Super-resolved Fluorescence Microscopy: Nobel Prize in Chemistry 2014 for Eric Betzig, Stefan Hell, and William E. Moerner

A big honor for small objects: The Nobel Prize in Chemistry 2014 was jointly awarded to Eric Betzig, Stefan Hell, and William E. Moerner “for the development of super‐resolved fluorescence microscopy”. This Highlight describes how the field of super‐resolution microscopy developed from the first detection of a single molecule in 1989 to the sophisticated techniques of today

Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single-Molecule Tracking Study

We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper-free click chemistry. Finally, these biotin-tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin-induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA-protein networks employing single-molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface

Microdomain Formation Controls Spatiotemporal Dynamics of Cell-Surface Glycoproteins

The effect of galectin-mediated microdomain formation on the spatiotemporal dynamics of glycosylated membrane proteins in human microvascular endothelial cells (HMEC-1) was studied qualitatively and quantitatively by high-resolution fluorescence microscopy and artificially mimicked by metabolic glycoprotein engineering. Two types of membrane proteins, sialic acid-bearing proteins (SABPs) and mucin-type proteins (MTPs), were investigated. For visualization they were metabolically labeled with azido sugars and then coupled to a cyclooctyne-conjugated fluorescent dye by click chemistry. Both spatial (diffusion) and temporal (residence time) dynamics of SABPs and MTPs on the membrane were investigated after treatment with exogenous galectin-1 or -3. Strong effects of galectin-mediated lattice formation were observed for MTPs (decreased spatial mobility), but not for SABPs. Lattice formation also strongly decreased the turnover of MTPs (increased residence time on the cell membrane). The effects of galectin-mediated crosslinking was accurately mimicked by streptavidin-mediated crosslinking of biotin-tagged glycoproteins and verified by single-molecule tracking. This technique allows the induction of crosslinking of membrane proteins under precisely controlled conditions, thereby influencing membrane residence time and the spatial dynamics of glycans on the cell membrane in a controlled way

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow

We investigated the properties of six Escherichia coli adhesion inhibitors under static and under flow conditions. On mannan-covered model substrates and under static conditions, all inhibitors were able to almost completely abolish lectin-mediated E. coli adhesion. On a monolayer of living human microvascular endothelial cells (HMEC-1), the inhibitors reduced adhesion under static conditions as well, but a large fraction of bacteria still managed to adhere even at highest inhibitor concentrations. In contrast, under flow conditions E. coli did not exhibit any adhesion to HMEC-1 not even at inhibitor concentrations where significant adhesion was detected under static conditions. This indicates that the presence of shear stress strongly affects inhibitor properties and must be taken into account when evaluating the potency of bacterial adhesion inhibitors. (C) 2016 Elsevier B.V. All rights reserved

Tuning Nanoparticle Uptake: Live-Cell Imaging Reveals Two Distinct Endocytosis Mechanisms Mediated by Natural and Artificial EGFR Targeting Ligand

Therapeutic nanoparticles can be directed to cancer cells by incorporating selective targeting ligands. Here, we investigate the epidermal growth factor receptor (EGFR)mediated endocytosis of gene carriers (polyplexes) either targeted with natural EGF or GE11, a short synthetic EGFR-binding peptide. Highly sensitive live-cell fluorescence microcopy with single particle resolution unraveled the existence of two different uptake mechanisms; EGF triggers accelerated nanoparticle endocytosis due to its dual active role in receptor binding and signaling activation, For GE11, an alternative EGFR signaling independent, actin-driven pathway is presented

The glycocalyx regulates the uptake of nanoparticles by human endothelial cells in vitro

Aim: To assess the role of the endothelial glycocalyx (eGCX) for the uptake of nanoparticles by endothelial cells. Methods: The expression of the eGCX on cultured human umbilical vein endothelial cells was determined by immunostaining of heparan sulfate. Enzymatic degradation of the eGCX was achieved by incubating the cells with eGCX-shedding enzymes. The uptake of 50-nm polystyrene nanospheres was quantified by confocal microscopy. Results: Human umbilical vein endothelial cells expressed a robust eGCX when cultured for 10 days. The uptake of both carboxylated and aminated polystyrene nanospheres was significantly increased in cells in which the glycocalyx was enzymatically degraded, while it remained at a low level in cells with an intact glycocalyx. Conclusion: The eGCX constitutes a barrier against the internalization of blood-borne nanoparticles by endothelial cells

Optoelektronik in Bakteriorhodopsin. Teilprojekt: Optische Untersuchungen Schlussbericht

Following the BMFT-project 0319231B, which showed potential applications of bacteriorhodopsin (BR) in the field of optical information processing, this project studies the use of BR and its genetically produced variants for applications in bio-technical hybrid systems. The light-induced charge separation, observed in electrophoretically deposited, oriented BR-films, can be used to switch liquid crystalls. Extensive investigations were carried out to determine orientation, surface quality, and photoelectric properties of the BR-films. With this experience it was possible to fabricate an OASLM prototype (OASLM = optically addressed spatial light modulator) on the basis of BR. However, technical applications are limited due to the rather slow response. Because of its large hyperpolarizability, BR is an interesting material for non-linear optical applications. Here, the #beta#-values of the (isolated) BR-chromophore retinal and its derivatives are of fundamental interest. In order to characterize these compounds, resonance- and fluorescence-free HRS-measurements (hyper-Rayleigh scattering) were carried out for the first time. The derived hyperpolarizabilities are one order of magnitude lower than those reported previously. The differences in hyperpolarizability of single photocycle intermediates are important for potential all-optical switching devices based on BR. To determine the ratio of the hyperpolarizabilities of the intermediates B_5_7_0 and M_4_1_0, HRS-measurements of BR suspensions were carried out as well as SHG measurements (second harmonic generation) of BR films. Both methods yielded a wavelength dependent hyperpolarizability ratio #beta#_B_5_7_0/#beta#_M_4_1_0>2. Thus non-linear optical applications based on the intermediates of the BR photocyclus seem possible. (orig.)Available from TIB Hannover: F98B1406+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Invasiveness of Cells Leads to Changes in Their Interaction Behavior with the Glycocalyx

Transendothelial migration is a crucial step during metastasis. Before circulating tumor cells enter the endothelium, they face the glycocalyx. While invasive migration of cancer cells is well studied, few investigations exist regarding their interaction with the glycocalyx. Here, the interaction of three breast cell lines with an endothelial glycocalyx is studied. Benign MCF-10A, noninvasive malign MCF-7, and invasive MDA-MB-231 cells penetrate the glycocalyx, just adhere to it or approach without even attaching to it. Remarkable fluctuations in these interaction modes are detected by time-resolved interaction profiles. Adhesion, migration, and invasion characteristics as well as combinations of interaction modes, cell shapes, and cell extensions are studied. The motility and penetration depth into the glycocalyx are analyzed. The invasive cells are the most flexible, penetrating the glycocalyx mostly with a round shape and feet-like membrane extensions. Noninvasive cancer cells penetrate the glycocalyx the deepest over time and benign cells integrate more likely into the endothelial cell layer underneath the glycocalyx

A Photoswitchable Trivalent Cluster Mannoside to Probe the Effects of Ligand Orientation in Bacterial Adhesion

We have recently demonstrated, by employing azobenzene glycosides, that bacterial adhesion to surfaces can be switched through reversible reorientation of the carbohydrate ligands. To investigate this phenomenon further, we have turned here to more complex-that is, multivalent-azobenzene glycoclusters. We report on the synthesis of a photosensitive trivalent cluster mannoside conjugated to an azobenzene hinge at the focal point. Molecular dynamics studies suggested that this cluster mannoside, despite the conformational flexibility of the azobenzene-glycocluster linkage, offers the potential for reversibly changing the glycocluster's orientation on a surface. Next, the photoswitchable glycocluster was attached to human cells, and adhesion assays with type 1 fimbriated Escherichia coli bacteria were performed. They showed marked differences in bacterial adhesion, dependent on the light-induced reorientation of the glycocluster moiety. These results further underline the importance of orientational effects in carbohydrate recognition and likewise the value of photoswitchable glycoconjugates for their study

Relative binding affinities of chlorophylls in peridinin-chlorophyll-protein reconstituted with heterochlorophyllous mixtures

Peridinin-chlorophyll-protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these "half-mers, homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.6 page(s