Tracking single serotonin transporter molecules at the endoplasmic reticulum and plasma membrane.
Anderluh, A., Klotzsch, E., Ries, J., Reismann, A.W., Weber, S., Folser, M., Koban, F., Freissmuth, M., Sitte, H.H. & Schutz, G.J.
Biophys J. 2014 May 6;106(9):L33-5. doi: 10.1016/j.bpj.2014.03.019.
Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma membrane, rendering total internal reflection microscopy of plasma membrane proximal ER regions feasible. Here we used single-molecule fluorescence microscopy to study the diffusion of the human serotonin transporter at the ER and the plasma membrane. We exploited the single-molecule trajectories to map out the structure of the ER close to the plasma membrane at subdiffractive resolution. Furthermore, our study provides a comparative picture of the diffusional behavior in both environments. Under unperturbed conditions, the majority of proteins showed similar mobility in the two compartments; at the ER, however, we found an additional 15% fraction of molecules moving with 25-fold faster mobility. Upon degradation of the actin skeleton, the diffusional behavior in the plasma membrane was strongly influenced, whereas it remained unchanged in the ER.
The yeast kinetochore - structural insights from optical microscopy.
Cieslinski, K. & Ries, J.
This is a review article.
Curr Opin Chem Biol. 2014 Apr 21;20C:1-8. doi: 10.1016/j.cbpa.2014.03.020.
In eukaryotic organisms each chromosome is captured by spindle microtubules. This interaction is mediated by an evolutionarily conserved, multi-protein complex called kinetochore. The main function of the kinetochore is to maintain correct chromosome segregation during cell division by transporting each sister chromatid to the spindle pole bodies localized at the opposite sites of the yeast nucleus. The kinetochore of budding yeast (Saccharomyces cerevisiae) is often used as a model system due to its simple composition compared to higher eukaryotes. This review highlights results obtained using optical imaging that revealed relative positions and stoichiometry of the major components of the budding yeast kinetochore.
Localization microscopy in yeast.
Mund, M., Kaplan, C. & Ries, J.
Methods Cell Biol. 2014;123C:253-271. doi: 10.1016/B978-0-12-420138-5.00014-8.
Conventional light and fluorescence microscopy techniques have offered tremendous insight into cellular processes and structures. Their resolution is however intrinsically limited by diffraction. Superresolution techniques achieve an order of magnitude higher resolution. Among these, localization microscopy relies on the position determination of single emitters with nanometer accuracy, which allows the subsequent reconstruction of an image of the target structure. In this chapter, we provide general guidelines for localization microscopy with a focus on Saccharomyces cerevisiae. Its different cellular architecture complicates efforts to directly transfer protocols established in mammalian cells to yeast. We compare different methodologies to label structures of interest and provide protocols for the respective sample preparation, which are not limited to yeast. Using these guidelines, nanoscopic subcellular structures in yeast can be investigated by localization microscopy, which perfectly complements live-cell fluorescence and electron microscopy.
A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of beta-amyloid production.
Udayar, V., Buggia-Prevot, V., Guerreiro, R.L., Siegel, G., Rambabu, N., Soohoo, A.L., Ponnusamy, M., Siegenthaler, B., Bali, J., Simons, M., Ries, J., Puthenveedu, M.A., Hardy, J., Thinakaran, G. & Rajendran, L.
Cell Rep. 2013 Dec 26;5(6):1536-51. doi: 10.1016/j.celrep.2013.12.005.
Alzheimer's disease (AD) is characterized by cerebral deposition of beta-amyloid (Abeta) peptides, which are generated from amyloid precursor protein (APP) by beta- and gamma-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Abeta levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Abeta production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled beta-secretase endosomal recycling to the plasma membrane and thus affected Abeta production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Abeta levels and show how systems biology approaches can unravel the molecular complexity underlying AD.
Superresolution imaging of amyloid fibrils with binding-activated probes.
Ries, J., Udayar, V., Soragni, A., Hornemann, S., Nilsson, K.P., Riek, R., Hock, C., Ewers, H., Aguzzi, A.A. & Rajendran, L.
ACS Chem Neurosci. 2013 Jul 17;4(7):1057-61. doi: 10.1021/cn400091m. Epub 2013Apr 22.
Protein misfolding into amyloid-like aggregates underlies many neurodegenerative diseases. Thus, insights into the structure and function of these amyloids will provide valuable information on the pathological mechanisms involved and aid in the design of improved drugs for treating amyloid-based disorders. However, determining the structure of endogenous amyloids at high resolution has been difficult. Here we employ binding-activated localization microscopy (BALM) to acquire superresolution images of alpha-synuclein amyloid fibrils with unprecedented optical resolution. We propose that BALM imaging can be extended to study the structure of other amyloids, for differential diagnosis of amyloid-related diseases and for discovery of drugs that perturb amyloid structure for therapy.
The bacterial SMC complex displays two distinct modes of interaction with the chromosome.
Kleine Borgmann, L.A., Ries, J., Ewers, H., Ulbrich, M.H. & Graumann, P.L.
Cell Rep. 2013 May 30;3(5):1483-92. doi: 10.1016/j.celrep.2013.04.005. Epub 2013May 9.
The bacterial SMC (structural maintenance of chromosomes) complex binds nonspecifically to DNA in vitro and forms two discrete subcellular centers in vivo, one in each cell half. How this distribution is maintained is unclear. We show by time-lapse imaging of single molecules that the localization is achieved through limited, yet rapid movement of the SMC subunits through the nucleoid. Accessory ScpAB subunits mediate the arrest of 20% of SMC molecules at the center of a cell half and do not move together with the 80% mobile SMC molecules. Only free SMC, but not the preformed SMC/ScpAB complex, was able to bind to DNA in vitro, revealing distinct functions of SMC fractions. Thus, whereas SMC alone dynamically interacts with many sites on the chromosome, it forms static assemblies together with ScpAB complex partners. Our findings reveal two distinct modes of interaction of SMC with the chromosome and indicate that limited diffusion within a confined space and transient arrest may be a general mechanism for positioning proteins within a chromosome and within a noncompartmentalized cell.
Tuning the "roadblock" effect in Kinesin-based transport.
Schmidt, C., Kim, B., Grabner, H., Ries, J., Kulomaa, M. & Vogel, V.
Nano Lett. 2012 Jul 11;12(7):3466-71. Epub 2012 Jun 14.
Major efforts are underway to harness motor proteins for technical applications. Yet how to best attach cargo to microtubules that serve as kinesin-driven "molecular shuttles" without compromising transport performance remains challenging. Furthermore, microtubule-associated proteins (MAPs) can block motor protein-powered transport in neurons, which can lead to neurodegenerative diseases. Again it is unclear how different physical roadblock parameters interfere with the stepping motion of kinesins. Here, we employ a series of MAPs, tailored (strept)avidins, and DNA as model roadblocks and determine how their geometrical, nanomechanical, and electrochemical properties can reduce kinesin-mediated transport. Our results provide insights into kinesin transport regulation and might facilitate the choice of appropriate cargo linkers for motor protein-driven transport devices.
A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.
Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H.
Nat Methods. 2012 Jun;9(6):582-4. doi: 10.1038/nmeth.1991. Epub 2012 Apr 29.
We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.
Fluorescence correlation spectroscopy.
Ries, J. & Schwille, P.
Bioessays. 2012 May;34(5):361-8. doi: 10.1002/bies.201100111. Epub 2012 Mar 13.
Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of fluorescence fluctuation spectroscopy.
Fluorescence Correlation Spectroscopy (FCS).
Ries, J., Weidemann, T. & Schwille, P.
Comprehensive Biophysics 2, Biophysical Techniques for Characterization of Cells, 210-245 (2012).
Binding-activated localization microscopy of DNA structures.
Schoen, I., Ries, J., Klotzsch, E., Ewers, H. & Vogel, V.
Nano Lett. 2011 Sep 14;11(9):4008-11. Epub 2011 Aug 18.
Many nucleic acid stains show a strong fluorescence enhancement upon binding to double-stranded DNA. Here we exploit this property to perform superresolution microscopy based on the localization of individual binding events. The dynamic labeling scheme and the optimization of fluorophore brightness yielded a resolution of approximately 14 nm (fwhm) and a spatial sampling of 1/nm. We illustrate our approach with two different DNA-binding dyes and apply it to visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy (BALM) can be extended to other dyes and targets such as protein structures.
Cxcl12 evolution--subfunctionalization of a ligand through altered interaction with the chemokine receptor.
Boldajipour, B., Doitsidou, M., Tarbashevich, K., Laguri, C., Yu, S.R., Ries, J., Dumstrei, K., Thelen, S., Dorries, J., Messerschmidt, E.M., Thelen, M., Schwille, P., Brand, M., Lortat-Jacob, H. & Raz
Development. 2011 Jul;138(14):2909-14.
The active migration of primordial germ cells (PGCs) from their site of specification towards their target is a valuable model for investigating directed cell migration within the complex environment of the developing embryo. In several vertebrates, PGC migration is guided by Cxcl12, a member of the chemokine superfamily. Interestingly, two distinct Cxcl12 paralogs are expressed in zebrafish embryos and contribute to the chemotattractive landscape. Although this offers versatility in the use of chemokine signals, it also requires a mechanism through which migrating cells prioritize the relevant cues that they encounter. Here, we show that PGCs respond preferentially to one of the paralogs and define the molecular basis for this biased behavior. We find that a single amino acid exchange switches the relative affinity of the Cxcl12 ligands for one of the duplicated Cxcr4 receptors, thereby determining the functional specialization of each chemokine that elicits a distinct function in a distinct process. This scenario represents an example of protein subfunctionalization--the specialization of two gene copies to perform complementary functions following gene duplication--which in this case is based on receptor-ligand interaction. Such specialization increases the complexity and flexibility of chemokine signaling in controlling concurrent developmental processes.
Principles and Applications of Fluorescence Correlation Spectroscopy (FCS).
Schwille, P. & Ries, J.
6385 (Biophotonics: Dordrecht, 2010).doi:10.1007/978-90-481-9977-8_4
Fluorescence correlation spectroscopy (FCS) is one of the various modern optical techniques that allow access to single fluorescently labeled biomolecules in aqueous solution. In contrast to other fluorescence measurements, however, the parameter of primary interest is not the emission intensity itself, but rather spontaneous intensity fluctuations caused by the minute deviations of a small ensemble of molecules from thermal equilibrium. In general, all physical parameters that give rise to fluctuations in the fluorescence signal are accessible by FCS. It is, for example, rather straightforward to determine local concentrations, mobility coefficients or characteristic rate constants of inter- or intramolecular reactions of fluorescently labeled biomolecules at nanomolar to micromolar concentrations.
DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells.
Jeyasekharan, A.D., Ayoub, N., Mahen, R., Ries, J., Esposito, A., Rajendra, E., Hattori, H., Kulkarni, R.P. & Venkitaraman, A.R.
Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21937-42. Epub 2010 Nov 22.
How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus.
Automated suppression of sample-related artifacts in Fluorescence Correlation Spectroscopy.
Ries, J., Bayer, M., Csucs, G., Dirkx, R., Solimena, M., Ewers, H. & Schwille, P.
Opt Express. 2010 May 24;18(11):11073-82. doi: 10.1364/OE.18.011073.
Fluorescence Correlation Spectroscopy (FCS) in cells often suffers from artifacts caused by bright aggregates or vesicles, depletion of fluorophores or bleaching of a fluorescent background. The common practice of manually discarding distorted curves is time consuming and subjective. Here we demonstrate the feasibility of automated FCS data analysis with efficient rejection of corrupted parts of the signal. As test systems we use a solution of fluorescent molecules, contaminated with bright fluorescent beads, as well as cells expressing a fluorescent protein (ICA512-EGFP), which partitions into bright secretory granules. This approach improves the accuracy of FCS measurements in biological samples, extends its applicability to especially challenging systems and greatly simplifies and accelerates the data analysis.
Scanning FCS for the characterization of protein dynamics in live cells.
Petrasek, Z., Ries, J. & Schwille, P.
Methods Enzymol. 2010;472:317-43.
Scanning fluorescence correlation spectroscopy (sFCS) is the generic term for a group of fluorescence correlation techniques where the measurement volume is moved across the sample in a defined way. The introduction of scanning is motivated by its ability to alleviate or remove several distinct problems often encountered in standard FCS, and thus, to extend the range of applicability of fluorescence correlation methods in biological systems. These problems include poor statistical accuracy in measurements with slowly moving molecules, photobleaching, optical distortions affecting the calibration of the measurement volume, membrane instabilities, etc. Here, we present an overview of sFCS methods, explaining their benefits, implementation details, requirements, and limitations, as well as relations to each other. Further, we give examples of different sFCS implementations as applied to cellular systems, namely large-circle sFCS to measure protein dynamics in embryo cortex and line sFCS to measure protein diffusion and interactions in unstable membranes.
A Comprehensive Framework for Fluorescence Cross-Correlation Spectroscopy.
J. Ries, Z. Petrasek, A. Garcia-Saez, and P. Schwille.
New Journal of Physics 12, 113009 (2010).
Membrane promotes tBID interaction with BCL(XL).
Garcia-Saez, A.J., Ries, J., Orzaez, M., Perez-Paya, E. & Schwille, P.
Nat Struct Mol Biol. 2009 Nov;16(11):1178-85. Epub 2009 Oct 11.
Two important questions on the molecular mechanism of the B cell CLL/lymphoma 2 (BCL2) proteins involve the interaction network between pro- and antiapoptotic members and the role of their translocation to the mitochondrial membrane during apoptosis. We used fluorescence correlation spectroscopy to quantify the molecular interactions of BH3-interacting domain death agonist (BID) and its truncated form tBID with the B cell lymphoma extra-large protein truncated at the C terminus (BCL(XL)DeltaCt) in solution and in membranes, and we found that (i) only the active form tBID binds to BCL(XL)DeltaCt and (ii) that the membrane strongly promotes binding between them. Particularly, a BH3 peptide from BID disrupts the tBID-BCL(XL) complex in solution, but only partially in lipid bilay
Detergent-activated BAX protein is a monomer.
Ivashyna, O., Garcia-Saez, A.J., Ries, J., Christenson, E.T., Schwille, P. & Schlesinger, P.H.
J Biol Chem. 2009 Sep 4;284(36):23935-46. Epub 2009 Jun 29.
BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation with detergents, such as n-octyl glucoside. During BAX activation with n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.
Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.
Yu, S.R., Burkhardt, M., Nowak, M., Ries, J., Petrasek, Z., Scholpp, S., Schwille, P. & Brand, M.
Nature. 2009 Sep 24;461(7263):533-6. Epub 2009 Sep 9.
It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.
Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.
Ries, J., Yu, S.R., Burkhardt, M., Brand, M. & Schwille, P.
Nat Methods. 2009 Sep;6(9):643-5. Epub 2009 Aug 2.
Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.
Accurate determination of membrane dynamics with line-scan FCS.
Ries, J., Chiantia, S. & Schwille, P.
Biophys J. 2009 Mar 4;96(5):1999-2008.
Here we present an efficient implementation of line-scan fluorescence correlation spectroscopy (i.e., one-dimensional spatio-temporal image correlation spectroscopy) using a commercial laser scanning microscope, which allows the accurate measurement of diffusion coefficients and concentrations in biological lipid membranes within seconds. Line-scan fluorescence correlation spectroscopy is a calibration-free technique. Therefore, it is insensitive to optical artifacts, saturation, or incorrect positioning of the laser focus. In addition, it is virtually unaffected by photobleaching. Correction schemes for residual inhomogeneities and depletion of fluorophores due to photobleaching extend the applicability of line-scan fluorescence correlation spectroscopy to more demanding systems. This technique enabled us to measure accurate diffusion coefficients and partition coefficients of fluorescent lipids in phase-separating supported bilayers of three commonly used raft-mimicking compositions. Furthermore, we probed the temperature dependence of the diffusion coefficient in several model membranes, and in human embryonic kidney cell membranes not affected by temperature-induced optical aberrations.
Fluorescence correlation spectroscopy in membrane structure elucidation.
Chiantia, S., Ries, J. & Schwille, P.
Biochim Biophys Acta. 2009 Jan;1788(1):225-33. Epub 2008 Aug 29.
This review describes the application of fluorescence correlation spectroscopy (FCS) for the study of biological membranes. Monitoring the fluorescence signal fluctuations, it is possible to obtain diffusion constants and concentrations for several membrane components. Focusing the attention on lipid bilayers, we explain the technical difficulties and the new FCS-based methodologies introduced to overcome them. Finally, we report several examples of studies which apply FCS on both model and biological membranes to obtain interesting insight in the topic of lateral membrane organization.
Plasma membranes are poised for activation of raft phase coalescence at physiological temperature.
Lingwood, D., Ries, J., Schwille, P. & Simons, K.
Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10005-10. Epub 2008 Jul 9.
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 degrees 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.
Total internal reflection fluorescence correlation spectroscopy: effects of lateral diffusion and surface-generated fluorescence.
Ries, J., Petrov, E.P. & Schwille, P.
Biophys J. 2008 Jul;95(1):390-9. Epub 2008 Mar 13.
Fluorescence correlation spectroscopy with total internal reflection excitation (TIR-FCS) is a promising method with emerging biological applications for measuring binding dynamics of fluorescent molecules to a planar substrate as well as diffusion coefficients and concentrations at the interface. Models for correlation functions proposed so far are rather approximate for most conditions, since they neglect lateral diffusion of fluorophores. Here we propose accurate extensions of previously published models for axial correlation functions taking into account lateral diffusion through detection profiles realized in typical experiments. In addition, we consider the effects of surface-generated emission in objective-based TIR-FCS. The expressions for correlation functions presented here will facilitate quantitative and accurate measurements with TIR-FCS.
New concepts for fluorescence correlation spectroscopy on membranes.
Ries, J. & Schwille, P.
Phys Chem Chem Phys. 2008 Jun 28;10(24):3487-97. Epub 2008 Mar 27.
Fluorescence correlation spectroscopy (FCS) is a powerful tool to measure useful physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in model and cell membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we assess the potential and limitations of standard confocal FCS on lipid membranes and present recent developments which facilitate accurate and quantitative measurements on such systems. In particular, we discuss calibration-free diffusion and concentration measurements using z-scan FCS and two focus FCS and present several approaches using scanning FCS to accurately measure slow dynamics. We also show how surface confined FCS enables the study of membrane dynamics even in presence of a strong cytosolic background and how FCS with a variable detection area can reveal submicroscopic heterogeneities in cell membranes.
Spatial regulators for bacterial cell division self-organize into surface waves in vitro.
Loose, M., Fischer-Friedrich, E., Ries, J., Kruse, K. & Schwille, P.
Science. 2008 May 9;320(5877):789-92.
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.
Role of ceramide in membrane protein organization investigated by combined AFM and FCS.
Chiantia, S., Ries, J., Chwastek, G., Carrer, D., Li, Z., Bittman, R. & Schwille, P.
Biochim Biophys Acta. 2008 May;1778(5):1356-64. Epub 2008 Feb 29.
Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.
Supercritical angle fluorescence correlation spectroscopy.
Ries, J., Ruckstuhl, T., Verdes, D. & Schwille, P.
Biophys J. 2008 Jan 1;94(1):221-9. Epub 2007 Sep 7.
We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.
Rho regulates membrane transport in the endocytic pathway to control plasma membrane specialization in oligodendroglial cells.
Kippert, A., Trajkovic, K., Rajendran, L., Ries, J. & Simons, M.
J Neurosci. 2007 Mar 28;27(13):3560-70.
Differentiation of oligodendrocytes is associated with dramatic changes in plasma membrane structure, culminating in the formation of myelin membrane sheaths. Previous results have provided evidence that regulation of endocytosis may represent a mechanism to control myelin membrane growth. Immature oligodendrocytes have a high rate of clathrin-independent endocytosis for the transport of membrane to late endosomes/lysosomes (LE/Ls). After maturation and receiving signals from neurons, endocytosis is reduced and transport of membrane from LE/Ls to the plasma membrane is triggered. Here, we show that changes in Rho GTPase activity are responsible for switching between these two modes of me
Combined AFM and two-focus SFCS study of raft-exhibiting model membranes.
Chiantia, S., Ries, J., Kahya, N. & Schwille, P.
Chemphyschem. 2006 Nov 13;7(11):2409-18.
Dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/cholesterol) model membranes exhibit liquid-liquid phase separation and therefore provide a physical model for the putative liquid-ordered domains present in cells. Here we present a combination of atomic force microscopy (AFM) imaging, force measurements, confocal fluorescence imaging and two-focus scanning fluorescence correlation spectroscopy (two-focus SFCS) to obtain structural and dynamical information about this model membrane system. Partition coefficients and diffusion coefficients in the different phases were measured with two-focus SFCS for numerous fluorescent lipid analogues and proteins, while being directly related to the lateral organization of the membrane and its mechanical properties probed by AFM. Moreover we show how the combination of these different approaches is effective in reducing artifacts resulting from the use of a single technique.
Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy.
Meacci, G., Ries, J., Fischer-Friedrich, E., Kahya, N., Schwille, P. & Kruse, K.
Phys Biol. 2006 Nov 28;3(4):255-63.
In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spectroscopy. Two distinct timescales are clearly visible in the correlation curves. While the faster timescale can be attributed to cytoplasmic diffusion, the slower timescale could result from diffusion of membrane-bound proteins or from protein exchange between the cytoplasm and the membrane. We determine the diffusion constant of cytoplasmic MinD to be approximately 16 microm(2) s(-1), while for MinE we find about 10 microm(2) s(-1), independently of the processes responsible for the slower time-scale. The implications of the measured values for the oscillation mechanism are discussed.
Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy.
Ries, J. & Schwille, P.
Biophys J. 2006 Sep 1;91(5):1915-24. Epub 2006 Jun 16.
Here we discuss the application of scanning fluorescence correlation spectroscopy (SFCS) using continuous wave excitation to analyze membrane dynamics. The high count rate per molecule enables the study of very slow diffusion in model and cell membranes, as well as the application of two-foci fluorescence cross-correlation spectroscopy for parameter-free determination of diffusion constants. The combination with dual-color fluorescence cross-correlation spectroscopy with continuous or pulsed interleaved excitation allows binding studies on membranes. Reduction of photobleaching, higher reproducibility, and stability compared to traditional FCS on membranes, and the simple implementation in a commercial microscopy setup make SFCS a valuable addition to the pool of fluorescence fluctuation techniques.
Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS.
Chiantia, S., Kahya, N., Ries, J. & Schwille, P.
Biophys J. 2006 Jun 15;90(12
The sphingolipid ceramides are known to influence lipid lateral organization in biological membranes. In particular, ceramide-induced alterations of microdomains can be involved in several cell functions, ranging from apoptosis to immune response. We used a combined approach of atomic force microscopy, fluorescence correlation spectroscopy, and confocal fluorescence imaging to investigate the effects of ceramides in model membranes of biological relevance. Our results show that physiological quantities of ceramide in sphingomyelin/dioleoylphosphatidylcholine/cholesterol supported bilayers lead to a significant rearrangement of lipid lateral organization. Our experimental setup allowed a simultaneous characterization of both structural and dynamic modification of membrane microdomains, induced by the presence of ceramide. Formation of similar ceramide-enriched domains and, more general, alterations of lipid-lipid interactions can be of crucial importance for the biological function of cell membranes.
Experimental vacuum squeezing in rubidium vapor via self-rotation.
Ries, J., Brezger, B. & Lvovsky, A.
Phys Rev a 68, (2003)
Quantum scissors: Teleportation of single-mode optical states by means of a nonlocal single photon
Ries, J. & Lvovsky, A.
EPL (Europhysics Letters) (2003).