Cholesterol Depletion Mimics the Effect of Cytoskeletal Destabilization on Membrane Dynamics of the Serotonin1A Receptor: A zFCS Study

Single-point fluorescence correlation spectroscopy (FCS) of membrane-bound molecules suffers from a number of limitations leading to inaccurate estimation of diffusion parameters. To overcome such problems and with the overall goal of addressing membrane heterogeneities, we performed z-scan FCS (zFCS) of the serotonin_1A receptor. We analyzed the results according to FCS diffusion laws that provide information on the organization of the diffusing species. Analysis of our results shows that the diffusion coefficients of the receptor and a fluorescently labeled phospholipid are similar when probed at length scales ∼210 nm. We discuss the significance of the spatiotemporal evolution of dynamics of membrane-bound molecules in the overall context of membrane domains and heterogeneity. Importantly, our results show that the serotonin_1A receptor exhibits confinement in cell membranes, possibly due to interaction with the actin cytoskeleton. Surprisingly, depletion of membrane cholesterol appears to reduce receptor confinement in a manner similar to that observed in the case of cytoskeletal destabilization, implying possible changes in the actin cytoskeleton induced upon cholesterol depletion. These results constitute the first report on G-protein-coupled receptor dynamics utilizing a combination of zFCS and the FCS diffusion laws, and present a convenient approach to explore cell membrane heterogeneity at the submicron level.     

Anomalous Diffusion in Inverted Variable-Lengthscale Fluorescence Correlation Spectroscopy

Using fluorescence correlation spectroscopy (FCS) to distinguish between different types of diffusion processes is often a perilous undertaking because the analysis of the resulting autocorrelation data is model dependant. Two recently introduced strategies, however, can help move toward a model-independent interpretation of FCS experiments: 1) the obtention of correlation data at different length scales and 2) their inversion to retrieve the mean-squared displacement associated with the process under study. We use computer simulations to examine the signature of several biologically relevant diffusion processes (simple diffusion, continuous-time random walk, caged diffusion, obstructed diffusion, two-state diffusion, and diffusing diffusivity) in variable-length-scale FCS. We show that, when used in concert, length-scale variation and data inversion permit us to identify non-Gaussian processes and, regardless of Gaussianity, to retrieve their mean-squared displacement over several orders of magnitude in time. This makes unbiased discrimination between different classes of diffusion models possible.     

FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange

Obtaining quantitative kinetic parameters from fluorescence recovery after photobleaching (FRAP) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries: stripes, and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry, which creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by parameterizing, more accurately, the initial postbleach state. This allows for reconstruction of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLC-δ1 in Caenorhabditis elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation, and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane.     

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.     

Salt stress triggers enhanced cycling of Arabidopsis root plasma-membrane aquaporins

Aquaporins of the plasma membrane intrinsic protein (PIP) subfamily are channels which facilitate the diffusion of water across the plant plasma membrane (PM). Although PIPs have been considered as canonical protein markers of this compartment, their endomembrane trafficking is still not well documented. We recently obtained insights into the constitutive cycling of PIPs in Arabidopsis root cells by means of fluorescence recovery after photobleaching (FRAP). This work also uncovered the behavior of the model isoform AtPIP2;1 in response to NaCl. The present addendum connects these findings to another recent work which describes the dynamic properties of AtPIP2;1 in the PM in normal and salt stress conditions by means of single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS). The results suggest that membrane rafts play an important role in the partitioning of AtPIP2;1 in normal conditions and that clathrin-mediated endocytosis is predominant. In salt stress conditions, the rate of AtPIP2;1 cycling was enhanced and endocytosis was cooperated by a membrane raft-associated salt-induced pathway and a clathrin-dependent pathway.     

Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP₂), several investigators have proposed that there are separate pools of PIP₂ in the plasma membrane. Recent experiments show the surface concentration of PIP₂ is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP₂ are also concentrated in these regions. However, how is the PIP₂ produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP₂ within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP₂. FCS measurements show that PIP₂ diffuses rapidly (D ∼ 1 μm²/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP₂ does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP₂ into and out of forming phagosomes.     

Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter

Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in E. coli. In previous studies, we created a mutant AcrB_P223G, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrB_P223G in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrB_P223G in E. coli cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrB_P223G-YPet in live E. coli cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrB_P223G exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes.     

Diffusion, Transport, and Cell Membrane Organization Investigated by Imaging Fluorescence Cross-Correlation Spectroscopy

Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid “platforms”). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), a widely used spectroscopic technique to measure membrane dynamics, has the required time resolution but lacks imaging capabilities. A promising solution is the recently introduced method known as imaging total internal reflection (ITIR)-FCS, which can probe diffusion phenomena in lipid membranes with good temporal and spatial resolution. In this work, we extend ITIR-FCS to perform ITIR fluorescence cross-correlation spectroscopy (ITIR-FCCS) between pixel areas of arbitrary shape and derive a generalized expression that is applicable to active transport and diffusion. ITIR-FCCS is applied to model systems exhibiting diffusion, active transport, or a combination of the two. To demonstrate its applicability to live cells, we observe the diffusion of a marker, the sphingolipid-binding domain (SBD) derived from the amyloid peptide Aβ, on live neuroblastoma cells. We investigate the organization and dynamics of SBD-bound lipid microdomains under the conditions of cholesterol removal and cytoskeleton disruption.     

Diffusion of green fluorescent protein in three cell environments in Escherichia coli

Surprisingly little is known about the physical environment inside a prokaryotic cell. Knowledge of the rates at which proteins and other cell components can diffuse is crucial for the understanding of a cell as a physical system. There have been numerous measurements of diffusion coefficients in eukaryotic cells by using fluorescence recovery after photobleaching (FRAP) and related techniques. Much less information is available about diffusion coefficients in prokaryotic cells, which differ from eukaryotic cells in a number of significant respects. We have used FRAP to observe the diffusion of green fluorescent protein (GFP) in cells of Escherichia coli elongated by growth in the presence of cephalexin. GFP was expressed in the cytoplasm, exported into the periplasm using the twin-arginine translocation (Tat) system, or fused to an integral plasma membrane protein (TatA). We show that TatA-GFP diffuses in the plasma membrane with a diffusion coefficient comparable to that of a typical eukaryotic membrane protein. A previous report showed a very low rate of protein diffusion in the E. coli periplasm. However, we measured a GFP diffusion coefficient only slightly smaller in the periplasm than that in the cytoplasm, showing that both cell compartments are relatively fluid environments.     

Size Dependence of Protein Diffusion in the Cytoplasm of Escherichia coli

Diffusion in the bacterial cytoplasm is regarded as the primary method of intracellular protein movement and must play a major role in controlling the rates of cell processes. A number of recent studies have used green fluorescent protein (GFP) tagging and fluorescence microscopy to probe the movement and distribution of proteins in the bacterial cytoplasm. However, the dynamic behavior of indigenous proteins must be controlled by a complex mixture of specific interactions, combined with the basic physical constraints imposed by the viscosity and macromolecular crowding of the cytoplasm. These factors are difficult to unravel in studies with indigenous proteins. To what extent the addition of a GFP tag might affect the movement of a protein through the cytoplasm has also remained unknown. To resolve these problems, we have carried out a systematic study of the size dependence of protein diffusion coefficients in the Escherichia coli cytoplasm, using engineered GFP multimers (from 2 to 6 covalently linked GFP molecules). Diffusion coefficients were measured using confocal fluorescence recovery after photobleaching (FRAP). At least up to 110 kDa (four linked GFP molecules), the diffusion coefficient varies with size roughly as would be predicted from the Einstein-Stokes equation for a classical (Newtonian) fluid. Thus, protein diffusion coefficients are predictable over this range. GFP tagging of proteins has little impact on the diffusion coefficient over this size range and therefore need not significantly perturb protein movement. Two indigenous E. coli proteins were used to show that their specific interactions within the cell are the main controllers of the diffusion rate.The use of fluorescence microscopic techniques to monitor macromolecular diffusion in eukaryotic (HeLa) cells showed that the diffusion of DNA is strongly size dependent but also that two fluorescently labeled dextrans (70 kDa and 580 kDa) can diffuse freely in the cytoplasm and nucleus (16). Within bacterial cells such as Escherichia coli, similar measurements are challenging because of the small dimensions of the cell. Nevertheless, studies of the mobility of fluorescently tagged proteins are starting to give powerful insights into the dynamics of processes occurring in living bacterial cells. Examples include studies of the mobility of signal transduction proteins in the E. coli cytoplasm (22), the mobility and distribution of transporters and respiratory complexes in the plasma membrane (14, 15), and the dynamic assembly/disassembly of the flagellar motor (13). All of these studies depend on the use of cells engineered to express fusion proteins in which the protein of interest is fused to a fluorescent protein tag, usually a variant of green fluorescent protein (GFP). In many cases, the fluorescent tag is comparable in size to or even larger than the protein of interest. For example, the chemotaxis signal transducer CheY (14 kDa) was tagged with yellow fluorescent protein (YFP), producing a fusion protein of about 41 kDa (3, 22) It remains an open question how much the addition of a substantial fluorescent tag might perturb the mobility of the protein of interest.The bacterial cytoplasm is a complex, crowded environment (5). The movement of proteins within the cytoplasm must be constrained by a combination of viscosity, macromolecular crowding, and specific interactions of the protein with other cell components (e.g., other proteins, nucleic acids, and the cytoplasmic membrane). Any indigenous protein is likely to have specific interactions with other cell components. Therefore, it is difficult to dissect out the specific aspects of its behavior from the more general physical constraints in the cytoplasm. The effects of crowding in the cytoplasm could be complex. For example, it is conceivable that macromolecules could form a molecular sieve imposing a distinct size limit on protein mobility (19). The diffusion of fluorescent proteins in the E. coli cytoplasm can conveniently be measured using fluorescence recovery after photobleaching (FRAP) (6, 11, 18). To resolve the question of the size dependence of protein diffusion in the E. coli cytoplasm, FRAP was used to measure diffusion coefficients (D) for a series of engineered GFP oligomers, ranging in size from 30 kDa (GFP monomers) to 165 kDa (six linked GFP molecules). The compact barrel-like structure of GFP (30) minimizes its interactions with other proteins. Diffusion in the cytoplasm is independent of the type and amount of coexpressed protein, and overcrowding of the cytoplasm does not seem to lead to self-interaction of GFP (24). Since GFP is not indigenous to E. coli and is unlikely to have specific interactions with other cell components, it can be assumed that the behavior of GFP oligomers reflects only the simple physical constraints controlling protein movement in the cytoplasm.     

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.     

Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization

To probe the complexity of the cell membrane organization and dynamics, it is important to obtain simple physical observables from experiments on live cells. Here we show that fluorescence correlation spectroscopy (FCS) measurements at different spatial scales enable distinguishing between different submicron confinement models. By plotting the diffusion time versus the transverse area of the confocal volume, we introduce the so-called FCS diffusion law, which is the key concept throughout this article. First, we report experimental FCS diffusion laws for two membrane constituents, which are respectively a putative raft marker and a cytoskeleton-hindered transmembrane protein. We find that these two constituents exhibit very distinct behaviors. To understand these results, we propose different models, which account for the diffusion of molecules either in a membrane comprising isolated microdomains or in a meshwork. By simulating FCS experiments for these two types of organization, we obtain FCS diffusion laws in agreement with our experimental observations. We also demonstrate that simple observables derived from these FCS diffusion laws are strongly related to confinement parameters such as the partition of molecules in microdomains and the average confinement time of molecules in a microdomain or a single mesh of a meshwork.     

Localization of Protein Aggregation in Escherichia coli Is Governed by Diffusion and Nucleoid Macromolecular Crowding Effect

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of “soft” intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.     

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.     

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.     

Line-FRAP,A Versatile Method to Measure Diffusion Rates In Vitro and In Vivo

The crowded cellular milieu affect molecular diffusion through hard (occluded space) and soft (weak, non-specific) interactions. Multiple methods have been developed to measure diffusion coefficients at physiological protein concentrations within cells, each with its limitations. Here, we show that Line-FRAP, combined with rigours data analysis, is able to determine diffusion coefficients in a variety of environments, from in vitro to in vivo. The use of Line mode greatly improves time resolution of FRAP data acquisition, from 20-100 Hz in the classical mode to 800 Hz in the line mode. This improves data analysis, as intensity and radius of the bleach at the first post-bleach frame is critical. We evaluated the method on different proteins labelled chemically or fused to YFP in a wide range of environments. The diffusion coefficients measured in HeLa and in E. coli were ~2.5-fold and 15-fold slower than in buffer, and were comparable to previously published data. Increasing the osmotic pressure on E. coli further decreases diffusion, to the point at which proteins virtually stop moving. The method presented here, which requires a confocal microscope equipped with dual scanners, can be applied to study a large range of molecules with different sizes, and provides robust results in a wide range of environments and protein concentrations for fast diffusing molecules.     

Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli

Fluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer‐Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane‐spanning alpha‐helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature‐dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.     

Anomalous protein diffusion in living cells as seen by fluorescence correlation spectroscopy

We investigate the challenges and limitations that are encountered when studying membrane protein dynamics in vivo by means of fluorescence correlation spectroscopy (FCS). Based on theoretical arguments and computer simulations, we show that, in general, the fluctuating fluorescence has a fractal dimension D(0) >or= 1.5, which is determined by the anomality alpha of the diffusional motion of the labeled particles, i.e., by the growth of their mean square displacement as (Deltax)(2) approximately t(alpha). The fractality enforces an initial power-law behavior of the autocorrelation function and related quantities for small times. Using this information, we show by FCS that Golgi resident membrane proteins move subdiffusively in the endoplasmic reticulum and the Golgi apparatus in vivo. Based on Monte Carlo simulations for FCS on curved surfaces, we can rule out that the observed anomalous diffusion is a result of the complex topology of the membrane. The apparent mobility of particles as determined by FCS, however, is shown to depend crucially on the shape of the membrane and its motion in time. Due to this fact, the hydrodynamic radius of the tracked particles can be easily overestimated by an order of magnitude.     

Simultaneous diffusion and brightness measurements and brightness profile visualization from single fluorescence fluctuation traces of GFP in living cells

Fluorescence correlation spectroscopy (FCS) and photon-counting histogram (PCH) analysis use the same experimental fluorescence intensity fluctuations, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH analysis yielding information about the molecular brightness of fluorescent species. Analysis of both FCS and PCH results in the molecular concentration of the sample. Using a previously described global analysis procedure we report here precise, simultaneous measurements of diffusion constants and brightness values from single fluorescence fluctuation traces of green-fluorescent protein (GFP, S65T) in the cytoplasm of Dictyostelium cells. The use of a polynomial profile in PCH analysis, describing the detected three-dimensional shape of the confocal volume, enabled us to obtain well fitting results for GFP in cells. We could visualize the polynomial profile and show its deviation from a Gaussian profile.