scholarly journals Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

10.3791/51074 ◽  
2014 ◽  
Author(s):  
Eva C. Arnspang ◽  
Jennifer S. Koffman ◽  
Saw Marlar ◽  
Paul W. Wiseman ◽  
Lene N. Nejsum
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Diffusion Coefficients ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Plasma Membrane Proteins ◽  
Image Correlation Spectroscopy ◽  
Space Image

scholarly journals Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single particle tracking

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sara Makaremi ◽  
Markus Rose ◽  
Suman Ranjit ◽  
Michelle A. Digman ◽  
Dawn M. E. Bowdish ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Particle Tracking ◽  
Diffusion Coefficients ◽  
Basal Membrane ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Live Cells ◽  
Raster Image ◽  
Image Correlation Spectroscopy

Abstract The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was significantly faster in the apical membrane than in the basal membrane, suggesting diffusion hindrance by the adhesion of the cells to the substrate. This finding highlights the importance of selecting the appropriate membrane (i.e., basal or apical) and corresponding method when measuring receptor diffusion in live cells. Accurately knowing the diffusion coefficient of two macrophage receptors involved in the response to pathogen insults will facilitate the study of changes that occur in signaling in these cells as a result of aging and disease.

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

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 568
Author(s):  
Jakob L. Kure ◽  
Thommie Karlsson ◽  
Camilla B. Andersen ◽  
B. Christoffer Lagerholm ◽  
Vesa Loitto ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Actin Polymerization ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Membrane Diffusion ◽  
Hek 293 ◽  
293 Cells ◽  
Aquaporin 9

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.

scholarly journals Measuring Lateral Diffusion of Receptors On Plasma Membrane of Macrophages Using Raster Image Correlation Spectroscopy

2018 ◽  
Vol 24 (S1) ◽  
pp. 1356-1357
Author(s):  
S. Makaremi ◽  
S. Ranjit ◽  
M.A. Digman ◽  
E. Gratton ◽  
D. M.E. Bowdish ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lateral Diffusion ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Raster Image ◽  
Image Correlation Spectroscopy

scholarly journals Anisotropic diffusion of fluorescently labeled ATP in rat cardiomyocytes determined by raster image correlation spectroscopy

2008 ◽  
Vol 295 (5) ◽  
pp. C1302-C1315 ◽  
Author(s):  
Marko Vendelin ◽  
Rikke Birkedal
Keyword(s):  
Diffusion Coefficient ◽  
Diffusion Coefficients ◽  
Anisotropic Diffusion ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Rat Cardiomyocytes ◽  
Intracellular Diffusion ◽  
Average Diffusion ◽  
Image Correlation Spectroscopy ◽  
Kinetics Of

A series of experimental data points to the existence of profound diffusion restrictions of ADP/ATP in rat cardiomyocytes. This assumption is required to explain the measurements of kinetics of respiration, sarcoplasmic reticulum loading with calcium, and kinetics of ATP-sensitive potassium channels. To be able to analyze and estimate the role of intracellular diffusion restrictions on bioenergetics, the intracellular diffusion coefficients of metabolites have to be determined. The aim of this work was to develop a practical method for determining diffusion coefficients in anisotropic medium and to estimate the overall diffusion coefficients of fluorescently labeled ATP in rat cardiomyocytes. For that, we have extended raster image correlation spectroscopy (RICS) protocols to be able to discriminate the anisotropy in the diffusion coefficient tensor. Using this extended protocol, we estimated diffusion coefficients of ATP labeled with the fluorescent conjugate Alexa Fluor 647 (Alexa-ATP). In the analysis, we assumed that the diffusion tensor can be described by two values: diffusion coefficient along the myofibril and that across it. The average diffusion coefficients found for Alexa-ATP were as follows: 83 ± 14 μm2/s in the longitudinal and 52 ± 16 μm2/s in the transverse directions ( n = 8, mean ± SD). Those values are ∼2 (longitudinal) and ∼3.5 (transverse) times smaller than the diffusion coefficient value estimated for the surrounding solution. Such uneven reduction of average diffusion coefficient leads to anisotropic diffusion in rat cardiomyocytes. Although the source for such anisotropy is uncertain, we speculate that it may be induced by the ordered pattern of intracellular structures in rat cardiomyocytes.

An image correlation analysis of the distribution of clathrin associated adaptor protein (AP-2) at the plasma membrane

1998 ◽  
Vol 111 (2) ◽  
pp. 271-281 ◽  
Author(s):  
C.M. Brown ◽  
N.O. Petersen
Keyword(s):  
Plasma Membrane ◽  
Adaptor Protein ◽  
Bimodal Distribution ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Spectroscopy Analysis ◽  
Coated Pits ◽  
Binding Characteristics ◽  
Image Correlation Spectroscopy ◽  
Number Counts

Clathrin associated adaptor protein is involved in endocytosis at the plasma membrane (AP-2) and protein sorting at the Golgi membrane (AP-1). There is a great deal of information available on the structure, function and binding characteristics of AP-2, however, there is little quantitative data on the AP-2 distribution at the membrane. Image correlation spectroscopy is a technique which yields number counts from an autocorrelation analysis of intensity fluctuations within confocal microscopy images. Image correlation spectroscopy analysis of the indirect immunofluorescence from AP-2 at the plasma membrane of CV-1 cells shows that AP-2 is in a bimodal distribution consisting of large coated pit associated aggregates of approximately 60 AP-2 molecules, and smaller aggregates containing approximately 20 AP-2 molecules, which we propose are coated pit nucleation sites. Following hypertonic treatment 25% of the AP-2 molecules dissociate from the large AP-2 aggregates and form AP-2 dimers, leaving the remaining AP-2 as large aggregates with approximately 45 molecules. The smaller AP-2 aggregates completely dissociate forming AP-2 dimers. Dispersion of AP-2 with hypertonic treatment is not seen qualitatively because the number of large AP-2 aggregates is unchanged, the aggregates are just 25% smaller. Change in temperature from 37 degrees C to 4 degrees C has no affect on the number of AP-2 aggregates or the AP-2 distribution between the two populations. These data and estimates of the coated pit size suggest that coated pits cover approximately 0.9% of the cell membrane. Combination of image correlation spectroscopy analysis and measurements of the CV-1 cell surface area show that there are approximately 6x10(5) AP-2 molecules per CV-1 cell with approximately 2x10(5) AP-2 molecules within coated pit structures.

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

2016 ◽  
Vol 22 (2) ◽  
pp. 290-299 ◽  
Author(s):  
Martina Laňková ◽  
Jana Humpolíčková ◽  
Stanislav Vosolsobě ◽  
Zdeněk Cit ◽  
Jozef Lacek ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Image Correlation ◽  
Fluorescence Recovery ◽  
Confocal Laser ◽  
Raster Image ◽  
Image Correlation Spectroscopy ◽  
Plant Plasma Membrane

AbstractA 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.

Investigating membrane protein dynamics in living cellsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

2006 ◽  
Vol 84 (6) ◽  
pp. 825-831 ◽  
Author(s):  
Ian R. Bates ◽  
Paul W. Wiseman ◽  
John W. Hanrahan
Keyword(s):  
Membrane Proteins ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Fluorescence Correlation ◽  
Transport Dynamics ◽  
Health And Disease ◽  
Image Correlation Spectroscopy ◽  
Selection Of

Live cell imaging is a powerful tool for understanding the function and regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based techniques for studying the transport dynamics of membrane proteins: fluorescence-correlation spectroscopy, image-correlation spectroscopy, fluorescence recovery after photobleaching, and single-particle and (or) molecule tracking. The advantages and limitations of each approach are illustrated using recent studies of an ion channel and cell adhesion molecules.

Sign in / Sign up

Export Citation Format

Share Document