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Biomedical subjects

Petra Schwille

Publications and source records attributed to Petra Schwille.

At least 19 recordsLinked to original sources
PubMed2006

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.

Algorithms↗
PubMed2005

Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform.

We have developed a filterless multicolor detection unit for fluorescence correlation spectroscopy (FCS). This grating-based setup is continuously tunable for multicolor separation and is thus a powerful alternative to the classical cascade of dichroic mirrors and filters. Our tailored platform allows for accommodation of up to 15 detection channels covering the entire visible spectral range. As a proof of principle, we successfully demonstrate simultaneous FCS of four distinct fluorescent quantum dot species being mixed in aqueous solution. Grating-based detection allows for spectral high-resolution FCS in a stable and compact setup and is a feasible tool for quantitative investigation of complexbiomolecular dynamics on a single molecule level.

Equipment Design↗
PubMed2005

Lipids as modulators of proteolytic activity of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro.

The beta-secretase, BACE, is a membrane spanning aspartic protease, which cleaves the amyloid precursor protein (APP) in the first step of proteolytic processing leading to the formation of the neurotoxic beta-amyloid peptide (Abeta). Previous results have suggested that the regulation of beta-secretase and BACE access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have expressed recombinant human full-length BACE in insect cells and purified milligram amounts to homogeneity. We have studied partitioning of fluorophor-conjugated BACE between the liquid ordered and disordered phases in giant (10-150 mum) unilamellar vesicles, and found approximately 20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquid-ordered phase increased upon cross-linking of raft lipids. To examine involvement of individual lipid species in modulating BACE activity, we have reconstituted the purified BACE in large ( approximately 100 nm) unilamellar vesicles, and determined its specific activity in vesicles of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1) neutral glycosphingolipids (cerebrosides), 2) anionic glycerophospholipids, and 3) sterols (cholesterol).

Amyloid Precursor Protein Secretases↗
PubMed2005

Determining protease activity in vivo by fluorescence cross-correlation analysis.

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions.

Algorithms↗
PubMed2005

Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances.

Atomic force microscopy (AFM) has been applied to characterize hydrated sphingomyelin/dioleoylphosphatidylcholine/cholesterol supported bilayers, after dehydration either in the absence or in the presence of several stabilizing substances. Such a study provides information about the effect of extreme environmental conditions on biological membranes and, in particular, on lipidic microdomains. Dehydration stress, indeed, is thought to cause both macroscopical damage and alterations of microdomains in biomembranes, leading to deleterious effects. These phenomena can be avoided if disaccharides are added during dehydration. In this work, we apply AFM imaging to directly visualize damage caused to supported lipid bilayers by water removal. We compare the efficiency of sucrose, trehalose, dextran, dimethyl sulfoxide, and glucose in preserving the structural integrity of domain-exhibiting model membranes. Finally, in addition to confirming previous findings, our results provide further insight into damage and alteration of microdomains in membranes as a consequence of stressful drying conditions.

Disaccharides↗
PubMed2005

Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles.

Much attention has recently been drawn to the hypothesis that cellular membranes organize in functionalized platforms called rafts, enriched in sphingolipids and cholesterol. The notion that glycosylphosphatidylinositol (GPI)-anchored proteins are strongly associated with rafts is based on their insolubility in nonionic detergents. However, detergent-based methodologies for identifying raft association are indirect and potentially prone to artifacts. On the other hand, rafts have proven to be difficult to visualize and investigate in living cells. A number of studies have demonstrated that model membranes provide a valuable tool for elucidating some of the raft properties. Here, we present a model membrane system based on domain-forming giant unilamellar vesicles (GUVs), in which the GPI-anchored protein, human placental alkaline phosphatase (PLAP), has been functionally reconstituted. Raft morphology, protein raft partitioning, and dynamic behavior have been characterized by fluorescence confocal microscopy and fluorescence correlation spectroscopy (FCS). Approximately 20-30% of PLAP associate with sphingomyelin-enriched domains. The affinity of PLAP for the liquid-ordered (l(o)) phase is compared to that of a nonraft protein, bacteriorhodopsin. Next, detergent extraction was carried out on PLAP-containing GUVs as a function of temperature, to relate the lipid and protein organization in distinct phases of the GUVs to the composition of detergent resistant membranes (DRMs). Finally, antibody-mediated cross-linking of PLAP induces a shift of its partition coefficient in favor of the l(o) phase.

Alkaline Phosphatase↗
PubMed2005

Differential lipid packing abilities and dynamics in giant unilamellar vesicles composed of short-chain saturated glycerol-phospholipids, sphingomyelin and cholesterol.

The ability of membrane components to arrange themselves heterogeneously within the bilayer induces the formation of microdomains. Much work has been devoted to mimicking domain-assembly in artificial bilayers and characterizing their physico-chemical properties. Ternary lipid mixtures composed of unsaturated phospholipids, sphingomyelin and cholesterol give rise to large, round domains. Here, we replaced the unsaturated phospholipid in the ternary mixture with sphingomyelin and cholesterol by saturated glycero-phospholipids of different chain length and characterized the critical role of cholesterol in sorting these lipids by confocal imaging and fluorescence correlation spectroscopy (FCS). More cholesterol is needed to obtain phase segregation in ternary mixtures, in which the unsaturated phospholipid is replaced by a saturated one. Finally, lipid dynamics in distinct phases is very low and astonishingly similar, thereby suggesting the poor ability of cholesterol in sorting short-chain saturated glycero-phospholipids and sphingomyelin.

Cholesterol↗
PubMed2005

An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy.

Site-specific exchange of genetic information is mediated by DNA recombinases, such as FLP or Cre, and has become a valuable tool in modern molecular biology. The so far low number of suitable recombinating enzymes has driven current research activities towards alteration of catalytic properties, such as thermostability or recognition sequences. However, identification and analysis of new mutants requires sensitive in vitro activity assays, which traditionally are based on gel electrophoresis. Here, we describe the development of a new sensitive DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy (DC-FCCS), which works in homogenous solution and does not require any separation step such as electrophoresis. The assay was validated with unlabeled FLP recombinase and different fluorescently labeled DNA substrates containing the FLP recognition target (FRT). This strategy fulfills all requirements for possible application in high throughput screening and engineering of new site-specific DNA recombinases starting from the FLP-FRT system, and is easily adjustable to other systems like Cre/loxP.

Color↗
PubMed2005

Two-photon cross-correlation analysis of intracellular reactions with variable stoichiometry.

We successfully demonstrate the effectiveness of two-photon fluorescence cross-correlation spectroscopy (TPCCS) to study the complex binding stoichiometry of calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaMKII). Practical considerations are made for developing an intracellular cross-correlation assay, including characterization of the fluorescent molecules involved, calibration procedures of the setup, and optimal measurement conditions. Potential pitfalls and artifacts are discussed, and the complex stoichiometry of the molecular system is accounted for by a new experimental and theoretical framework for TPCCS. Our tailored model accommodates up to 12 red-labeled CaMs binding to a single green-labeled dodecameric CaMKII holoenzyme and accounts for the probability distributions of bound ligand as well as the respective changes in fluorescence emission upon binding. The model was experimentally demonstrated both in solution and in living cells by analyzing the binding of Alexa 633(C2)CaM to eGFP-CaMKII under different biochemical conditions known to induce the basal, activated, and autophosphorylated forms of the enzyme. Key binding parameters, such as binding degree, concentrations of reactants, and binding affinities, were determined under varying conditions with certain assumptions. TPCCS thus offers the unique ability to test our biochemical understanding of protein dynamics in the intracellular milieu.

Biophysical Phenomena↗
PubMed2005

New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking.

We report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1-2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes.

Lipid Metabolism↗
PubMed2005

Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes.

The existence of lipid rafts in biological membranes in vivo is still debated. In contrast, the formation of domains in model systems has been well documented. In giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol, a clear separation of liquid-disordered and sphingomyelin-enriched, liquid-ordered phases could be observed. This phase separation can lead to the fission of the liquid-ordered phase from the vesicle. Here we show that in cholesterol-containing GUVs, the phase separation can involve dynamic redistribution of lipids from one phase into another as a result of a cross-linking perturbation. We found that the molecular structure of a sterol used for the preparation of GUVs determines (i) its ability to induce phase separation and (ii) the curvature (positive or negative) of the formed liquid-ordered phase. As a consequence, the latter can pinch off to the outside or inside of the vesicle. Remarkably, some mixtures of sterols induce liquid-ordered domains exhibiting both positive and negative curvature, which can lead to a new type of budding behavior in GUVs. Our findings could have implications for the role of sterols in various cell-biological processes such as budding of secretory vesicles, endocytosis, or formation of multivesicular bodies.

Cholera Toxin↗
PubMed2005

Breakdown of axonal synaptic vesicle precursor transport by microglial nitric oxide.

The mechanism of axonal injury in inflammatory brain diseases is still unclear. Increased microglial production of nitric oxide (NO) is a common early sign in neuroinflammatory diseases. We found by fluorescence correlation spectroscopy that synaptophysin tagged with enhanced green fluorescence protein (synaptophysin-EGFP) moves anterogradely in axons of cultured neurons. Activated microglia focally inhibited the axonal movement of synaptophysin-EGFP in a NO synthase-dependent manner. Direct application of a NO donor to neurons resulted in inhibition of axonal transport of synaptophysin-EGFP and synaptotagmin I tagged with EGFP, mediated via phosphorylation of c-jun NH2-terminal kinase (JNK). Thus, overt production of reactive NO by activated microglia blocks the axonal transport of synaptic vesicle precursors via phosphorylation of JNK and could cause axonal and synaptic dysfunction.

Animals↗
PubMed2005

A novel homogenous assay for topoisomerase II action and inhibition.

Topoisomerase II is the only enzyme able to cleave and religate double-stranded DNA; this makes it essential for many vital functions during normal cell growth. Increased expression of topoisomerase II is a common occurrence in neoplasia, and different topoisomerase II inhibitors have indeed been proven to be powerful anticancer drugs. For this reason, the topoisomerase II catalytic cycle has attracted strong interest, but only a few techniques contributing to studies in this field have emerged. All of the currently used conventional methods to elucidate the action and inhibition of topoisomerase II require separation steps and are therefore unsatisfactory in terms of sensitivity, speed, and throughput. Here, for the first time, we present an assay that works in homogenous solution. The assay is based on dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) and allows monitoring of topoisomerase II action and, especially, detection and discrimination of different topoisomerase II inhibitor classes. The effectiveness of our new assay was confirmed by measuring the effects of a catalytic inhibitor (novobiocin) and a topoisomerase poison (m-AMSA) with bacteriophage T4 topoisomerase as a model system, thus showing the strategy to be easy, fast, and extremely sensitive. Further development of the DC-FCCS-based assay and subsequent application in high-throughput drug screening of new anticancer drugs is proposed and discussed.

Catalysis↗
PubMed2005

Fluorescence correlation spectroscopy with autofluorescent proteins.

Fluorescence correlation spectroscopy (FCS) is a versatile technique operating at the single-molecule level, that successfully meets many challenges of modern biological research. Based on the detection of mobile fluorescent molecules diffusing in and out of a diffraction-limited laser focus, the method allows to resolve particle dynamics within cells and their compartments. Previous FCS studies have described various parameters of protein function, namely mobility, transport and localization phenomena, enzymatic turnovers of biochemical substrates and molecular association and dissociation reactions. Recent progress in the application of FCS to intracellular systems has particularly taken advantage of detecting autofluorescent proteins and their genetically encoded fusions to cellular proteins. This review discusses recent applications of FCS analysis with and on fluorescent proteins, particularly highlighting chemical and physical properties. Inherent limitations of the presented approaches are discussed in detail and strategies for optimisation of experimental systems outlined.

Luminescent Proteins↗
PubMed2004

Characterization of interaction between cationic lipid-oligonucleotide complexes and cellular membrane lipids using confocal imaging and fluorescence correlation spectroscopy.

Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers.

Biological Transport↗
PubMed2004

SNAREs prefer liquid-disordered over "raft" (liquid-ordered) domains when reconstituted into giant unilamellar vesicles.

Membrane domains ("rafts") have received great attention as potential platforms for proteins in signaling and trafficking. Because rafts are believed to form by cooperative lipid interactions but are not directly accessible in vivo, artificial phase-separating lipid bilayers are useful model systems. Giant unilamellar vesicles (GUVs) offer large free-standing bilayers, but suitable methods for incorporating proteins are still scarce. Here we report the reconstitution of two water-insoluble SNARE proteins into GUVs without fusogenic additives. Following reconstitution, protein functionality was assayed by confocal imaging and fluorescence auto- and cross-correlation spectroscopy. Incorporation into GUVs containing phase-separating lipids revealed that, in the absence of other cellular factors, both proteins exhibit an intrinsic preference for the liquid-disordered phase. Although the picture from detergent resistance assays on whole cells is ambiguous, reconstitutions of components of the exocytic machinery into GUVs by this new approach should yield insight into the dynamics of protein complex associations with hypothesized liquid-ordered phase microdomains, the correspondence between detergent-resistant membranes and liquid-ordered phase, and the mechanism of SNARE-mediated membrane fusion.

Exocytosis↗
PubMed2004

Lipid domain formation and dynamics in giant unilamellar vesicles explored by fluorescence correlation spectroscopy.

Lipids in eukaryotic cell membranes have been shown to cluster in "rafts" with different lipid/protein compositions and molecular packing. Model membranes such as giant unilamellar vesicles (GUVs) provide a key system to elucidate the physical mechanisms of raft assembly. Despite the large amount of work devoted to the detection and characterization of rafts, one of the most important pieces of information still missing in the picture of the cell membrane is dynamics: how lipids organize and move in rafts and how they modulate membrane fluidity. This missing element is of crucial importance for the trafficking at and from the periphery of the cell regulated by endo- and exocytosis and, in general, for the constant turnover which redistributes membrane components. Here, we review studies of combined confocal fluorescence microscopy and fluorescence correlation spectroscopy on lipid dynamics and organization in rafts assembled in GUVs prepared from various lipid mixtures which are relevant to the problem of raft formation.

Biological Transport↗