Raster Image Correlation Spectroscopy Research Articles

3D raster image correlation spectroscopy based on ultra-continuous laser STED system

Stimulated emission depletion is a kind of super resolution imaging technology widely used in biomedical fields, which improves far-field optical resolution to less than 100nm. In this paper, 3D raster image correlation spectroscopy which is based on ultra-continuous laser STED system is introduced. The optimized system will be discussed from the aspects of precise collinear alignment, pulse time delay and central intensity of STED spot. Experiments show that the resolution is about 60nm. The laser reduces the effects of photobleaching and dark state. Moreover, the combination of STED and 3D raster image correlation spectroscopy can extend applications of STED into molecular dynamics. Simplified but rigorous fitting equations were rewritten according to the benefits from the laser. Diffusion coefficients of particles in different situations are obtained.

Distinct Mechanisms Regulate Lck Spatial Organization in Activated T Cells.

Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation.

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Arbitrary-Region Image Correlation Spectroscopy

Combining imaging with correlation spectroscopy (ICS), as in raster image correlation spectroscopy (RICS), makes it possible to extract molecular translational diffusion constants (D), absolute concentrations (c), and intermolecular binding constants from single or multicolor confocal laser-scanning microscopy (CLSM) images. The difficulty with performing quantitative fluctuation imaging in living cells is complications due to aggregates or vesicles moving with the image. Also, current algorithms require square or rectangular regions-of-interest (ROI), which is often problematic in a live-cell context. Here, we describe a new algorithm for performing ICS with an arbitrary ROI. This enables whole-cell and even cell-organelle specific analyses to be performed. We describe the theory behind arbitrary region ICS (ARICS) as well as ways of automatically generating the ROI, given some simple threshold parameters. Automatic ROI generation significantly reduces analysis time, opening the door for medium-throughput automated analysis, and allows threshold-specific analyses. We demonstrate the advantages of ARICS on both simulated data and from measurements in live HeLa and E.coli cells. Arbitrary region ICS has the potential of becoming the new standard for extracting biophysical parameters from confocal fluorescence images.

Restricted Diffusion of cAMP is Mediated by Mitochondrial Localized PKA Buffering in Living Cells

Different G-protein coupled receptors can elicit unique functional responses by selectively utilizing specific subsets of spatially restricted cAMP signaling domains. Previous studies have concluded that this can only be achieved if the rate of cAMP movement within cells is much slower than the rate of free diffusion. However, this prediction has not been demonstrated experimentally. In this study, we directly test the hypothesis that the cytoplasmic diffusion of cAMP is indeed slow and then address the long standing question of what regulates its movement. We used Raster Image Correlation Spectroscopy (RICS) to directly measure the diffusion coefficients of various molecules in intact HEK 293 cells and adult ventricular cardiac myocytes to evaluate the effect of particle size and cell morphology on cAMP movement. Fluorescein and pharos-450 (φ450), fluorescent molecules similar in size to cAMP, exhibited diffusion coefficients 3-4 fold slower than their expected rates of free diffusion. The diffusion coefficient of φ450-labeled cAMP (φ450-cAMP) was >6 fold slower than fluorescein or φ450 free dye alone. This extremely slow rate of diffusion could not be explained by its size. In cardiac myocytes, φ450-cAMP co-localized with the type II regulatory (RII) subunit of protein kinase A (PKA). In addition, disruption of RII subunit anchoring to A kinase anchoring proteins (AKAPs) significantly increased the diffusion coefficients of both RII subunits and φ450-cAMP. Furthermore, RII subunits and φ450-cAMP co-localized with mitochondria. These results demonstrate that the cytosolic movement of cAMP is indeed much slower than the predicted rate of free diffusion and that this is due to interactions with PKA regulatory subunits associated specifically with mitochondria. These findings have important implications with respect to cAMP signaling in all cell types.

The E.coli Sec Pathway under a Single-Molecule Loupe

Protein targeting and secretion is an essential biological process in all life forms. In E. coli, the Sec translocase machinery consists of a conserved protein-conducting channel (SecYEG), which associates with cytoplasmic partners such as SecA. Although busily studied, important mechanistic details on bacterial protein targeting and secretion are still lacking. Since protein conformation and dynamics seems to be key to the function of translocases, we therefore carried out quantitative studies on the dynamic conformation of SecA in vitro using single-pair Forster resonance energy transfer (FRET) on a home-built multiparameter fluorescence detection microscope with pulsed interleaved excitation (MFD-PIE). Using FRET restrained structural modeling (FPS), we selected suitable FRET pairs on SecA. The different positions were mutated to Cysteines, and optimal conditions for labeling with maleimide dyes were determined. Preliminary FRET measurements were performed that provide insight in the stoichiometry of SecA in solution. The perspective is to study all the SecA mutants to create a dynamic 3D model of SecA. In a second part, we perform a biophysical analysis of the Sec pathway in vivo in E. coli. To do this, we have selected plasmids that allow controllable expression of the proteins. Then, we employed number and brightness analysis and raster image correlation spectroscopy to study the stoichiometry and dynamics of SecA in E. coli using a confocal laser scanning microscope.

Mechanisms Restricting Diffusion of Intracellular cAMP.

Although numerous receptors stimulate cAMP production in a wide array of cells, many elicit distinct, highly localized responses, implying that the subcellular distribution of cAMP is not uniform. One often used explanation is that phosphodiesterases, which breakdown cAMP, act as functional barriers limiting diffusion. However, several studies refute the notion that this is sufficient, suggesting that phosphodiesterase-independent movement of cAMP must occur at rates slower than free diffusion. But, until now this has never been demonstrated. Using Raster Image Correlation Spectroscopy (RICS), we measured the diffusion coefficient of a fluorescently-labeled cAMP derivative (φ450-cAMP) as well as other fluorescent molecules in order to investigate the role that molecular size, cell morphology, and buffering by protein kinase A (PKA) play in restricting cAMP mobility in different cell types. Our results demonstrate that cytosolic movement of cAMP is indeed much slower than the rate of free diffusion and that interactions with PKA, especially type II PKA associated with mitochondria, play a significant role. These findings have important implications with respect to cAMP signaling in all cells.

Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers.

Monitoring few molecular binding events in scalable confined aqueous compartments by raster image correlation spectroscopy (CADRICS).

The assembly of scalable liquid compartments for binding assays in array formats constitutes a topic of fundamental importance in life sciences. This challenge can be addressed by mimicking the structure of cellular compartments with biological native conditions. Here, inkjet printing is employed to develop up to hundreds of picoliter aqueous droplet arrays stabilized by oil-confinement with mild surfactants (Tween-20). The aqueous environments constitute specialized compartments in which biomolecules may exploit their function and a wide range of molecular interactions can be quantitatively investigated. Raster Image Correlation Spectroscopy (RICS) is employed to monitor in each compartment a restricted range of dynamic intermolecular events demonstrated through protein-binding assays involving the biotin/streptavidin model system.

Interplay between flow and diffusion in capillary alginate hydrogels.

Alginate gels with naturally occurring macroscopic capillaries have been used as a model system to study the interplay between laminar flow and diffusion of nanometer-sized solutes in real time. Calcium alginate gels that contain homogeneously distributed parallel-aligned capillary structures were formed by external addition of crosslinking ions to an alginate sol. The effects of different flow rates (0, 1, 10, 50 and 100 μl min(-1)) and three different probes (fluorescein, 10 kDa and 500 kDa fluorescein isothiocyanate-dextran) on the diffusion rates of the solutes across the capillary wall and in the bulk gel in between the capillaries were investigated using confocal laser scanning microscopy. The flow in the capillaries was produced using a syringe pump that was connected to the capillaries via a tube. Transmission electron microscopy revealed an open aggregated structure close to the capillary wall, followed by an aligned network layer and the isotropic network of the bulk gel. The most pronounced effect was observed for the 1 nm-diameter fluorescein probe, for which an increase in flow rate increased the mobility of the probe in the gel. Fluorescence recovery after photobleaching confirmed increased mobility close to the channel, with increasing flow rate. Mobility maps derived using raster image correlation spectroscopy showed that the layer with the lowest mobility corresponded to the anisotropic layer of ordered network chains. The combination of microscopy techniques used in the present study elucidates the flow and diffusion behaviors visually, qualitatively and quantitatively, and represents a promising tool for future studies of mass transport in non-equilibrium systems.

Inhibition of Receptor Dimerization as a Novel Negative Feedback Mechanism of EGFR Signaling.

Dimerization of the epidermal growth factor receptor (EGFR) is crucial for initiating signal transduction. We employed raster image correlation spectroscopy to continuously monitor the EGFR monomer-dimer equilibrium in living cells. EGFR dimer formation upon addition of EGF showed oscillatory behavior with a periodicity of about 2.5 min, suggesting the presence of a negative feedback loop to monomerize the receptor. We demonstrated that monomerization of EGFR relies on phospholipase Cγ, protein kinase C, and protein kinase D (PKD), while being independent of Ca2+ signaling and endocytosis. Phosphorylation of the juxtamembrane threonine residues of EGFR (T654/T669) by PKD was identified as the factor that shifts the monomer-dimer equilibrium of ligand bound EGFR towards the monomeric state. The dimerization state of the receptor correlated with the activity of an extracellular signal-regulated kinase, downstream of the EGFR. Based on these observations, we propose a novel, negative feedback mechanism that regulates EGFR signaling via receptor monomerization.

Raster image cross-correlation analysis for spatiotemporal visualization of intracellular degradation activities against exogenous DNAs.

Reducing intracellular DNA degradation is critical to enhance the efficiency of gene therapy. Exogenous DNA incorporation into cells is strictly blocked by the defense machinery of intracellular nuclease activity. Raster image correlation spectroscopy (RICS) and raster image cross-correlation spectroscopy (cross-correlation RICS; ccRICS) are image-based correlation methods. These powerful tools allow the study of spatiotemporal molecular dynamics. Here we performed spatiotemporal ccRICS analyses of fluorescent DNA and directly monitored the process of exogenous DNA degradation in living cell cytoplasm. Such direct monitors of DNA degradation allow us to determine the fate of the exogenous DNA in living cells. On comparing the process in living cells, our study shows that cytoplasmic nuclease activity differs between cell lines; therefore, we propose that the difference of nuclease activity in cytoplasm dictates a different resistance to exogenous DNA incorporation. New insight on efficient gene delivery can be provided with our study.

Characterization of exogenous DNA mobility in live cells through fluctuation correlation spectroscopy.

The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm and further inactive transportation along the peri-nuclear region. This study provides the first real-time insight into the trafficking of DNA delivered through lipofection using image-based fluctuation correlation spectroscopy approaches. Thereby, gaining information with single particle sensitivity to develop a deeper understanding of DNA lipoplex delivery through the cell.

Characterisation of Stress-Induced Aggregate Size Distributions and Morphological Changes of a Bi-Specific Antibody Using Orthogonal Techniques

A critical step in monoclonal antibody (mAb) screening and formulation selection is the ability of the mAb to resist aggregation following exposure to environmental stresses. Regulatory authorities welcome not only information on the presence of micron-sized particles, but often any information on sub-visible particles in the size range obtained by orthogonal sizing techniques. The present study demonstrates the power of combining established techniques such as dynamic light scattering (DLS) and micro-flow imaging (MFI), with novel analyses such as raster image correlation spectroscopy (RICS) that offer to bridge existent particle sizing gaps in this area. The influence of thermal and freeze–thaw stress treatments on particle size and morphology was assessed for a bi-specific antibody (mAb2). Aggregation of mAb2 was confirmed to be concentration- and treatment-dependent following thermal stress and freeze–thaw cycling. Particle size and count data show concentration- and treatment-dependent behaviour of aggregate counts, morphological descriptors and particle size distributions. Complementarity in particle size output was observed between all approaches utilised, where RICS bridged the analytical size gap (∼0.5–5μm) between DLS and MFI. Overall, this study highlights the potential of orthogonal image analyses such as RICS (analytical size gap) and MFI (particle morphology) for formulation screening.

Photothermal raster image correlation spectroscopy of gold nanoparticles in solution and on live cells.

Raster image correlation spectroscopy (RICS) measures the diffusion of fluorescently labelled molecules from stacks of confocal microscopy images by analysing correlations within the image. RICS enables the observation of a greater and, thus, more representative area of a biological system as compared to other single molecule approaches. Photothermal microscopy of gold nanoparticles allows long-term imaging of the same labelled molecules without photobleaching. Here, we implement RICS analysis on a photothermal microscope. The imaging of single gold nanoparticles at pixel dwell times short enough for RICS (60 μs) with a piezo-driven photothermal heterodyne microscope is demonstrated (photothermal raster image correlation spectroscopy, PhRICS). As a proof of principle, PhRICS is used to measure the diffusion coefficient of gold nanoparticles in glycerol : water solutions. The diffusion coefficients of the nanoparticles measured by PhRICS are consistent with their size, determined by transmission electron microscopy. PhRICS was then used to probe the diffusion speed of gold nanoparticle-labelled fibroblast growth factor 2 (FGF2) bound to heparan sulfate in the pericellular matrix of live fibroblast cells. The data are consistent with previous single nanoparticle tracking studies of the diffusion of FGF2 on these cells. Importantly, the data reveal faster FGF2 movement, previously inaccessible by photothermal tracking, and suggest that inhomogeneity in the distribution of bound FGF2 is dynamic.

Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments.

Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens.

Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues.

Mitochondrial Buffering of cAMP in Adult Cardiac Myocytes

Compartmentation of the diffusible second messenger cAMP plays a key role in producing unique responses to different G‐protein coupled receptors. It has been proposed that this can only happen if the movement of cAMP within the cell occurs at rates well below those of free diffusion. To directly test this hypothesis we used Raster Image Correlation Spectroscopy (RICS) to measure the diffusion coefficient of fluorescently labeled cAMP, 8‐[Pharos‐450]‐cAMP (P450‐cAMP) in live cells. Confocal imaging showed that incubation with the membrane permeable P450‐cAMP led to a distinct longitudinal pattern of fluorescence accumulation in adult rat ventricular myocytes. This was in contrast to the uniform distribution of cAMP‐free dye (P450), suggesting that P450‐cAMP binds to immobilized target. Interestingly, expression of PKA type II regulatory subunit labeled with CFP (RII‐CFP) exhibited a pattern similar to P450‐cAMP, which corresponded with the extra‐myofibrillar space populated by mitochondria, as verified by labeling with MitoTracker Red. RICS analysis found the diffusion coefficient (μm2/s) of P450‐cAMP (9.7±1.6, n=37) to be significantly slower than that of P450 (17.6 ± 1.73, n=14), but similar to that of RII‐CFP (12.1±1.85, n=11). Furthermore, Ht31, a peptide that disrupts interactions between RII and A kinase anchoring proteins (AKAPs), caused the distribution pattern of both P450‐cAMP and RII‐CFP to become more diffuse. Correspondingly, the diffusion coefficients for P450‐cAMP (34.4±4.37, n=15) and RII‐CFP (32.0±4.53, n=8) increased significantly, while that of P450 did not. These data suggest that the movement of cAMP in adult cardiac myocytes is limited by interactions with PKA RII subunits associated specifically with mitochondria.

Nuclear Actin Dynamics Regulate Nuclear Organization and Transcription

2716-Pos Board B146 Nuclear Actin Dynamics Regulate Nuclear Organization and Transcription Leo Serebryannyy 1 , Megan Parilla 1 , Paolo Annibale 2 , Christina Cruz 1 , Dmitri Kudryashov 3 , Enrico Gratton 2 , Cara J. Gottardi 4 , Primal de Lanerolle 1 . Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA, 2 Laboratory of Fluorescence Dynamics, University of California at Irvine, Irvine, CA, USA, 3 Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA, 4 Medicine, Northwestern University, Chicago, IL, USA. Actin is an important and elegant mechanotransducer that transfers messages from the extracellular environment into the nucleus, thus conferring changes in both the physical properties and the genetic program of the cell. Although actin in the nucleus has been established in a growing number of functions, the form of nuclear actin remains poorly understood as there are no apparent actin filaments in the nucleus. Therefore, we tested how disrupting the form of nuclear actin impacts function and what effects this may have on the nucleus. To do so, we formed stable nuclear actin filaments using a variety of methods, including the nuclear enrichment of actin binding proteins supervillin and a-E-catenin and the expression of a mutant form of skeletal a-actin. Using fluorescence recovery after photobleaching (FRAP), we found that stabiliza- tion of nuclear actin filaments significantly impairs actin dynamics within the nucleus. The formation of nuclear actin filaments coincides with striking changes in nuclear structure and overall nuclear topography as determined by confocal microscopy and raster image correlation spectroscopy. Using a com- bination of FRAP analysis, structured illumination microscopy and immuno- logical assays, we found that nuclear actin filaments reduce the association of actin with RNA polymerase II and this correlates with impaired RNA poly- merase II dynamics, localization and gene recruitment. Moreover, we were able to recapitulate our findings in purified nuclear extract by using in vitro transcription assays with the covalent actin crosslinking domain (ACD) of V. cholerae MARTX toxin. Based on our data, which help explain the absence of nuclear actin filaments in the interphase mammalian nucleus, we propose a model where nuclear actin dynamics are critical for maintaining proper nu- clear function.

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a “membrane fence” model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more “fluid” and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family.In conclusion: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R–Gq/G11α protein coupling.