scholarly journals Revealing Plasma Membrane Nano-Domains with Diffusion Analysis Methods

Membranes ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 314
Author(s):  
Jakob L. Kure ◽  
Camilla B. Andersen ◽  
Kim I. Mortensen ◽  
Paul W. Wiseman ◽  
Eva C. Arnspang
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Signalling ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Protein Oligomerization ◽  
Diffusion Analysis ◽  
Lipid Diffusion

Nano-domains are sub-light-diffraction-sized heterogeneous areas in the plasma membrane of cells, which are involved in cell signalling and membrane trafficking. Throughout the last thirty years, these nano-domains have been researched extensively and have been the subject of multiple theories and models: the lipid raft theory, the fence model, and the protein oligomerization theory. Strong evidence exists for all of these, and consequently they were combined into a hierarchal model. Measurements of protein and lipid diffusion coefficients and patterns have been instrumental in plasma membrane research and by extension in nano-domain research. This has led to the development of multiple methodologies that can measure diffusion and confinement parameters including single particle tracking, fluorescence correlation spectroscopy, image correlation spectroscopy and fluorescence recovery after photobleaching. Here we review the performance and strengths of these methods in the context of their use in identification and characterization of plasma membrane nano-domains.

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 Characterization of blinking dynamics in quantum dot ensembles using image correlation spectroscopy

2006 ◽  
Vol 99 (6) ◽  
pp. 064503 ◽  
Author(s):  
Alexia I. Bachir ◽  
Nela Durisic ◽  
Benedict Hebert ◽  
Peter Grütter ◽  
Paul W. Wiseman
Keyword(s):  
Quantum Dot ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Image Correlation Spectroscopy

scholarly journals Characterization of TSET, an ancient and widespread membrane trafficking complex

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jennifer Hirst ◽  
Alexander Schlacht ◽  
John P Norcott ◽  
David Traynor ◽  
Gareth Bloomfield ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Scaffolding Proteins ◽  
Eukaryotic Evolution ◽  
Bioinformatics Tool ◽  
Membrane Turnover ◽  
Evolutionary Path ◽  
The Core ◽  
Eukaryotic Supergroup

The heterotetrameric AP and F-COPI complexes help to define the cellular map of modern eukaryotes. To search for related machinery, we developed a structure-based bioinformatics tool, and identified the core subunits of TSET, a 'missing link' between the APs and COPI. Studies in Dictyostelium indicate that TSET is a heterohexamer, with two associated scaffolding proteins. TSET is non-essential in Dictyostelium, but may act in plasma membrane turnover, and is essentially identical to the recently described TPLATE complex, TPC. However, whereas TPC was reported to be plant-specific, we can identify a full or partial complex in every eukaryotic supergroup. An evolutionary path can be deduced from the earliest origins of the heterotetramer/scaffold coat to its multiple manifestations in modern organisms, including the mammalian muniscins, descendants of the TSET medium subunits. Thus, we have uncovered the machinery for an ancient and widespread pathway, which provides new insights into early eukaryotic evolution.

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.

scholarly journals Insulin-triggered repositioning of munc18c on syntaxin-4 in GLUT4 signalling

2008 ◽  
Vol 410 (2) ◽  
pp. 255-260 ◽  
Author(s):  
Natalie P. Smithers ◽  
Conrad P. Hodgkinson ◽  
Matt Cuttle ◽  
Graham J. Sale
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Cell Signalling ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Vesicle Docking ◽  
Fluorescence Correlation ◽  
Syntaxin 4 ◽  
Single Living Cells ◽  
Single Living

One of the most important actions of insulin is the stimulation of the uptake of glucose into fat and muscle cells. Crucial to this response is the translocation of GLUT4 (glucose transporter-4) to the plasma membrane. The insulin-stimulated GLUT4 vesicle docking at the plasma membrane requires an interaction between VAMP-2 (vesicle-associated membrane protein-2) on the GLUT4 vesicle and syntaxin-4 in the plasma membrane. In the basal state, munc18c is thought to preclude GLUT4 vesicle docking by inhibiting this interaction. Here, we have used FCS (fluorescence correlation spectroscopy) in single living cells to show that munc18c binds to syntaxin-4 in both the basal and insulin-stimulated states. We show that munc18c contains two binding sites for syntaxin-4, one of which is disrupted by insulin, while the other is activated by insulin. Insulin-triggered repositioning of munc18c on syntaxin-4 in this way in turn allows syntaxin-4 to adopt its ‘open’ conformation and bind VAMP-2, resulting in the docking of the GLUT4 vesicle at the cell surface. The results also demonstrate the utility of using FCS in intact single living cells to elucidate cell signalling events.

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.

scholarly journals Quantitative characterization of Tetraspanin 8 homointeractions in the plasma membrane

2021 ◽  
Author(s):  
Daniel Wirth ◽  
Ece Özdemir ◽  
Christopher King ◽  
Lena Ahlswede ◽  
Dirk Schneider ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Dissociation Constant ◽  
Membrane Trafficking ◽  
Dissociation Constants ◽  
Cell Communication ◽  
Live Cells ◽  
Functional Protein ◽  
Quantitative Fluorescence ◽  
Dissociation Constant Kd

The spatial distribution of proteins in cell membranes is crucial for signal transduction, cell communication and membrane trafficking. Members of the Tetraspanin family organize functional protein clusters within the plasma membrane into so-called Tetraspanin-enriched microdomains (TEMs). Direct interactions between Tetraspanins are believed to be important for this organization. However, studies thus far have utilized mainly co-immunoprecipitation methods that cannot distinguish between direct and indirect, through common partners, interactions. Here we study Tetraspanin 8 homointeractions in living cells via quantitative fluorescence microscopy. We demonstrate that Tetraspanin 8 exists in a monomer-dimer equilibrium in the plasma membrane. Tetraspanin 8 dimerization is described by a high dissociation constant (Kd = 14700 ± 1100 Tspan/μm2), one of the highest dissociation constants measured for membrane proteins in live cells. We propose that this high dissociation constant, and thus the short lifetime of the Tetraspanin 8 dimer, is critical for Tetraspanin 8 functioning as a master regulator of cell signaling.

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