Fluorescence recovery after photobleaching analysis after DNA damage induction in UV-irradiated cells.LiMRE11-GFP is recruited to DNA damage sites in human MRE1
The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an
We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although ...
Nucleostemin, a protein found in the nucleoli of highly proliferative cells (such as stem cells and some cancer cell lines), may help regulate cell proliferation. Tsai and McKay, who previously identified nucleostemin, have now investigated the mechanisms whereby it is targeted to the nucleolus. The authors used fluorescence recovery after photobleaching (FRAP) and inverse FRAP (iFRAP) to show that nucleostemin tagged with green fluorescent protein shuttled rapidly and bidirectionally between the nucleolus and the nucleoplasm of cultured CHO and U2 OS cells. Mutation of a GTP-binding motif (G1) that blocked the ability of nucleostemin to bind GTP also blocked its nucleolar localization, as did deletion of an N-terminal basic (B) domain. FRAP, together with further mutational analysis, indicated that GTP binding relieved the inhibitory action of a domain between G1 and the C terminal on nucleolar localization of the B domain and was required for long-term retention of nucleostemin in the nucleus. ...
A LinkedIn group for those interested in or currently using Fluorescence Recovery After Photobleaching (FRAP) for pre-screening crystallization conditions.
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Using fluorescence recovery after photobleaching (FRAP), we have previously shown that the GFP-tagged glucocorticoid receptor is bound at a specific promoter fo...
Growth factors transmit biological signals for the stimulation of cell growth and their stimulation may be involved in turnourigenesis. It is therefore of great importance to understand growth factor receptor reactions in response to stimuli such as calcium depletion or ultraviolet radiation, which normal human cells are invariably exposed to during their growth cycle. We have studied human skin cells i.e. fibroblasts, lceratinocytes and melanocytes and their growth factor receptor expression on the surface of cells, reactions in the plane of the cell membrane, intracellular trafficlcing, and gene expression after exposure to their ligand, serum, UVB radiation and calcium depletion. We have used Fluorescence recovery after photo bleaching (FRAP) to assess receptor characteristics in cell membranes, confocal laser scanning microscopy to visualize receptor internalization, Ratio imaging for calcium studies, Northern blot for detection of the gene for the epidermal growth factor receptor (EGF-R) ...
The NHERFs are involved with anchoring NHE3 to the cytoskeleton. Under basal conditions, brush border NHE3 has a limited mobile fraction (∼30%) and this requires the presence of the NHERFs (Cha et al., 2004). Mobile fraction refers to the percentage of apical domain GFP tagged NHE3 which recovers after being bleached, as studied by `fluorescence recovery after photobleaching. A two amino acid point mutation of NHE3 between amino acids 586 and 605 abolishes NHERF association with NHE3 and increases the mobile fraction to ∼75%, a similar value to glycosylphosphatidylinositol (GPI) which is present only in the outer leaflet of the plasma membrane and is a control for free membrane mobility. As part of stimulation (LPA) and inhibition (elevation of Ca2+) of NHE3, the NHE3 mobility transiently increases, presumably freeing up the NHE3 initially in the microvilli to allow endocytosis and to accept further NHE3 trafficking to the brush border free from the NHERFs and cytoskeleton (Cha et al., ...
In two-photon laser-scanning microscopy using femtosecond laser pulses, the dependence of the photobleaching rate on excitation power may have a quadratic, cubic or even biquadratic order. To date, there are still many open questions concerning this so-called high-order photobleaching. We studied the photobleaching kinetics of an intrinsic (enhanced Green Fluorescent Protein (eGFP)) and an extrinsic (Hoechst 33342) fluorophore in a cellular environment in two-photon microscopy. Furthermore, we examined the correlation between bleaching and the formation of reactive oxygen species. We observed bleaching-orders of three and four for eGFP and two and three for Hoechst increasing step-wise at a certain wavelength. An increase of reactive oxygen species correlating with the bleaching over time was recognized. Comparing our results to the mechanisms involved in intracellular ablation with respect to the amount of interacting photons and involved energetic states, we found that a low-density plasma is ...
(= FRAP) Many fluorochromes are bleached by exposure to exciting light. If, for example, the cell surface is labelled with a fluorescent probe and an area bleached by laser illumination, then the bleached patch that starts off as a dark area will
Question 4: [8 pts]
Data for membrane mobility of three different proteins (X, Y, and Z) using fluorescent recovery after
photobleaching (FRAP) are shown
Molecular diffusion, often called simply diffusion, is the thermal motion of all (liquid or gas) particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size (mass) of the particles. Diffusion explains the net flux of molecules from a region of higher concentration to one of lower concentration, but it is important to note that diffusion also occurs when there is no concentration gradient. The result of diffusion is a gradual mixing of material. In a phase with uniform temperature, absent external net forces acting on the particles, the diffusion process will eventually result in complete mixing.. Diffusive equilibrium is reached when the concentrations of the diffusing substance in the two compartments becomes equal.. Consider two systems; S1 and S2 at the same temperature and capable of exchanging particles. If there is a change in the potential energy of a system; for example μ1>μ2 (μ is Chemical potential) an ...
photobleaching. Aug. 24, 2017Applications ASIs Dual Inverted Selective Plane Illumination Microscopy (diSPIM) ASIs diSPIM system is an extremely cell friendly for imaging live specimens. It
Equation 1). where Fs, Fb, Fc, and Fc0 represent the fluorescent intensities of the bleach ROI, background, control ROI before photobleaching, and control ROI after photobleaching, respectively. F was expressed as the percentage of initial intensity. The data (n ≥ 20 cells from 4 independent experiments) were averaged (mean values ± SEM) and, by nonlinear regression, fitted to the 1-phase exponential equation Y = Y0 + (plateau - Y0) × (1 - exp(-K × x)), where Y0 is the Y value when X (time) is 0. The plateau is the Y value at infinite times, and K is the rate constant expressed in the reciprocal of seconds. The mobile fraction was given by the plateau, and the recovery half-time was computed as T1/2 = ln(2)/K.. Golgi cisternal stack quantification. Electron microscopy was performed as previously described (59, 60). Sections of 60 nm were mounted on Formvar-coated nickel grids and double contrasted with 2% uranyl acetate for 5 minutes and 3% lead citrate for 5 minutes. Grids were imaged ...
Hormone binding nuclear receptors are transcription factors that activate gene transcription by binding to specific DNA sequences in the gene promoter region and then recruiting the necessary coactivator proteins for transcriptional activation. The knowledge of the function of these receptors, e.g. how they activate or repress transcription are deduced mainly from in vitro biochemical experiments. A general model has been proposed that these receptors and the recruited coactivator proteins form relatively stable complexes with gene promoters. Only recently, in the last four years, additional in vivo data measured with fluorescence recovery after photobleaching (FRAP) have shown that many hormone receptors and coactivators are very mobile inside the nucleus and their association to chromatin is very dynamic, much faster than what has been anticipated from earlier biochemical studies. Although the mobility is reported to be high for the hormone binding receptors, there are consistent reports about ...
Measurement of huntingtin chromatin recruitment dynamics by fluorescence recovery after photobleaching (FRAP) of the YFP-tagged huntingtin-specific intrabody, nucHCB2, under conditions of oxidative stress and PARP inhibition
The Malvern Zetasizer Nano range for characterization of particle size, zeta potential, molecular weight, protein mobility and rheological properties.
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A special interest-funded analysis found seven EPA regulations would negatively impact the coal-based electricity industry and reduce US employment by 1.5 million jobs over the next four years. The report, "Economic Implications of Recent and Anticipated EPA Regulations Affecting the Electricity Sector," was conducted by National Economic Research Associates on behalf … Read more ». ...
A comparison of FRAP in spiny and nonspiny regions showed that AMPAR movement was slower in spines [Ashby et al. (2006), their Table 1 (http://www.jneurosci.org/cgi/content/full/26/26/7046/T1)]. The slow diffusion rate of AMPARs on the spiny membrane could be attributable to special spine structures. To test this hypothesis, the authors focused on the narrow neck region of spines and monitored fluorescence loss in photobleaching from adjacent regions of the spine head. If the neck region acts as a barrier to AMPAR diffusion, one would expect a different time course of fluorescence loss between neck and head. Indeed, continually bleaching the spine head caused rapid fluorescence loss in regions adjacent to the spine head. In contrast, spine necks showed a relatively slow loss of fluorescence [Ashby et al. (2006), their Fig. 4A (http://www.jneurosci.org/cgi/content/full/26/26/7046/F4)], suggesting a role for the spine neck in restricting lateral diffusion of AMPARs.. If narrow spine necks actually ...
The establishment of cell polarity involves positive-feedback mechanisms that concentrate polarity regulators, including the conserved GTPase Cdc42p, at the "front" of the polarized cell. Previous studies in yeast suggested the presence of two parallel positive-feedback loops, one operating as a diffusion-based system, and the other involving actin-directed trafficking of Cdc42p on vesicles. F-actin (and hence directed vesicle traffic) speeds fluorescence recovery of Cdc42p after photobleaching, suggesting that vesicle traffic of Cdc42p contributes to polarization. We present a mathematical modeling framework that combines previously developed mechanistic reaction-diffusion and vesicle-trafficking models. Surprisingly, the combined model recapitulated the observed effect of vesicle traffic on Cdc42p dynamics even when the vesicles did not carry significant amounts of Cdc42p. Vesicle traffic reduced the concentration of Cdc42p at the front, so that fluorescence recovery mediated by Cdc42p flux ...
7.7. NUMERICAL METHODS FOR UNSTEADY-STATE MOLECULAR DIFFUSION 7.7A. Introduction Unsteady-state diffusion often occurs in inorganic, organic, and biological solid materials. If the boundary conditions are constant with time ... - Selection from Transport Processes and Separation Process Principles (Includes Unit Operations) Fourth Edition [Book]
Baran, T. M. and Foster, T. H. (2012), Fluence Rate-Dependent Photobleaching of Intratumorally Administered Pc 4 Does not Predict Tumor Growth Delay. Photochemistry and Photobiology, 88: 1273-1279. doi: 10.1111/j.1751-1097.2012.01171.x ...
Despite the availability of rigorous physical models of microscopy point spread functions (PSFs), approximative PSFs, particularly separable Gaussian approximations are widely used in practical microscopic data processing. In fact, compared with a physical PSF model, which usually involves non-trivial terms such as integrals and infinite series, a Gaussian function has the advantage that it is much simpler and can be computed much faster. Moreover, due to its special analytical form, a Gaussian PSF is often preferred to facilitate the analysis of theoretical models such as Fluorescence Recovery After Photobleaching (FRAP) process and of processing algorithms such as EM deconvolution. However, in these works, the selection of Gaussian parameters and the approximation accuracy were rarely investigated. In this paper, we present a comprehensive study of Gaussian approximations for diffraction-limited 2D/3D paraxial/non-paraxial PSFs of Wide Field Fluorescence Microscopy (WFFM), Laser Scanning ...
Multi-dimensional fluorescence imaging of live animal models demands strong optical sectioning, high spatial resolution, fast image acquisition, and minimal photobleaching. While conventional laser scanning microscopes are capable of deep penetration and sub-cellular resolution, they are generally too slow and causing excessive photobleaching for volumetric or time-lapse imaging. We demonstrate the performance of an augmented line-scan focal modulation microscope (aLSFMM), a high-speed imaging platform that affords above video-rate imaging speed by the use of line scanning. Exceptional background rejection is accomplished by combining a confocal slit with focal modulation. The image quality is further improved by merging the information from simultaneously acquired focal modulation and confocal images. Such a hybrid imaging scheme makes it possible to use very low power excitation light in high-speed imaging, and therefore leads to reduced photobleaching that is desirable for three-dimensional ...
Inhibition of myosin XI-2 and XI-K reduces the efficiency by which MP is targeted to PD.A-C, Kyomographs showing examples of fluorescence recovery during the ti
The discovery reveals the role of a growth factor and endothelial cells in thymus repair, and could have implications for chemotherapy and radiation patients recovery following treatment.. 0 Comments. ...
In mice, injected fragments of a naturally occurring protein boost memory in young and old animals and improve cognition and mobility in a model of neurodegenerative disease. 0 Comments. ...
Uno, K.; Bossi, M. L.; Irie, M.; Belov, V. N.; Hell, S. W.: Reversibly photoswitchable fluorescent diarylethenes resistant against photobleaching in aqueous solutions. Journal of the American Chemical Society 141 (41), pp. 16471 - 16478 (2019 ...
The orbital velocity of the satellite depends on its altitude above Earth. The nearer to Earth, the faster the required orbital velocity. At an altitude of 124 miles (200 kilometers), the required orbital velocity is a little more than 17,000 mph (about 27,400 kph). To maintain an orbit that is 22,223 miles (35,786 kilometers) above Earth, the satellite must orbit at a speed of about 7,000 mph (11,300 kph). That orbital speed and distance permit the satellite to make one revolution in 24 hours. Since Earth also rotates once in 24 hours, a satellite at 22,223 miles altitude stays in a fixed position relative to a point on Earths surface. Because the satellite stays right over the same spot all the time, this kind of orbit is called geostationary. Geostationary orbits are ideal for weather satellites and communications satellites.. In general, the higher the orbit, the longer the satellite can stay in orbit. At lower altitudes, a satellite runs into traces of Earths atmosphere, which creates ...
Voltage-gated calcium channels are multi-subunit membrane proteins which transduce depolarization into cellular functions like excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary β subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether β subunits can reversibly interact with, and thus differentially modulate channels in the membrane is still unresolved. Here we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and β subunits expressed in dysgenic myotubes to study the relative dynamics of these calcium channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct rates dynamic interactions. Whereas the skeletal muscle β1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle β2a and β4b isoforms dynamically interacted with both ...
We have established a robust and versatile analytical platform for probing membrane protein function in a defined lipid environment on solid supports. This approach is based on vesicle capturing onto an ultrathin poly(ethylene glycol) (PEG) polymer brush functionalized with fatty acid moieties and subsequent vesicle fusion into a contiguous membrane. In order to ensure efficient formation of these tethered polymer-supported membranes (PSM), very small unilamellar vesicles (VSUV) containing fluorescent lipids or model transmembrane proteins were generated by detergent depletion with cyclodextrin. Thus, very rapid reconstitution of membrane proteins into PSM was possible in a format compatible with microfluidics. Moreover, surfaces could be regenerated with detergent solution and reused multiple times. Lipid and protein diffusion in these membranes was investigated by fluorescence recovery after photobleaching, single molecule tracking, and fluorescence correlation spectroscopy. Full mobility of ...
We report the development and analysis of a velocimetry technique based on the short time displacement of molecular tracers, tagged thanks to photobleaching. We use confocal microscopy to achieve a good resolution transverse to the observation field in the direction of the velocity gradient. The intensity profiles are fitted by an approximate analytical model which accounts for hydrodynamic dispersion, and allow access to the local velocity. The method is validated using pressure driven flow in microfluidic slits having a thickness of a few tens of micrometers. We discuss the main drawbacks of this technique which is an overestimation of the velocity close to the walls due to the combination of molecular diffusion and shear. We demonstrate that this error, limited to a near wall region of a few micrometers thick, could be controlled by limiting the diffusion of fluorophore molecules or minimizing the bleaching time. The presented technique could be combined with standard particle imaging ...
Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (similar to 0.7 s) and the other half for longer time periods (similar to 2.3 s). A similar pattern of mobility was seen for the MR activated ...
TY - JOUR. T1 - Protein diffusion and long-term adsorption states at charged solid surfaces. AU - Kubiak-Ossowska, Karina. AU - Mulheran, Paul A. PY - 2012/11/6. Y1 - 2012/11/6. N2 - The diffusion pathways of lysozyme adsorbed to a model charged ionic surface are studied using fully atomistic steered molecular dynamics simulation. The simulations start from existing protein adsorption trajectories, where it has been found that one particular residue, Arg128 at the N,C-terminal face, plays a crucial role in anchoring the lysozyme to the surface [ Langmuir 2010 , 26 , 15954 - 15965 ]. We first investigate the desorption pathway for the protein by pulling the Arg128 side chain away from the surface in the normal direction, and its subsequent readsorption, before studying diffusion pathways by pulling the Arg128 side chain parallel to the surface. We find that the orientation of this side chain plays a decisive role in the diffusion process. Initially, it is oriented normal to the surface, aligning ...
The cell assay group establishes and runs cell based assays for the different epigenetic targets to test the in vitro characterised tool compounds for cellular activity. We first focus on demonstrating the on-target effect of the characterised compounds in cells to provide a well validated compound. The characterised inhibitors will then be used to explore biology of the targets and dissect findings obtained by genetic methods. Well characterised inhibitors provide detailed knowledge regarding the role of specific domains of a protein or on catalytic versus scaffolding functions of an enzyme, complementing findings obtained using siRNA, shRNA or related methods.. The group works on cellular assays for bromodomains as well as demethylases. Fluorescence Recovery After Photobleaching (FRAP) is the most direct assay to interrogate if an inhibitor displaces binding of the bromodomain from chromatin. Chromatin associated proteins typically show low mobility due to their tight immobilization on ...
We have compared the lateral diffusion of intact transmembrane proteins, wild-type H-2Ld antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type Ld antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules. ...
An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the ...
As briefly stated in a previous section on the fluid mosaic model of biological membranes, proteins and phospholipids diffuse both laterally and, to a lesser extent transversely, through the entire span of a membrane. This sort of behavior can be characterized by fluorescence microscopy. This particular technique is called FRAP or fluorescence recovery after photo bleaching. In a typical procedure, a specific portion of a cellular membrane is first tagged with a fluorescent chromophore. Next, an intense light is pulsed over a small part of the fluorescent-marked region and viewed under a microscope. As a result of the exposure to the powerful laser-light, the fluorescent molecules are bleached (destroyed). The bleached region is then monitored over time for the recovery of fluorescence (as neighboring unbleached molecules moving towards the bleached areas).This can determine the availability state of a protein - whether it is free to diffuse or is already bounded. The rate at which the recovery ...
Teruhiko Wakayama is the author of this article in the Journal of Visualized Experiments: Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development
Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic centriole conversion is regulated, but it requires Plk1/Polo kinase. Here we show that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before
GATICA, Y.A.; SALINAS, C. H. and ANANIAS, R.A.. Modeling conventional one-dimensional drying of radiata pine based on the effective diffusion coefficient. Lat. Am. appl. res. [online]. 2011, vol.41, n.2, pp. 183-189. ISSN 0327-0793.. We modeled conventional one-dimensional drying of radiata pine (Pinus radiata) wood using the concept of effective diffusion. The experimentally determined effective diffusion coefficients for the radial and tangential directions were related exponentially to the moisture content. These coefficients were characterized by two parameters that were determined through optimization within the context of an inverse problem. One-dimensional drying experiments were carried out under constant drying 44/36 (°C/°C) in order to determine transitory spatial distributions of moisture and drying curves, which were used then to determine the model parameters and validate the model. The mathematical model consisted of a partial, non-linear, differential equation of the second ...
[email protected] Abstract - We modeled conventional one-dimensional drying of radiata pine (Pinus radiata) wood using the concept of effective diffusion. The experimentally determined effective diffusion coefficients for the radial and tangential directions were related exponentially to the moisture content. These coefficients were characterized by two parameters that were determined through optimization within the context of an inverse problem. One-dimensional drying experiments were carried out under constant drying 44/36 (°C/°C) in order to determine transitory spatial distributions of moisture and drying curves, which were used then to determine the model parameters and validate the model. The mathematical model consisted of a partial, non-linear, differential equation of the second order and was characterized by coefficients that varied exponentially with moisture content; this later was integrated numerically through the finite volume method. Simulations of the transitory distribution ...
A coated substrate and a method of forming a diffusion barrier coating system between a substrate and a MCrAl coating, including a diffusion barrier coating deposited onto at least a portion of a substrate surface, wherein the diffusion barrier coating comprises a nitride, oxide or carbide of one or more transition metals and/or metalloids and a MCrAl coating, wherein M includes a transition metal or a metalloid, deposited on at least a portion of the diffusion barrier coating, wherein the diffusion barrier coating restricts the inward diffusion of aluminum of the MCrAl coating into the substrate.
The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common
We combine Fluorescence Recovery After Photobleaching (FRAP) experiments with mathematical modelling to study the dynamics inside the nucleus of both the TGF-β-sensitive transcriptional regulator Smad2, and Green-Fluorescent Protein (GFP). We show how combining modelling with bleaching strips of different areas allows a rigorous test of whether or not a protein is moving via diffusion as a single species. As noted recently by others, it is important to consider diffusion during the bleaching process. Neglecting it can cause serious error. Also, it is possible to use the bleaching process itself to provide an extra consistency test to the models predicting the recovery. With our method we show that the dynamics of GFP are consistent with it diffusing as a single species in a uniform environment in which flow is negligible. In contrast, the dynamics of the intracellular signal transducer Smad2 are never consistent with it moving as a single species via simple diffusion in a homogeneous ...
Background Different types of membrane microdomains (rafts) have been postulated to be present in the rear and front of polarized migrating T-lymphocytes. reorganization in human being T-lymphocytes and possible roles of flotillins in lymphocyte polarization. Results We studied flotillin reorganization and lateral mobility at the plasma membrane using immunofluorescence staining and FRAP (fluorescence recovery after photobleaching). We show that flotillins redistribute early upon chemokine stimulation and form very stable caps in the uropods of human peripheral blood T-lymphocytes colocalizing with the adhesion molecule PSGL-1 and activated ezrin/radixin/moesin (ERM) proteins. Chemokine-induced formation of stable flotillin caps requires Haloperidol (Haldol) integrity and dynamics of the actin cytoskeleton but is not abolished by inhibitors suppressing Rho-kinase or myosin II activity. Tagged flotillin-2 and flotillin-1 coexpressed in T-lymphocytes but Haloperidol (Haldol) not singly expressed ...
Proof-of-concept studies that display the potential of using a glucose-sensitive hydrogel as a continuous glucose sensor are presented. The swelling ratio, porosity, and diffusivity of the hydrogel increased with glucose concentration. In glucose solutions of 50, 100, 200, and 300 mg/dL, the hydrogel swelling ratios were 4.9, 12.3, 15.9, and 21.7, respectively, and the swelling was reversible. The impedance across the hydrogel depended solely on the thickness and had an average increase of 47 W/mm. The hydrogels exposed to a hyperglycemic solution were more porous than the hydrogels exposed to a normal glycemic solution. The diffusivity of 390 Da MW fluorescein isothiocyanate in hydrogels exposed to normal and hyperglycemic solutions was examined using fluorescence recovery after photobleaching and was found to be 9.3 × 10−14 and 41.4 × 10−14 m2/s, respectively, compared to 6.2 × 10−10 m2/s in glucose solution. There was no significant difference between the permeability of hydrogels in normal
The results of our study are consistent with a model in which synaptic vesicles have a restricted lateral mobility within presynaptic vesicle clusters (Henkel and Betz, 1995, Henkel et al., 1996b). However, they demonstrate that during recycling, newly reformed synaptic vesicles are incorporated in the cluster at random in the lateral plane. This intermixing is inhibited by staurosporine.. The restricted lateral mobility of synaptic vesicles within presynaptic vesicle clusters was demonstrated previously in FM1-43 photobleaching experiments at the frog neuromuscular junction (Henkel and Betz, 1995;Henkel et al., 1996b). Our present study extends this conclusion to CNS synapses in cultures, and complements it with information on the mobility of synaptic vesicles during the endocytic limb of their cycle. These new data have been obtained by the combined use of FM1-43, which labels synaptic vesicles through a single cycle (Betz and Bewick, 1992;Henkel et al., 1996a; Ryan et al., 1996), and ...