytosis of sub-apical vesicles containing the water channel aquaporin-2 (AQP2). Protein Kinase A (PKA) phosphorylates AQP2 on serine-256 (S256), which triggers plasma membrane accumulation of AQP2. This mediates insertion of AQP2 into the apical plasma membrane, increasing water permeability of the collecting duct. AQP2 is a homo-tetramer. When S256 on all four monomers is changed to the phosphomimic aspartic acid (S256D), AQP2-S256D localizes to the plasma membrane and internalization is decreased. In contrast, when S256 is mutated to alanine (S256A) to mimic non-phosphorylated AQP2, AQP2-S256A localizes to intracellular vesicles as well as the plasma membrane, with increased internalization from the plasma membrane. S256 phosphorylation is not necessary for exocytosis and dephosphorylation is not necessary for endocytosis, however, the degree of S256 phosphorylation is hypothesized to regulate the kinetics of AQP2 endocytosis and thus, retention time in the plasma membrane. Using k-space Image Correlation Spectroscopy (kICS), we determined how the number of phosphorylated to non-phosphorylated S256 monomers in the AQP2 tetramer affects diffusion speed of AQP2 in the plasma membrane. When all four monomers mimicked constitutive phosphorylation (AQP2-S256D), diffusion was faster than when all four were non-phosphorylated (AQP2-S256A). AQP2-WT diffused at a speed similar to that of AQP2-S256D. When an average of two or three monomers in the tetramer were constitutively phosphorylated, the average diffusion coefficients were not significantly different to that of AQP2-S256D. However, when only one monomer was phosphorylated, diffusion was slower and similar to AQP2-S256A. Thus, AQP2 with two to four phosphorylated monomers has faster plasma membrane kinetics, than the tetramer which contains just one or no phosphorylated monomers. This difference in diffusion rate may reflect behavior of AQP2 tetramers destined for either plasma membrane retention or endocytosis.

Opposing Effects of cAMP and T259 Phosphorylation on Plasma Membrane Diffusion of the Water Channel Aquaporin-5 in Madin-Darby Canine Kidney Cells.

Aquaporin-5 (AQP5) facilitates passive water transport in glandular epithelia in response to secretory stimuli via intracellular pathways involving calcium release, cAMP and protein kinase A (PKA). In epithelial plasma membranes, AQP5 may be acutely regulated to facilitate water transport in response to physiological stimuli by changes in protein modifications, interactions with proteins and lipids, nanoscale membrane domain organization, and turnover rates. Such regulatory mechanisms could potentially be associated with alteration of diffusion behavior, possibly resulting in a change in the plasma membrane diffusion coefficient of AQP5. We aimed to test the short-term regulatory effects of the above pathways, by measuring lateral diffusion of AQP5 and an AQP5 phospho-mutant, T259A, using k-space Image Correlation Spectroscopy of quantum dot- and EGFP-labeled AQP5. Elevated cAMP and PKA inhibition significantly decreased lateral diffusion of AQP5, whereas T259A mutation showed opposing effects; slowing diffusion without stimulation and increasing diffusion to basal levels after cAMP elevation. Thus, lateral diffusion of AQP5 is significantly regulated by cAMP, PKA, and T259 phosphorylation, which could be important for regulating water flow in glandular secretions.

Get Your kICS by Measuring Membrane Protein Dynamics

Akin to thepuckon an ice hockey rink,the lateral dynamics of a representativemembrane protein exhibits rich be-havior(1).Dependingontheinteractionwiththehockeyplayersandotherobsta-cles (membrane constituents), the puck(protein)candart aroundthe ice(mem-brane surface) or be stalled momen-tarily (transient confinement zone). Ifwe watched the motion of a singlepuck, we would need to watch manyhockey games beforewe might eventu-allyworkouttherulesofthegame.Butwhat if we could watch many games inparallel? This is where the suite of im-age correlation spectroscopy (ICS)techniques and its newest member, k-space image correlation spectroscopy(kICS) comes in Abu-Arish et al. (2).ICSandkICSaremembersofafam-ily of techniques which began withfluorescence correlation spectroscopy(3). Fluorescence fluctuation analysisprovides the statistical mechanicalfoundation with correlations betweenfluorescence fluctuations measuredrelative to a given lag vector. Correla-tions can be made as a function oftime (fixed space) called FCS (3), fluc-tuationsinspace(fixedtime)calledICS(4), and space-time fluctuations calledSTICS (5). The ergodic principle en-sures equivalence between occupancyfluctuations whether in time or space.In temporal ICS or TICS, one essen-tially images in time (using a laserscanning fluorescence confocal micro-scope) many molecules in parallel asthey change positions from one imageto the next image. If the molecules donot move between the first image andthe second image, the spatial correla-tion between image one and imagetwo will be high. However, if move-ment does occur between image oneand imagetwo,there isaloss of spatialcorrelation. By analyzing the correla-tion between images collected atdifferent times, information on proteinmotioncouldbeobtained.Thismethodprovidesinformation from manymole-cules without the need for tracking in-dividual particles. A problem endemicto all these methods is that fluctuationsin fluorescence are measured asopposed to particle fluctuations whichare the desired quantities in evaluatingtransport properties.In kICS (6), images collected from atime-seriesarefirstFouriertransformedinto k-space before image cross-corre-lation is performed. This has twodistinct advantages over (real space)ICS. First, fluctuations from photophy-sics (e.g., cis-trans isomerization, (de)protonation, reversible dark statequenching, reversible photobleaching)do not contribute to the determinationof transport coefficients (6). Second,determinationofthelateralpointspreadfunction dimension is not required, un-like conventional fluorescence fluctua-tion approaches (6).In this issueof the Biophysical Jour-nal, the pioneer of ICS and kICS, PaulWisemanhasteamedupwithAsmahanAbu-Arish,ElvisPandzic,JulieGoepp,Elizabeth Matthes, and John Hanrahan(2) to gain new insights into the dy-namics of a biomedically importantmembrane protein, the cystic fibrosistransmembrane conductance regulator.Using the kICS technique, the authorshaveprovidedevidencefortwopopula-tions of cystic fibrosis transmembraneconductance regulator (CFTR) mole-cules, which differed in degree ofconfinement and lateral motion on thecell surface. Impressively, they wereable to extract information on the dy-namics of CFTR inside domains,CFTR dynamicsoutside(andbetween)domains, fractional populations, anddegree of confinement (within do-mains). ICS analysis delivered clusterdensities and mean number of mole-cules per cluster.The authors examined the effect ofcholesterol on dynamics and clusteringbehavior of CFTR. Cholesterol has aremarkable effect on membrane pro-tein assembly, dynamics and function(7–9). Depletion of cholesterol causedthe confinedfractionand averagenum-ber of CFTR molecules per cluster todecrease, whereas increase in choles-terol were found to be associated withincrease in clustering and increasedconfined fraction. Interestingly, viralinfection was shown to increase clus-tering further into larger platformswith reduced CFTR mobility. Theseobservations and analyses suggestthat cholesterol-influenced membranedomains play an important role in thecell surface behavior and pathologyof CFTR.Aside from the important new in-sights into this anion channel, the re-sults of this study revealed howcomplex cell surface dynamics andclustering can be measured usingpowerful fluorescence microscopytechniques. With these new methodsin hand, biophysicists can sit back,relax and enjoy the hockey game.REFERENCES

Cholesterol Modulates CFTR Confinement in the Plasma Membrane of Primary Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function.

A high fat diet containing saturated but not unsaturated fatty acids enhances T cell receptor clustering on the nanoscale

Cell culture studies show that the nanoscale lateral organization of surface receptors, their clustering or dispersion, can be altered by changing the lipid composition of the membrane bilayer. However, little is known about similar changes in vivo, which can be effected by changing dietary lipids. We describe the use of a newly developed method, k-space image correlation spectroscopy, kICS, for analysis of quantum dot fluorescence to show that a high fat diet can alter the nanometer-scale clustering of the murine T cell receptor, TCR, on the surface of naive CD4+ T cells. We found that diets enriched primarily in saturated fatty acids increased TCR nanoscale clustering to a level usually seen only on activated cells. Diets enriched in monounsaturated or n-3 polyunsaturated fatty acids had no effect on TCR clustering. Also none of the high fat diets affected TCR clustering on the micrometer scale. Furthermore, the effect of the diets was similar in young and middle aged mice. Our data establish proof-of-principle that TCR nanoscale clustering is sensitive to the composition of dietary fat.

The Role of ROS in Tethering CFTR within Ceramide Platforms at the Plasma Membrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel which is expressed at the apical surface of many secretory epithelia. CFTR and reactive oxygen species (ROS) are both involved in innate immune responses against bacterial pathogens, however the link between them during host defense is not well understood. Here we study the surface expression and dynamics of CFTR in epithelial cells during protein kinase C (PKC) stimulation using fluorescence microscopy and k-space image correlation spectroscopy (kICS). kICS analysis was used to measure the confined dynamics of CFTR under control conditions and during PKC stimulation while simultaneously monitoring CFTR expression at the cell surface as total fluorescence intensity. During PKC stimulation, CFTR became aggregated into large (1 −2 µm diameter) structures on the plasma membrane called platforms. PKC induced a significant (1.5-fold) increase in CFTR surface expression and increased the CFTR confinement within platforms, as indicated by a decline in its confined diffusion coefficient from 0.011±0.001 µm2/s to 0.0033±0.0002 µm2/s. Pharmacological inhibition of ROS or acid sphingomyelinase (ASM) prevented the PKC-induced aggregation and tethering of CFTR. This suggests a scheme in which PKC stimulates the production of ROS, which activates ASM and ceramide synthesis, leading to the formation of platforms in the outer leaflet of the plasma membrane. Ceramide platforms may help stabilize CFTR in the membrane so that its surface expression and channel function are increased during secretagogue stimulation.

Measuring localization and diffusion coefficients of basolateral proteins in lateral versus basal membranes using functionalized substrates and kICS analysis

Micropatterning enabled semiquantitation of basolateral proteins in lateral and basal membranes of the same cell. Lateral diffusion coefficients of basolateral aquaporin-3 (AQP3-EGFP) and EGFP-AQP4 were extracted from “lateral” and “basal” membranes using identical live-cell imaging and k-space Image Correlation Spectroscopy (kICS).To simultaneously image proteins in “lateral” and “basal” membranes, micropatterning with the extracellular domain of E-cadherin and collagen, to mimic cell–cell and cell-extracellular matrix (ECM) adhesion, respectively, was used. In kidney collecting duct principal cells AQP3 localizes lateral and basal whereas AQP4 localizes mainly basal. On alternating stripes of E-cadherin and collagen, AQP3-EGFP was predominantly localized to “lateral” compared to “basal” membranes, whereas Orange-AQP4 was evenly distributed. Average diffusion coefficients were extracted via kICS analysis of rapid time-lapse sequences of AQP3-EGFP and EGFP-AQP4 on uniform substrates of either E-cadherin or collagen. AQP3-EGFP was measured to 0.022±0.010μm2/s on E-cadherin and 0.019±0.004μm2/s on collagen, whereas EGFP-AQP4 was measured to 0.044±0.009μm2/s on E-cadherin and 0.037±0.009μm2/s on collagen, thus, diffusion did not differ between substrates. Cholesterol depletion by methyl-beta-cyclodextrin (MBCD) reduced the AQP3-EGFP diffusion coefficient by 43% from 0.024±0.007μm2/s (water) to 0.014±0.003μm2/s (MBCD) (p<0.05) on collagen surfaces, and by 41% from 0.023±0.011μm2/s (water) to 0.014±0.005μm2/s (MBCD) (p<0.05) on E-cadherin surfaces. Thus, protein patterning enables the semiquantitation of protein distribution between the “lateral” and “basal” membranes as well as measurements of lateral diffusion coefficients.

Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-Space Image Correlation Spectroscopy.

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)(1)was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a "slope of slopes" plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

Elevated cAMP increases aquaporin-3 plasma membrane diffusion

Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water exits via basolateral AQP3 and AQP4. Upon long-term stimulation with AVP or during thirst, expression levels of both AQP2 and AQP3 are increased; however, there is so far no evidence for short-term AVP regulation of AQP3 or AQP4. To facilitate the increase in transepithelial water transport, AQP3 may be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts, and/or changes in steady-state turnover, which could result in changes in the diffusion behavior of AQP3. Thus we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin to reveal if AQP3 could be short-term regulated by AVP. k-Space image correlation spectroscopy (kICS) analysis of time-lapse image sequences of basolateral enhanced green fluorescent protein-tagged AQP3 (AQP3-EGFP) revealed that the forskolin-mediated elevation of cAMP increased the diffusion coefficient by 58% from 0.0147 ± 0.0082 μm(2)/s (control) to 0.0232 ± 0.0085 μm(2)/s (forskolin, P < 0.05). Quantum dot-conjugated antibody labeling also revealed a significant increase in AQP3 diffusion upon forskolin treatment by 44% [0.0104 ± 0.0040 μm(2)/s (control) vs. 0.0150 ± 0.0016 μm(2)/s (forskolin, P < 0.05)]. Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption.

Measuring Ligand-Receptor Binding Rates with K-Space Image Correlation Spectroscopy: Theory and Experimental Applications

The ability to measure accurate kinetic binding rates of ligand-receptor and receptor-receptor interactions is important in order to study the triggering of signaling events in cell membranes. We apply an image analysis technique, k-space image correlation spectroscopy (kICS), to measure the kinetic binding rates of ligand-receptor interactions from fluorescence video-microscopy data occurring within a thin plane of illumination. We consider two different kinds of experiments where: (1) fluorescently tagged ligands in solution bind and unbind dynamically to unlabeled receptors which are allowed to diffuse in the imaging plane and, (2) fluorescently tagged, diffusing receptors in the imaging plane interact with other (possibly non-fluorescing) receptors and interconvert between two diffusive states. For system (1), by measuring image intensity fluctuations of fluorescently labeled human doublecortin proteins in solution (2.5 nM), binding to microtubules adhering on a sample substrate, the kICS method accurately determines kinetic dissociation rates (kd = 1.6 ± 0.2 s−1) as well as diffusion coefficients (0.38 ± 0.19 μm2/s) of the proteins consistent with single-particle tracking data. For system (2), we perform drug-trial experiments on living COS-7 cells to show that kICS can quantify the degree to which the drugs will affect kinetic binding/unbinding rates. By labeling the glycolipid membrane receptor GM1 with a fluorescent cholera toxin B-subunit, we show that cytoskeleton perturbations with the drug cytochalasin D result in significant changes (ku = 0.013 ± 0.004 s−1 to 0.08 ± 0.02 s−1) to the undocking rate of GM1 to the actin cytoskeleton. With these two proof-of-concept experiments, we pave the way to using kICS as an efficient and reliable method to elucidate kinetic binding rates and transport dynamics parameters for studies in biomembranes.

Bridging the Gap between Single Molecule and Ensemble Methods for Measuring Lateral Dynamics in the Plasma Membrane

The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed for analyzing lateral dynamics in samples that are labeled at high densities, can also be used for fast and accurate analysis of single molecule density data of lipids and proteins labeled with quantum dots (QDs). We have further used kICS to investigate the effect of the label size and by comparing the results for a biotinylated lipid labeled at high densities with Atto647N-strepatvidin (sAv) or sparse densities with sAv-QDs. In this latter case, we see that the recovered diffusion rate is two-fold greater for the same lipid and in the same cell-type when labeled with Atto647N-sAv as compared to sAv-QDs. This data demonstrates that kICS can be used for analysis of single molecule data and furthermore can bridge between samples with a labeling densities ranging from single molecule to ensemble level measurements.

Measuring ligand–receptor binding kinetics and dynamics using k-space image correlation spectroscopy

Accurate measurements of kinetic rate constants for interacting biomolecules are crucial for understanding the mechanisms underlying intracellular signalling pathways. The magnitude of binding rates plays a very important molecular regulatory role which can lead to very different cellular physiological responses under different conditions. Here, we extend the k-space image correlation spectroscopy (kICS) technique to study the kinetic binding rates of systems wherein: (a) fluorescently labelled, free ligands in solution interact with unlabelled, diffusing receptors in the plasma membrane and (b) systems where labelled, diffusing receptors are allowed to bind/unbind and interconvert between two different diffusing states on the plasma membrane. We develop the necessary mathematical framework for the kICS analysis and demonstrate how to extract the relevant kinetic binding parameters of the underlying molecular system from fluorescence video-microscopy image time-series. Finally, by examining real data for two model experimental systems, we demonstrate how kICS can be a powerful tool to measure molecular transport coefficients and binding kinetics.

Ligand Receptor Binding Rate Kinetics via K-Space Image Correlation Spectroscopy: An in Silico Study

To achieve a fundamental understanding of intra-cellular signalling pathways, it will be necessary to measure the rates of reaction between chemically reacting and interacting macromolecules. The magnitude of binding rates plays a very important molecular regulatory role in a range of systems including the binding of antigens to T-cell receptors, which can lead to differing immune-responses, as well as rates of hydrolysis of GTPs with Rac or Rho proteins, which lead to temporal changes in actin filament lengths and cell motility. Here, we develop an image correlation technique which may be used to study such systems in conjunction with fluorescence microscopy. We simulate receptors diffusing in 2D with diffusion coefficients varying from 0 to 10 pixels2/frame. Receptors are only visible as point emitters when a fluorescent ligand is bound to the receptor; ligand binding kinetics are created by turning receptors ‘on' or ‘off' with average life-times varying from 3 to 20 frames drawn from a negative exponential distribution. Photobleaching is similarly incorporated by permanently turning diffusing receptors ‘off' after average times varying from 20 to 400 frames. The image time-series is generated by convolving a Gaussian function of fixed radius with the point emitter distribution to create 128x128 pixel images with background noise added to give a signal to noise ratio of 3. We use k-space image correlation spectroscopy (kICS) and develop a two-state kinetic binding model with freely diffusing receptors on a 2D membrane to capture the membrane binding kinetics of the image time-series. We show that kICS can accurately recover on/off binding rates at the 95% confidence level for over 75% of simulations. Typically, percentage errors are less than 30% for weak photobleaching and within 60% for strong photobleaching effects.

Role of Cftr in Host Defence Against Pseudomonas Aeruginosa

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial anion channel which is mutated in people with cystic fibrosis (CF). Loss of CFTR function in the airways leads to bacterial infection, chronic inflammation and eventually respiratory failure. Interactions between CFTR and the most common CF pathogen, Pseudomonas aeruginosa (PA), are not well understood. In this study we have studied the dynamical and functional effects of bacterial infection on CFTR in human primary airway epithelial cells. A new analysis based on the well-established k-space image correlation spectroscopy technique (kICS) was used to extract the mean-square-displacement (MSD) of fluorescently labeled CFTR (GFP-CFTR) molecules in time. Under resting conditions, GFP-CFTR formed sub-resolution clusters that were homogeneously distributed in sub-confluent cells. kICS analysis revealed the presence of two dynamically distinct populations, one which was freely diffusing (Ddiff = 1.5 ±0.3 x10-2 μm2/s) and other which was spatio-temporally confined (Dconfine = 10 ±2 x10-3 μm2/s). The confined population was dependent on cholesterol and sphingomyelin. When epithelial cells were exposed to PA, small CFTR clusters aggregated into large platforms (0.5-3 μm). This was accompanied by a significant reduction in CFTR dynamics (from Ddiff = 0.2 ±0.2 x10-2 μm2/s to Dconfine = 2.5 ±0.4 x10-3 μm2/s). Pre-treatment with lipid raft disrupting reagents prevented platforms from forming upon treatment with PA and the subsequent reduction of CFTR dynamics. CFTR platform formation is also triggered by several host defence mechanisms and inflammatory factors even in the absence of bacteria, therefore it may help protect the host epithelial cell against invasion by pathogens.

High dose antigen stimulation leads to binding defects caused by persistent spatial segregation of TCR and CD8 in cytotoxic T cells (178.9)

Abstract The lateral organization of surface receptors controls ligand binding and activation. Our group has shown that activated T Cells exhibit persistent clustering of T Cell Receptor (TCR) several days after stimulation, leading to enhanced binding of soluble Major Histocompatibility Complex (MHC) dimers at low concentrations. In contrast, other groups have shown a complete loss of binding in activated T Cells during the same time period. Here, we resolve this contradiction by demonstrating that persistent binding behavior is controlled by the dose of antigen during primary activation. T Cells from TCR transgenic mice stimulated with high but not low dose of peptide are unable to specifically bind soluble Kb-SIY or Db-GP100 MHC dimer four days after stimulation. The behavior is not due to differences in the surface expression of TCR, CD8alpha, or CD8beta. Cytokine production mirrors the loss of binding. However, both high and low dose stimulated cells are able to bind the so-called ‘CD8 independent’ Ld-QL9 dimer, implicating the coreceptor in the binding defect. Using biophysical binding assays, k-space Image Correlation Spectroscopy, and Forster Resonance Energy Transfer between TCR and CD8, we demonstrate that TCR and CD8 are segregated, and TCR unclustered, on high dose activated T Cells, impairing CD8-dependent binding. Thus, membrane organization derived binding defects represent a mechanism for controlling T Cell responses to supraoptimal antigen.

Quantum Dot Fluorescence Characterizes the Nanoscale Organization of T Cell Receptors for Antigen

Changes in the clustering of surface receptors modulate cell responses to ligands. Hence, global measures of receptor clustering can be useful for characterizing cell states. Using T cell receptor for antigen as an example, we show that k-space image correlation spectroscopy of quantum dots blinking detects T cell receptor clusters on a scale of tens of nanometers and reports changes in clustering after T cell activation. Our results offer a general approach to the global analysis of lateral organization and receptor clustering in single cells, and can thus be applied when the cell type of interest is rare.

Motility and Segregation of Hsp104-Associated Protein Aggregates in Budding Yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.

Persistent Changes in T Cell Receptor Surface Organization Visualized by Quantum Dot Probes with k-Space Image Correlation Spectroscopy (50.2)

Abstract There is growing evidence that the surface pattern and spatial distribution of T cell receptors (TCRs) on cell surfaces plays a critical role in T Cell antigen detection and effector function, from micron-sized Supramolecular Adhesion Clusters to microclusters 100’s of nanometers in diameter. To date, TCR organization patterns have been primarily examined in the context of antigen stimulation or immediately thereafter, on a timescale of seconds to minutes, despite evidence from binding studies that shows changes persist many days post-activation. Here, we present a method for imaging long-term TCR lateral surface organization on live cells at two relevant length scales: 10’s and 100’s of nms. TCR are labeled with monomeric biotin-MHC/peptide or antibody fragments, followed by streptavidin-quantum dots (QD). We have found that QD “blinking” is a useful parameter for detecting TCR organization on a nm scale, since electronic coupling between blinking states of adjacent QDs reports single or small clusters of receptors. Simultaneously, we quantify organization at the scale of 100’s of nms by epifluorescense. As proof of principle, we have demonstrated that in vitro activated T cells have enhanced clustering at the 10’s and 100’s nm scales compared to naïve counterparts, supporting results from MHC dimer binding assays. Clustering can also be modulated by IL-2 signaling for active, but not naïve, T cells.