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

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.

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

Scientific Reports ◽

10.1038/s41598-020-76272-2 ◽

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.

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Raster image correlation spectroscopy as a novel tool to study interactions of macromolecules with nanofiber scaffolds

Acta Biomaterialia ◽

10.1016/j.actbio.2011.07.012 ◽

2011 ◽

Vol 7 (12) ◽

pp. 4195-4203 ◽

Cited By ~ 15

Author(s):

S.C.P. Norris ◽

J. Humpolíčková ◽

E. Amler ◽

M. Huranová ◽

M. Buzgo ◽

...

Keyword(s):

Correlation Spectroscopy ◽

Image Correlation ◽

Raster Image ◽

Nanofiber Scaffolds ◽

Image Correlation Spectroscopy

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Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope

Journal of Microscopy ◽

10.1111/j.1365-2818.2007.01871.x ◽

2008 ◽

Vol 229 (1) ◽

pp. 78-91 ◽

Cited By ~ 128

Author(s):

C. M. BROWN ◽

R. B. DALAL ◽

B. HEBERT ◽

M. A. DIGMAN ◽

A. R. HORWITZ ◽

...

Keyword(s):

Protein Dynamics ◽

Laser Scanning ◽

Confocal Microscope ◽

Correlation Spectroscopy ◽

Image Correlation ◽

Laser Scanning Confocal Microscope ◽

Raster Image ◽

Image Correlation Spectroscopy

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Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells

Nature Communications ◽

10.1038/ncomms3093 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 63

Author(s):

Per Niklas Hedde ◽

René M. Dörlich ◽

Rosmarie Blomley ◽

Dietmar Gradl ◽

Emmanuel Oppong ◽

...

Keyword(s):

Correlation Spectroscopy ◽

Stimulated Emission ◽

Image Correlation ◽

Live Cells ◽

Raster Image ◽

Biomolecular Dynamics ◽

Image Correlation Spectroscopy ◽

Stimulated Emission Depletion

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Measurement of Protein 53 Diffusion Coefficient in Live HeLa Cells Using Raster Image Correlation Spectroscopy (RICS)

Journal of Biomaterials and Nanobiotechnology ◽

10.4236/jbnb.2010.11004 ◽

2010 ◽

Vol 01 (01) ◽

pp. 31-36 ◽

Cited By ~ 5

Author(s):

Sungmin Hong ◽

Ying-Nai Wang ◽

Hirohito Yamaguchi ◽

Harinibytaraya Sreenivasappa ◽

Chao-Kai Chou ◽

...

Keyword(s):

Diffusion Coefficient ◽

Hela Cells ◽

Correlation Spectroscopy ◽

Image Correlation ◽

Raster Image ◽

Image Correlation Spectroscopy

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

Journal of Cell Science ◽

10.1242/jcs.111.2.271 ◽

1998 ◽

Vol 111 (2) ◽

pp. 271-281 ◽

Cited By ~ 1

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.

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