A novel viscosity-sensitive fluorescent probe for monitoring the changes of mitochondrial viscosity

As a fundamental physical parameter, viscosity influences the diffusion in biological processes. The changes in intracellular viscosity led to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Here, we devised and synthesized a viscosity-sensitive fluorescent probe LBX-1 . LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement in fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 could localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes in mitochondrial viscosity in complex biological systems.     

Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy

This study evaluated the hypothesis that, due to functional and structural differences, the apparent elastic modulus and viscous behavior of cardiac and skeletal muscle and vascular endothelium would differ. To accurately determine the elastic modulus, the contribution of probe velocity, indentation depth, and the assumed shape of the probe were examined. Hysteresis was observed at high indentation velocities arising from viscous effects. Irreversible deformation was not observed for endothelial cells and hysteresis was negligible below 1 μm/s. For skeletal muscle and cardiac muscle cells, hysteresis was negligible below 0.25 μm/s. Viscous dissipation for endothelial and cardiac muscle cells was higher than for skeletal muscle cells. The calculated elastic modulus was most sensitive to the assumed probe geometry for the first 60 nm of indentation for the three cell types. Modeling the probe as a blunt cone–spherical cap resulted in variation in elastic modulus with indentation depth that was less than that calculated by treating the probe as a conical tip. Substrate contributions were negligible since the elastic modulus reached a steady value for indentations above 60 nm and the probe never indented more than 10% of the cell thickness. Cardiac cells were the stiffest (100.3±10.7 kPa), the skeletal muscle cells were intermediate (24.7±3.5 kPa), and the endothelial cells were the softest with a range of elastic moduli (1.4±0.1 to 6.8±0.4 kPa) depending on the location of the cell surface tested. Cardiac and skeletal muscle exhibited nonlinear elastic behavior. These passive mechanical properties are generally consistent with the function of these different cell types.     

Substituted 4-[4-(dimethylamino)styryl]pyridinium salt as a fluorescent probe for cell microviscosity

In aqueous solution, 4-[4-(dimethylamino)styryl]pyridine (DMASP) derivatives displayed dual fluorescence, in which excitation at either 469 or 360 nm produced an emission band near 600 nm. Increasing the viscosity of the environment intensified the fluorescence emission obtained at the longer wavelength of excitation, whereas the emission at the lower wavelength of excitation showed little change in intensity. Thus, using the ratio of the 600 nm emission obtained by exciting at 469 nm to that obtained with 360 nm excitation, it is possible to obtain a value related to the local viscosity that does not depend on the system parameters. The fluorescence emission of the dye in aqueous solution, as well as in living cells, is well suited for use with visible fluorescence spectroscopy. The N-carboxymethyl butyl ester DMASP derivative (1) was found to be irreversibly loaded into living smooth muscle cells, presumably because it is hydrolyzed by cellular esterases, transforming it into a membrane-impermeable fluorescent carboxylate DMASP derivative. (2) After calibrating 2 against glycerol/water and sucrose/water mixtures of known viscosity, the fluorescence ratio generated from cultured smooth muscle cells in dual-excitation mode gave an average intracellular viscosity of 4.5 cP. This value corresponds to those reported in the literature.     

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.^1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.^4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.^6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the Förster Hoffman equation.⁹ This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.^6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.     

NO synthesis, unlike respiration, influences intracellular oxygen tension.

We have developed a new phosphorescent probe, PdTCPPNa(4), whose luminescence properties are affected by local variations of intracellular oxygen tension (PO(2)). Spectrofluorometric measurements on living human umbilical venous endothelial cells loaded with this molecule show that a decrease in extracellular oxygen tension induces a decrease of PO(2), illustrating the phenomenon of oxygen diffusion and validating the use of this probe in living cells. Moreover, KCN- or 2,4-dinitrophenol-induced modifications of respiration do not lead to detectable PO(2) variations, probably because O(2) diffusion is sufficient to allow oxygen supply. On the contrary, activation by acetylcholine or endothelial nitric oxide synthase (eNOS), which produces NO while consuming oxygen, induces a significant decrease in PO(2), whose amplitude is dependent on the acetylcholine dose, i.e., the eNOS activity level. Hence, activated cytosolic enzymes could consume high levels of oxygen which cannot be supplied by diffusion, leading to PO(2) decrease. Other cell physiology mechanisms leading to PO(2) variations can now be studied in living cells with this probe.     

Effect of viscosity on swimming by the lateral and polar flagella of Vibrio alginolyticus.

By using mutants of Vibrio alginolyticus with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+), we examined the relationship between swimming speed and the viscosity of the medium for each flagellar system. Pof+ Laf- cells could not swim in the high-viscosity environment (ca. 200 cP) in which Pof- Laf+ cells swam at 20 microns/s. The Pof- Laf+ cells swam at about 20 microns/s at normal viscosity (1 cP) without the viscous agent, and the speed increased to 40 microns/s at about 5 cP and then decreased gradually as the viscosity was increased further. These results show the functional difference between polar and lateral flagella in viscous environments.     

Simultaneous detection of SO2 and viscosity in drug-induced inflammation in live cells and zebrafish

The viscosity within cells is a crucial microenvironmental factor, and sulfur dioxide (SO₂) has essential functions in regulating cellular apoptosis and inflammation. Some evidence has been confirmed that changes in viscosity and overexposure of SO₂ within the cell may cause detrimental effects including, but not limited to, respiratory and cardiovascular illnesses, inflammation, fatty liver, and various types of cancer. Therefore, precise monitoring of SO₂ and viscosity in biological entities holds immense practical importance. Therefore, in this research, we developed a versatile fluorescent TCF-Cou that enables the dual detection of SO₂ and viscosity in the living system. Probe TCF-Cou possessed a response to viscosity and SO₂ through red and green emissions. The alteration of SO₂ and viscosity levels in live cells and zebrafish were also monitored using probe TCF-Cou. We hope that this fluorescent probe could be a potential tool for revealing the related pathological and physiological processes through monitoring the changes in SO₂ and viscosity.     

Single cell measurement of micro-viscosity by ratio imaging of fluorescence of styrylpyridinium probe

In aqueous solution, compounds containing the styrylpyridinium group showed dual fluorescence, in which excitation at either 469 or 360 nm each produced an emission band around 600 nm. The ratio of fluorescence intensities of the two bands (R = I469/I360) was sensitive to local viscosity. The N-carboxymethyl butyl ester of DMASP was found to be able to irreversibly load into a living cell; presumably by hydrolysis involving cellular lipases it was transformed to a membrane-impermeable fluorescent carboxylate. A map of the ratio, R, from a single cell was generated using fluorescence imaging microscopy with a spectrofluorimeter in dual-excitation single-emission mode. After calibrating the ratio for the probe in water/glycerol solutions, the intracellular viscosities were obtained for a single cell of smooth muscle of a rat embryonic thoracic aorta. The intracellular viscosity is differentiated inside the cell and the obtained values 18-7 cP obey all the values reported by other laboratories. Fluorescence emission of the probe (500-650 nm) is in a very favourable region for its use with visible fluorescence microscopy, without interferences from cell or tissue auto-fluorescence. The results present ability to detect and follow small changes in the ratio of fluorescence intensities, and apparently of the micro-viscosity.     

A dual-emission fluorescent probe for discriminating cysteine from homocysteine and glutathione in living cells and zebrafish models

Cellular biothiols function crucially and differently in physiological and pathological processes. However, it is still challenging to detect and discriminate thiols within a single one molecule, especially for cysteine (Cys) and homocysteine (Hcy). In this study, a simple two-emission turn-on fluorescent biothiol probe (ICN-NBD) was rationally designed and synthesized through a facile ether bond linking 7-nitro-1,2,3-benzoxadiazole (NBD) and phenanthroimidazole containing a cyano tail. The probe in the presence of Cys elicited two fluorescence responses at 470 nm and 550 nm under excitation at 365 nm and 480 nm, respectively, because of the concomitant generation of both the fluorophore and NBD-N-Cys. In contrast, addition of Hcy and glutathione (GSH) could result in only a blue fluorescence enhancement at 470 nm. which was reasonably attributed to rearrangement from NBD-S-Hcy/GSH to NBD-N-Hcy/GSH as a result of geometrical constraints or solvent effects. Therefore, the fluorescent probe with the NBD scaffold could detect biothiols and simultaneously discriminate Cys from Hcy/GSH in both blue and green channels. The probe has been successfully applied for visualizing biothiols in living cells and zebrafish.     

Isolation of nuclei from epidermis cells of larvae of the blowfly Calliphora vicina R.-D

Rapid microspectrofluorometry has been used to evaluate 1-pyrene-butyric acid as an oxygen probe in single living EL2 ascites tissue culture cells. Despite instrumental conditions preventing detection of the pyrene butyric acid maxima at 380 and 400 nm, the probe having penetrated the cell can be easily identified (maximum around 440 nm in unconnected spectra) from the fluorescence emission spectrum, as compared with NAD(P)H emission in controls (maximum around 460 nm). Fluorescence changes during gradually increasing anaerobiosis under nitrogen flow, are compatible with a linear relationship between the reciprocal of the fluorescence intensity and the intracellular oxygen concentration (increase in 430, 434, 442/461 nm ratios at anaerobiosis). The cells having absorbed the probe continue to catabolize glycolytic substrate, but some inhibition is noticeable (e.g. from the amplitude of the NAD(P)H fluorescence increase spectrum due to intracellular addition of glucose-6-P). In principle rapid microspectrofluorometry allows a multiprobe (e.g. 1-pyrene-butyric acid for oxygen, vs NAD(P)H for metabolism) exploration of the living cell.     

Micropipette aspiration of living cells

The mechanical behavior of living cells is studied with micropipette suction in which the surface of a cell is aspirated into a small glass tube while tracking the leading edge of its surface. Such edges can be tracked in a light microscope to an accuracy of +/-25 nm and suction pressures as small as 0.1-0.2 pN/microm2 can be imposed on the cell. Both soft cells, such as neutrophils and red cells, and more rigid cells, such as chondrocytes and endothelial cells, are studied with this technique. Interpretation of the measurements with basic continuum models leads to values for a cell's elastic and viscous properties. In particular, neutrophils are found to behave as a liquid drop with a cortical (surface) tension of about 30 pN/microm and a viscosity on the order of 100 Pa s. On the other hand, chondrocytes and endothelial cells behave as solids with an elastic modulus of the order of 500 pN/microm2 (0.5 kPa).     

Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose

Never-dried and once-dried hardwood celluloses were oxidized by a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated system, and highly crystalline and individualized cellulose nanofibers, dispersed in water, were prepared by mechanical treatment of the oxidized cellulose/water slurries. When carboxylate contents formed from the primary hydroxyl groups of the celluloses reached approximately 1.5 mmol/g, the oxidized cellulose/water slurries were mostly converted to transparent and highly viscous dispersions by mechanical treatment. Transmission electron microscopic observation showed that the dispersions consisted of individualized cellulose nanofibers 3-4 nm in width and a few microns in length. No intrinsic differences between never-dried and once-dried celluloses were found for preparing the dispersion, as long as carboxylate contents in the TEMPO-oxidized celluloses reached approximately 1.5 mmol/g. Changes in viscosity of the dispersions during the mechanical treatment corresponded with those in the dispersed states of the cellulose nanofibers in water.     

Measurement of the Internal Frictional Drag of the Bacterial Flagellar Motor by Fluctuation Analysis

The bacterial flagellar motor generates the torque that drives the rotation of bacterial flagellar filaments. The torque it generates depends sensitively on the frictional viscous drag on the motor, which includes the frictional viscous drag on the filaments (external load) and the internal frictional viscous drag on the rotor (internal load). The internal load was roughly estimated previously by modeling it as a sphere of a radius of 20 nm rotating in a lipid of viscosity of 100 cp but was never measured experimentally. Here, we measured the internal load by fluctuation analysis of the motor velocity traces. A similar approach should be applicable to other molecular motors.     

Fluorescent sensors based on quinoline‐containing styrylcyanine: determination of ferric ions,hydrogen peroxide,and glucose,pH‐sensitive properties and bioimaging

A novel styrylcyanine‐based fluorescent probe 1 was designed and synthesized via facile methods. Ferric ions quenched the fluorescence of probe 1, whereas the addition of ferrous ions led to only small changes in the fluorescence signal. When hydrogen peroxide was introduced into the solution containing probe 1 and Fe²⁺, Fe²⁺ was oxidized to Fe³⁺, resulting in the quenching of the fluorescence. The probe 1/Fe²⁺ solution fluorescence could also be quenched by H₂O₂ released from glucose oxidation by glucose oxidase (GOD), which means that probe 1/Fe²⁺ platform could be used to detect glucose. Probe 1 is fluorescent in basic and neutral media but almost non‐fluorescent in strong acidic environments. Such behaviour enables it to work as a fluorescent pH sensor in both the solution and solid states and as a chemosensor for detecting volatile organic compounds with high acidity and basicity. Subsequently, the fluorescence microscopic images of probe 1 in live cells and in zebrafish were achieved successfully, suggesting that the probe has good cell membrane permeability and a potential application for imaging in living cells and living organisms. Copyright © 2014 John Wiley & Sons, Ltd.     

Assessment of Fura-2 for measurements of cytosolic free calcium

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: Effects of cholesterol depletion and apoptosis

Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.     

Polysaccharide reserve material in the acetotrophic methanogen,Methanosarcina thermophila strain TM-1: accumulation and mobilization

The ability of Methanosarcina thermophila strain TM-1 to store a reserve polysaccharide was studied using both biochemical methods and thin-section electron microscopy. When grown under conditions of excess carbon and energy (either methanol or acetate) and limiting nitrogen, M. thermophila accumulated a polysaccharide which could be hydrolyzed to glucose by the enzyme amyloglucosidase. This polysaccharide reached levels of 20 mg polysaccharide per g protein in nitrogen-limited cells, while cells limited for carbon, as well as cells in the exponential phase of growth, did not accumulate significant amounts of this polysaccharide. Thin-section electron micrographs of M. thermophila showed glycogen-like inclusion granules in nitrogen-limited cells but not in carbon-limited or exponential-phase cells. These granules were stained by a polysaccharide-specific staining procedure, the PATO stain. The polysaccharide was purified from cell extracts, the iodine-polysaccharide complex gave a maximum absorption at between 500 and 510 nm. The polysaccharide was mobilized within 21 h by cells starved for a carbon/energy source. N-Limited (polysaccharide-containing) acetategrown cells could shift to methanogenesis from methanol more quickly than did C-limited acetate-grown cells lacking polysaccharide, and ATP levels remained higher in N-limited cells. The results are consistant with the hypothesis that this polysaccharide can provide carbon and energy for metabolic shifts but other storage compounds, such as polyphosphate, may also play a similar role.     

In situ control of cell adhesion using photoresponsive culture surface

A photoresponsive culture surface (PRCS) allowing photocontrol of cell adhesion was prepared with a novel polymer material composed of poly(N-isopropylacrylamide) having spiropyran chromophores as side chains. Cell adhesion of the surface was drastically enhanced by the irradiation with ultraviolet (UV) light (wavelength: 365 nm); after subsequent cooling and washing on ice, many cells remained in the irradiated region, whereas most cells were removed from the nonirradiated region. The cell adhesion of the PRCS, which had been enhanced by previous UV irradiation, was reset by the visible light irradiation (wavelength 400-440 nm) and the annealing at 37 degrees C for 2 h. Also it was confirmed that the regional control of cell adhesion was induced several times by repeating the same series of operations. Further, living cell patterning with the 200 microm line width was produced readily by projecting UV light along a micropattern on the PRCS on which the living cells had been seeded uniformly in advance. By using a fluorescent probe that stains living cells only, it was confirmed that the cells maintained sufficient viability even after UV light irradiation followed by cooling and washing.     

Influence of phosphate and disinfection on the composition of biofilms produced from drinking water, as measured by fluorescence in situ hybridization

Biofilms were grown in annular reactors supplied with drinking water enriched with 235 microg C/L. Changes in the biofilms with ageing, disinfection, and phosphate treatment were monitored using fluorescence in situ hybridization. EUB338, BET42a, GAM42a, and ALF1b probes were used to target most bacteria and the alpha (alpha), beta (beta), and gamma (gamma) subclasses of Proteobacteria, respectively. The stability of biofilm composition was checked after the onset of colonization between T = 42 days and T = 113 days. From 56.0% to 75.9% of the cells detected through total direct counts with DAPI (4'-6-diamidino-2-phenylindole) were also detected with the EUB338 probe, which targets the 16S rRNA of most bacteria. Among these cells, 16.9%-24.7% were targeted with the BET42a probe, 1.8%-18.3% with the ALF1b probe, and <2.5% with the GAM42a probe. Phosphate treatment induced a significant enhancement to the proportion of gamma-Proteobacteria (detected with the GAM42a probe), a group that contains many health-related bacteria. Disinfection with monochloramine for 1 month or chlorine for 3 days induced a reduction in the percentage of DAPI-stained cells that hybridized with the EUB338 probe (as expressed by percentages of EUB338 counts/DAPI) and with any of the ALF1b, BET42a, and GAM42a probes. The percentage of cells detected by any of the three probes (ALF1b+BET42a+GAM42a) tended to decrease, and reached in total less than 30% of the EUB338-hybridized cells. Disinfection with chlorine for 7 days induced a reverse shift; an increase in the percentage of EUB338 counts targeted by any of these three probes was noted, which reached up to 87%. However, it should be noted that the global bacterial densities (heterotrophic plate counts and total direct counts) tended to decrease over the duration of the experiment. Therefore, those bacteria that could be considered to resist 7 days of chlorination constituted a small part of the initial biofilm community, up to the point at which the other bacterial groups were destroyed by chlorination. The results suggest that there were variations in the kinetics of inactivation by disinfectant, depending on the bacterial populations involved.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.