Methods to measure the lateral diffusion of membrane lipids and proteins

In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are presented in that order. FRAP and FCS methodologies are described for a dedicated wide field microscope although many confocal microscopes now have software permitting these measurement to be made; nevertheless, the principles of the measurement are the same for a wide field or confocal microscope. SPT can be applied to trace the movements of single fluorescent molecules in membranes but this aspect will not be treated in detail.     

Evidence for lack of damage during photobleaching measurements of the lateral mobility of cell surface components.

Fluorescence recovery after photobleaching (FRAP) experiments to measure the mobility of cell surface components require a brief, but intense, pulse of light to photobleach the fluorescence in a restricted area of the cell. We studied possible photodamage to the cell surface during the photobleaching step using light and scanning electron microscopy (SEM) and various FRAP measurements themselves. The cell membrane was left impermeable to trypan blue after photobleaching. SEM studies show that the morphology of the cell surface is not altered by photobleaching. Cells can be repeatedly photobleached and/or photobleached using longer bleach times and greater intensities without systematically altering FRAP kinetics. Singlet oxygen quenchers or free radical traps designed to inhibit putative photoreagents produced during photobleaching do not markedly affect the results. Fluorescein and rhodamine labels give similar results. All of these results, obtained with several different monolayer cultures, suggest that photodamage induced during photobleaching is not a serious artefact in the cellular FRAP results obtained to date.     

Fluorescence correlation spectroscopy and photobleaching recovery of multiple binding reactions. II. FPR and FCS measurements at low and high DNA concentrations

The preceding paper develops the theory for the interpretation of fluorescence photobleaching recovery (FPR) measurements of multiple binding of a ligand to a multivalent substrate molecule. Based on a reasonable assumption about the mechanism of the photobleaching process, this analysis shows that the observed behavior of a multivalent system should be practically identical to that of a univalent binding system. This is in contrast to the expected and observed behavior of fluorescence correlation spectroscopy (FCS) measurments. Experimental FPR measurements of multivalent binding of ethidium bromide to DNA confirm these conclusions. The FCS and FPR measurements also reveal an apparently enhanced diffusion of ethidium at high DNA concentration. This enhancement might result from direct transfer of ethidium among DNA molecules.     

A quantitative approach to analyze binding diffusion kinetics by confocal FRAP

Most of the important types of interactions that occur in cells can be characterized as binding-diffusion type processes, and can be quantified by kinetic rate constants such as diffusion coefficients (D) and binding rate constants (k_on and k_off). Confocal FRAP is a potentially important tool for the quantitative analysis of intracellular binding-diffusion kinetics, but how to dependably extract accurate kinetic constants from such analyses is still an open question. To this end, in this study, we developed what we believe is a new analytical model for confocal FRAP-based measurements of intracellular binding-diffusion processes, based on a closed-form equation of the FRAP formula for a spot photobleach geometry. This approach incorporates a binding diffusion model that allows for diffusion of both the unbound and bound species, and also compensates for binding diffusion that occurs during photobleaching, a critical consideration in confocal FRAP analysis. In addition, to address the problem of parametric multiplicity, we propose a scheme to reduce the number of fitting parameters in the effective diffusion subregime when D's for the bound and unbound species are known. We validate this method by measuring kinetic rate constants for the CAAX-mediated binding of Ras to membranes of the endoplasmic reticulum, obtaining binding constants of k_on ∼ 255/s and k_off ∼ 31/s.     

Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching

To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of approximately 3.6 x 10(-9) cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-beta2AR (beta2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the Vmax and KM values for [3H]dopamine uptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity.     

Expanding the scope of quantitative FRAP analysis

In this study, new mathematical models were developed for analysis of fluorescence recovery after photobleaching (FRAP) data to account for features not represented in previous analysis: conical photobleaching geometry, spatial variations in binding of fluorescent molecules, and directed transport of fluorescent molecules. To facilitate computations in conical geometry, a fast computational method for calculation of fluorescence recovery is presented. Two approximations are presented to aid in FRAP analysis when binding varies spatially, one applying to cases of relatively fast diffusion and slow binding and the other to binding of molecules to small cellular structures. Numerical results show that using a model that represents the influential physical processes and that is formulated in the appropriate geometry can substantially improve the accuracy of FRAP calculations.     

Fluorescence Correlation Spectroscopy Measurements of the Membrane Protein TetA in Escherichia coli Suggest Rapid Diffusion at Short Length Scales

Structural inhomogeneities in biomembranes can lead to complex diffusive behavior of membrane proteins that depend on the length or time scales that are probed. This effect is well studied in eukaryotic cells, but has been explored only recently in bacteria. Here we used fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) to study diffusion of the membrane protein TetA-YFP in E. coli. We find that the diffusion constant determined from FRAP is comparable to other reports of inner membrane protein diffusion constants in E. coli. However, FCS, which probes diffusion on shorter length scales, gives a value that is almost two orders of magnitude higher and is comparable to lipid diffusion constants. These results suggest there is a population of TetA-YFP molecules in the membrane that move rapidly over short length scales (∼ 400 nm) but move significantly more slowly over the longer length scales probed by FRAP.     

Using FCS to accurately measure protein concentration in the presence of noise and photobleaching

Quantitative cell biology requires precise and accurate concentration measurements, resolved both in space and time. Fluorescence correlation spectroscopy (FCS) has been held as a promising technique to perform such measurements because the fluorescence fluctuations it relies on are directly dependent on the absolute number of fluorophores in the detection volume. However, the most interesting applications are in cells, where autofluorescence and confinement result in strong background noise and important levels of photobleaching. Both noise and photobleaching introduce systematic bias in FCS concentration measurements and need to be corrected for. Here, we propose to make use of the photobleaching inevitably occurring in confined environments to perform series of FCS measurements at different fluorophore concentration, which we show allows a precise in situ measurement of both background noise and molecular brightness. Such a measurement can then be used as a calibration to transform confocal intensity images into concentration maps. The power of this approach is first illustrated with in vitro measurements using different dye solutions, then its applicability for in vivo measurements is demonstrated in Drosophila embryos for a model nuclear protein and for two morphogens, Bicoid and Capicua.     

Cholesterol modulation of nicotinic acetylcholine receptor surface mobility

Nicotinic acetylcholine receptor (AChR) function and distribution are quite sensitive to cholesterol (Chol) levels in the plasma membrane (reviewed by Barrantes in J Neurochem 103 (suppl 1):72–80, 2007). Here we combined confocal fluorescence recovery after photobleaching (FRAP) and confocal fluorescence correlation spectroscopy (FCS) to examine the mobility of the AChR and its dependence on Chol content at the cell surface of a mammalian cell line. Plasma membrane AChR exhibited limited mobility and only ~55% of the fluorescence was recovered within 10 min after photobleaching. Depletion of membrane Chol by methyl-β-cyclodextrin strongly affected the mobility of the AChR at the plasma membrane; the fraction of mobile AChR fell from 55 to 20% in Chol-depleted cells, whereas Chol enrichment by methyl-β-cyclodextrin-Chol treatment did not reduce receptor mobility at the cell surface. Actin depolymerization caused by latrunculin A partially restored receptor mobility in Chol-depleted cells. In agreement with the FRAP data, scanning FCS experiments showed that the diffusion coefficient of the AChR was about 30% lower upon Chol depletion. Taken together, these results suggest that membrane Chol modulates AChR mobility at the plasma membrane through a Chol-dependent mechanism sensitive to cortical actin.     

Improved FRAP Measurements on Biofilms

We expand the standard fluorescence recovery after photobleaching (FRAP) model introduced by Axelrod et al. in 1976. Our goal is to capture some of the following common artifacts observed in the fluorescence measurements obtained with a confocal laser scanning microscope in biofilms: 1) linear drift, 2) exponential decrease (due to bleaching during the measurements), 3) stochastic Gaussian noise, and 4) uncertainty in the exact time point of the onset of fluorescence recovery. To fit the resulting stochastic model to data from FRAP measurements and to estimate all unknown model parameters, we apply a suitably adapted Metropolis-Hastings algorithm. In this way, a more accurate estimation of the diffusion coefficient of the fluorophore is achieved. The method was tested on data obtained from FRAP measurements on a cultivated biofilm.     

Fluorescence correlation spectroscopy and photobleaching recovery of multiple binding reactions. I. Theory and FCS measurements

Fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR) are two methods that may be used to measure diffusion and chemical reaction kinetics in small, labile systems such as biological cells. These methods are here applied to systems in which a fluorescent ligand can bind to a polyvalent substrate molecule in a multistep reaction sequence. The analytical theory for both FCS and FPR is extended to allow analysis of these kinds of systems. Experimental measurements of the binding of ethidium bromide to DNA by FCS confirm the theoretical analysis. (FPR measurements on the same system are reported in the accompanying paper.) The analysis shows that FCS and FPR perceive multivalent binding reactions differently. This difference results from the selective effect of the photobleaching process in the chemical reaction system. The development and results we report could have useful applications to a wide range of biopolymeric binding and assembly process.     

Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.     

Protein diffusion in mammalian cell cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.     

Investigating complexity of protein-protein interactions in focal adhesions

The formation of focal adhesions governs cell shape and function; however, there are few measurements of the binding kinetics of focal adhesion proteins in living cells. Here, we used the fluorescence recovery after photobleaching (FRAP) technique, combined with mathematical modeling and scaling analysis to quantify dissociation kinetics of focal adhesion proteins in capillary endothelial cells. Novel experimental protocols based on mathematical analysis were developed to discern the rate-limiting step during FRAP. Values for the dissociation rate constant k_OFF ranged over an order of magnitude from 0.009 ± 0.001/s for talin to 0.102 ± 0.010/s for FAK, indicating that talin is bound more strongly than other proteins in focal adhesions. Comparisons with in vitro measurements reveal that multiple focal adhesion proteins form a network of bonds, rather than binding in a pair-wise manner in these anchoring structures in living cells.