Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5.

Cellular functions rely on proper actions of organelles such as peroxisomes. These organelles rely on the import of proteins from the cytosol. The peroxisomal import receptor PEX5 takes up target proteins in the cytosol and transports them to the peroxisomal matrix. However, its cytosolic molecular interactions have so far not directly been disclosed. Here, we combined advanced optical microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy and stimulated emission depletion microscopy with biochemical tools to present a detailed characterization of the cytosolic diffusion and interaction dynamics of PEX5. Among other features, we highlight a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol as well as highlighting the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol by studying their diffusion dynamics.

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly.

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1-infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly.

Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy.

Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50 ± 15 nm and 120 ± 40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

Self-organizing actin patterns shape membrane architecture but not cell mechanics.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells.

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration.

We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range.

Fluorescence fluctuation spectroscopy in reduced detection volumes.

Fluorescence fluctuation spectroscopy is a versatile technique applied to in vitro and in vivo investigations of biochemical processes such as interactions, mobilities or densities with high specifity and sensitivity. The prerequisite of this dynamical fluorescence technique is to have, at a time, only few fluorescent molecules in the detection volume in order to generate significant fluorescence fluctuations. For usual confocal fluorescence microscopy this amounts to a useful concentration in the nanomolar range. The concentration of many biomolecules in living cell or on cell membranes is, however, often quite high, usually in the micro- to the millimolar range. To allow fluctuation spectroscopy and track intracellular interaction or localization of single fluorescently labeled biomolecules in such crowded environments, development of detection volumes with nanoscale resolution is necessary. As diffraction prevents this in the case of light microscopy, new (non-invasive) optical concepts have been developed. In this mini-review article we present recent advancements, implemented to decrease the detection volume below that of normal fluorescence microscopy. Especially, their combination with fluorescence fluctuation spectroscopy is emphasized.

Comparison of different fluorescence fluctuation methods for their use in FRET assays: monitoring a protease reaction.

We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

New fluorescence techniques for high-throughput drug discovery.

The rapid increase of compound libraries as well as new targets emerging from the Human Genome Project require constant progress in pharmaceutical research. An important tool is High-Throughput Screening (HTS), which has evolved as an indispensable instrument in the pre-clinical target-to-IND (Investigational New Drug) discovery process. HTS requires machinery, which is able to test more than 100,000 potential drug candidates per day with respect to a specific biological activity. This calls for certain experimental demands especially with respect to sensitivity, speed, and statistical accuracy, which are fulfilled by using fluorescence technology instrumentation. In particular the recently developed family of fluorescence techniques, FIDA (Fluorescence Intensity Distribution Analysis), which is based on confocal single-molecule detection, has opened up a new field of HTS applications. This report describes the application of these new techniques as well as of common fluorescence techniques--such as confocal fluorescence lifetime and anisotropy--to HTS. It gives experimental examples and presents advantages and disadvantages of each method. In addition the most common artifacts (auto-fluorescence or quenching by the drug candidates) emerging from the fluorescence detection techniques are highlighted and correction methods for confocal fluorescence read-outs are presented, which are able to circumvent this deficiency.

Data registration and selective single-molecule analysis using multi-parameter fluorescence detection.

A general strategy to identify and quantify sample molecules in dilute solution employing a new spectroscopic method for data registration and specific burst analysis denoted as multi-parameter fluorescence detection (MFD) was recently developed. While keeping the experimental advantage of monitoring single molecules diffusing through the microscopic open volume element of a confocal epi-illuminated set-up as in experiments of fluorescence correlation spectroscopy, MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the four-dimensional fluorescence information (intensity, F; lifetime, tau; anisotropy, r; and spectral range, lambda(r)) in real time and allows for exclusion of extraneous events for subsequent analysis. In this review, the versatility of this technique in confocal fluorescence spectroscopy will be presented by identifying freely diffusing single dyes via their characteristic fluorescence properties in homogenous assays, resulting in significantly reduced misclassification probabilities. Major improvements in background suppression are demonstrated by time-gated autocorrelation analysis of fluorescence intensity traces extracted from MFD data. Finally, applications of MFD to real-time conformational dynamics studies of fluorescence labeled oligonucleotides will be presented.

The psychosocial implications of live-related kidney donation.

The aim of the study was to explore the donor's experience of the live related transplantation process whilst seeking to identify the external and internal resources and ethical considerations which were reflected in the decision-making process. The outcome would indicate that altruism and a sense of moral obligation were the prime motivational influences with the spontaneity of decision-making casting doubt on the ability to give informed consent. These findings are also discussed in relation to implications for professional practice and would suggest that potential donors benefit from having access to a counsellor during the donor-search and throughout the transplant work up programme.

Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence.

Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F1 part of the H+-ATPase from Escherichia coli, EF1, carrying the gammaT106-C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355-21359] in aqueous solution upon excitation with a mode-locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3 x 10(-7) cm2 s(-1). In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational changes.

Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy.

A recently developed, real-time spectroscopic technique, burst-integrated fluorescence lifetime (BIFL), is shown to be well suited for monitoring the individual molecular conformational dynamics of a single molecule diffusing through the microscopic, open measurement volume (approximately 10 fl) of a confocal epi-illuminated set-up. In a highly diluted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhodamine (TMR), fluorescence bursts indicating traces of individual molecules are registered and further subjected to selective burst analysis. The two-dimensional BIFL data allow the identification and detection of different temporally resolved conformational states. A complementary autocorrelation analysis was performed on the time-dependent fluctuations in fluorescence lifetime and intensity. The consistent results strongly support the hypothesized three-state model of the conformational dynamics of the TMR-DNA duplex with a polar, a nonpolar, and a quenching environment of TMR.

Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis.

The photostability of fluorescent dyes is of crucial importance for the statistical accuracy of single-molecule detection (SMD) and for the image quality of scanning confocal microscopy. Concurrent results for the photostability were obtained by two different experimental techniques. First, the photostabilities of several coumarin and rhodamine derivatives in aqueous solution were obtained by monitoring the steady-state fluorescence decay in a quartz cell. Furthermore, an epi-illuminated microscope, continuous wave (CW) excitation at 514.5 nm, and fluorescence correlation spectroscopy (FCS) with a newly developed theory were used to study the photobleaching characteristics of rhodamines under conditions used for SMD. Depending on the rhodamine structure, the probability of photobleaching, p(b), is in the order of 10(-)(6)-10(-)(7) for irradiances below 10(3) W/cm(2). However, a considerable increase of p(b) for irradiances above this level was observed which can only be described by photobleaching reactions from higher excited states (two-step photolysis). In view of these observations, the probability of photobleaching, p(b), as well as a closed expression of its dependence on the CW excitation irradiance considering a five-level molecular electronic state model with the possibility of photobleaching from higher excited electronic states, is derived. From this model, optimal conditions for SMD with respect to the number of emitted fluorescence photons and to the signal-to-background ratio are discussed, taking into account both saturation and photobleaching. The additional photobleaching due to two-step photolysis limits the applicable irradiance.