Petra Schwille

Ceramide Triggers Budding of Exosome Vesicles into Multivesicular Endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.

Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.

The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude.

Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation.

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.

Fluorescence cross-correlation spectroscopy in living cells

Cell biologists strive to characterize molecular interactions directly in the intracellular environment. The intrinsic resolution of optical microscopy, however, allows visualization of only coarse subcellular localization. By extracting information from molecular dynamics, fluorescence cross-correlation spectroscopy (FCCS) grants access to processes on a molecular scale, such as diffusion, binding, enzymatic reactions and codiffusion, and has become a valuable tool for studies in living cells. Here we review basic principles of FCCS and focus on seminal applications, including examples of intracellular signaling and trafficking. We consider FCCS in the context of fluorescence resonance energy transfer and multicolor imaging techniques and discuss application strategies and recent technical advances.

Fluorescence correlation spectroscopy with single molecule sensitivity on cell and model membranes.

We report on the successful application of fluorescence correlation spectroscopy (FCS) to the analysis of single fluorescently labeled lipid analogue molecules diffusing laterally in lipid bilayers, as exemplified by time traces of fluorescence bursts of individual molecules entering and leaving the excitation area. FCS measurements performed on lipid probes in rat basophilic leukemia cell membranes showed deviations from two-dimensional Brownian motion with a single uniform diffusion constant. Giant unilamellar vesicles were employed as model systems to characterize diffusion of fluorescent lipid analogues in both homogeneous and mixed lipid phases with diffusion heterogeneity. Comparing the results of cell membrane diffusion with the findings on the model systems suggests possible explanations for the observations: (a) anomalous subdiffusion in which evanescent attractive interactions with disparate mobile molecules modifies the diffusion statistics; (b) alternatively, probe molecules are localized in microdomains of submicroscopic size, possibly in heterogeneous membrane phases.

Plasma membranes are poised for activation of raft phase coalescence at physiological temperature.

Cell membranes are not randomly organized, but rather are populated by fluctuating nanoassemblies of increased translational order termed lipid rafts. This lateral heterogeneity can be biophysically extended because cooling formaldehyde-isolated plasma membrane preparations results in separation into phases similar to the liquid-ordered (Lo) and liquid-disordered (Ld) states seen in model membrane systems [Baumgart T, et al. (2007) Proc Natl Acad Sci USA 104:3165–3170]. In this work we demonstrate that raft clustering, i.e., amplifying underlying raft-based connectivity to a larger scale, makes an analogous capacity accessible at 37°C. In plasma membranes at this temperature, cholera toxin-mediated cross-linking of the raft ganglioside GM1 induced the sterol-dependent emergence of a slower diffusing micrometer-scale phase that was enriched in cholesterol and selectively reorganized the lateral distribution of membrane proteins. Although parallels can be drawn, we argue that this raft coalescence in a complex biological matrix cannot be explained by only those interactions that define Lo formation in model membranes. Under this light, our induction of raft-phase separation suggests that plasma membrane composition is poised for selective and functional raft clustering at physiologically relevant temperature.

Fluorescence correlation spectroscopy and its potential for intracellular applications.

Fluorescence correlation spectroscopy (FCS) is a time-averaging fluctuation analysis of small molecular ensembles, combining maximum sensitivity with high statistical confidence. Among a multitude of physical parameters that are, in principle, accessible by FCS, it most conveniently allows to determine local concentrations, mobility coefficients, and characteristic rate constants of fast-reversible and slow-irreversible reactions of fluorescently labeled biomolecules at very low (nanomolar) concentrations, under equilibrium conditions and without physical separation. Its presently most popular instrumentation by confocal-microscope setups allows for a spatial resolution of fractions of femtoliters for the measurement volumes, containing sparse or even single molecules at any time, and encourages the adaptation of the solution-based technique for cellular applications. The scope of this review is thus, to introduce the FCS technique in particular to the reader with biological background, searching for new methods for a precise quantification of physical parameters governing cellular mechanisms and dynamics, especially if high sensitivity and fast dynamic resolution are required. After a short theoretical introduction, examples are given for the so far most important experimental applications, with respect to their implementation in cellular systems. As an interesting alternative to the confocal instrumentation, two-photon excitation will be introduced, offering a number of important advantages especially in cellular systems with high-noise and low-signal levels.

Spatial regulators for bacterial cell division self-organize into surface waves in vitro.

In the bacterium Escherichia coli, the Min proteins oscillate between the cell poles to select the cell center as division site. This dynamic pattern has been proposed to arise by self-organization of these proteins, and several models have suggested a reaction-diffusion type mechanism. Here, we found that the Min proteins spontaneously formed planar surface waves on a flat membrane in vitro. The formation and maintenance of these patterns, which extended for hundreds of micrometers, required adenosine 5′-triphosphate (ATP), and they persisted for hours. We present a reaction-diffusion model of the MinD and MinE dynamics that accounts for our experimental observations and also captures the in vivo oscillations.

GM1 structure determines SV40-induced membrane invagination and infection

Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells.

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.