Mobility measurement by analysis of fluorescence photobleaching recovery kinetics

Axelrod, Daniel; Koppel, Dennis E.; Schlessinger, Joseph; Elson, Elliot L.; Webb, Watt W.

doi:10.1016/s0006-3495(76)85755-4

Cited by 2,518 publications

(2,332 citation statements)

References 14 publications

(19 reference statements)

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“…1C). To calculate effective diffusion coefficients, D, recovery curves were individually fit to the solution to the diffusion equation in one dimension based on the work of Axelrod et al (1976) and Soumpasis (1983) and detailed in the Appendix.…”

Section: Frap Acquisition and Analysismentioning

confidence: 99%

“…Unfortunately, the few techniques available to directly measure these parameters are often limited, particularly in their application to the plasma membrane of living cells (Edidin 2003). However, quantifying the translational diffusion or lateral mobility of molecules in the plane of the bilayer is a useful method to directly measure membrane fluidity and to indirectly assess changes in other interconnected membrane material properties in live, native specimens (Axelrod et al 1976;Edidin 1987Edidin , 1994Cullis et al 1996). Lateral mobility measurements are readily made using fluorescence recovery after photobleaching (FRAP) techniques.…”

Section: Introductionmentioning

confidence: 99%

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Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

Organ

Raphael

2009

JARO

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The outer hair cell (OHC) lateral plasma membrane houses the transmembrane protein prestin, a necessary component of the yet unknown molecular mechanism (s) underlying electromotility and the exquisite sensitivity and frequency selectivity of mammalian hearing. The importance of the plasma membrane environment in modulating OHC electromotility has been substantiated by recent studies demonstrating that membrane cholesterol alters prestin activity in a manner consistent with cholesterol-induced changes in auditory function. Cholesterol is known to affect membrane material properties, and measurements of lipid lateral mobility provide a method to asses these changes in living OHCs. Using fluorescence recovery after photobleaching (FRAP), we characterized regional differences in the lateral diffusion of the lipid analog di-8-ANEPPS in OHCs and investigated whether lipid mobility, which reflects membrane fluidity, is sensitive to membrane cholesterol. FRAP experiments revealed quantitative differences in lipid lateral mobility among the apical, lateral, and basal regions of the OHC and demonstrated that diffusion in individual regions is uniquely sensitive to cholesterol manipulations. Interestingly, in the lateral region, both cholesterol depletion and loading significantly reduced the effective diffusion coefficient from control values. Thus, the fluidity of the OHC lateral plasma membrane is regulated by cholesterol levels in a non-monotonic manner, suggesting that the overall material properties of the lateral plasma membrane are optimally tuned for OHC function in the native state. These results support the idea that the cholesteroldependent regulation of prestin function and electromotility correlates with changes in the properties of the lipid environment that surrounds and supports prestin.

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Section: Frap Acquisition and Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

Organ

Raphael

2009

JARO

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“…41,42 The diffusion constant for the NBD-PE lipid was 3.4 ± 0.8 μm 2 /s and the mobile fraction was ~95 ± 2%. Moreover, individual SLBs were completely confined within their protein boundaries.…”

Section: Resultsmentioning

confidence: 98%

“…Fluorescence recovery after photobleaching (FRAP) 41,42 experiments were conducted to check the quality and fluidity of the SLBs. This was done using an inverted epifluorescence Nikon Eclipse TE2000-U microscope equipped with a 10× objective.…”

Section: Epifluorescence and Tirfmmentioning

confidence: 99%

Multiplexing Ligand−Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

2008

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A method has been designed for patterning supported phospholipid bilayers (SLBs) on planar substrates and inside microfluidic channels. To do this, bovine serum albumin (BSA) monolayers were formed via adsorption at the liquid/solid interface. Next, this interfacial protein film was selectively patterned by using deep UV lithography. Subsequently, SLBs could be deposited in the patterned locations by vesicle fusion. By cycling through this process several times, spatially addressed bilayer arrays could be formed with intervening protein molecules serving as twodimensional corrals. By employing this method, phospholipid bilayers containing various concentrations of ganglioside GM1 were addressed along the length of individual microfluidic channels. Therefore, the binding of GM1 with pentameric cholera toxin B (CTB) subunits could be probed. A seven-channel microfluidic device was fabricated for this purpose. Each channel was simultaneously patterned with four chemically distinct SLBs containing 0, 0.2, 0.5, and 2.0 mol % GM1, respectively. Varying concentrations of CTB were then introduced into each of the channels. With the use of total internal reflection fluorescence microscopy, it was possible to simultaneously abstract multiple equilibrium dissociation constants as a function of ligand density for the CTB-GM1 system in a single shot.Multivalent ligand-receptor interactions are ubiquitous on cell surfaces. They have a wide variety of consequences including the modulation of equilibrium dissociation constants, high receptor selectivity, and receptor clustering. 1 Multivalency can also play a direct role in signal transduction processes. 2 Examination of the underlying thermodynamics of ligandreceptor binding may lead to a greater understanding of its biological role and could provide insight into biomedical applications involving inhibitory drug design. 1,3,4 Unfortunately, high-throughput, low-protein consumption assays are not presently well enough developed in this field to afford rapid, systematic studies of binding data at two-dimensionally fluid bilayer interfaces. 5 In previous work it has been demonstrated that microfluidic devices can be designed for measuring binding affinities in multivalent systems. [5][6][7][8][9][10][11][12][13][14] In our previous setup, ligands were incorporated into supported lipid bilayers (SLBs) coated on the walls and floors of polydimethylsiloxane/glass microchannels. Linear arrays of channels were then monitored by total internal reflection fluorescence microscopy (TIRFM) 15 to obtain equilibrium dissociation constants in one-shot assays. This could be done by using the same surface chemistry in each microchannel while varying the solution concentration of the aqueous proteins. Such a setup made these assays relatively rapid, afforded high accuracy, and used only a few microliters of protein solution. Nevertheless, such platforms could be markedly improved by measuring multiple binding constants with various membrane chemistries simultaneously. This could be achie...

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“…Translational or lateral diffusion has been quantified in synthetic membrane systems using the standard fluorescence recovery after photobleaching (FRAP) technique which consists in measuring the recovery of fluorescence in a determined region of interest of a membrane (ROI) that was exposed to photobleaching 69. The motion of unbleached lipids in the membrane allows a recovery of fluorescence in the bleached ROI with kinetics that can be monitored and fitted with appropriate models.…”

Section: Resultsmentioning

confidence: 99%

Asymmetric Hybrid Polymer–Lipid Giant Vesicles as Cell Membrane Mimics

et al. 2017

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Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion‐centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)‐b‐poly(ethylene oxide) (PBut‐b‐PEO) and outer monolayer of 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half‐life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(lissamine rhodamine B sulfonyl) (DOPE‐rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 μm2 s−1 at 25 °C and D = 2.3 ± 0.7 μm2 s−1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell‐like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.

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Mobility measurement by analysis of fluorescence photobleaching recovery kinetics

Cited by 2,518 publications

References 14 publications

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

Multiplexing Ligand−Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

Asymmetric Hybrid Polymer–Lipid Giant Vesicles as Cell Membrane Mimics

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