Au cours des dernières années, des efforts importants ont été déployés pour développer des métasurfaces (MSs) électromagnétiques avec la possibilité de changer de manière abrupte les propriétés de la lumière. Ces avancées ont ouvert une nouvelle gamme de possibilités pour contrôler la lumière en utilisant des dispositifs optiques ultra-minces. Dans ce contexte, et plus spécifiquement dans le spectre visible, les applications en bio-imagerie s’avèrent particulièrement intéressantes. Une technique qui est particulièrement bien adaptée à l'étude de molécules proches d'une membrane cellulaire est la microscopie à fluorescence par réflexion interne (TIRFM), qui repose sur un champ évanescent d'excitation. Dans ce cas la lumière incidente est totalement réfléchie sur une interphase (typiquement verre/eau) en raison de son angle d'incidence élevé. À ce jour, la TIRFM est généralement mise en œuvre à l'aide d'objectifs volumineux de grande ouverture numérique et de petit champ de vision.Dans ce travail de thèse, nous réalisons de substrats pour la microscopie TIRF à base de métasurfaces constituées de réseaux périodiques de structures asymétriques fabriquées en dioxyde de titane (TiO2) sur du verre borosilicaté. Ces structures, aussi petites que 48 nm, ont été optimisées à l’aide de simulations numériques "Rigorous coupled-wave analysis” (RCWA) dans le but de coupler de 50 à 90% de la lumière incidente dans le premier ordre de diffraction avec des angles élevés (θ > 63deg). Le fait de pouvoir utiliser des objectifs de faible grossissement et d'avoir une grande zone de champ évanescent fournit des conditions TIRF uniques qui ne sont pas accessibles par les méthodes traditionnelles. De plus, ces structures sont compatibles avec la lithographie par nanoimpression UV, ce qui permet d’envisager une fabrication à bas coût et à grande échelle. Outre la conception, et la fabrication, dans cette thèse nous aboutissons à une preuve de principe de la microscopie TIRF basée sur des métasurfaces en milieu biologique en imageant notamment des membranes fluorescentes de cellules souches. Ces métasurfaces permettent ainsi l’implémentation TIRFM à contraste élevé et à faible photo-blanchissement compatible avec des microscopes à champ large peu coûteux. / In recent years there has been a significant effort to push electromagnetic metasurfaces with the ability to abruptly change light properties into visible wavelengths. These advancements have opened a new range of possibilities to reshape light using ultra-thin optical devices and there is one field that is starting to gather attention: bioimaging. One technique particularly well suited for the study of molecules near a cell membrane is Total Internal Reflection Fluorescence (TIRF) microscopy, which relies on an evanescence field created by light being totally internally reflected within a glass substrate due to its high incidence angle. As of today, TIRF is generally implemented using bulky high-NA, small field of view oil objectives.In this project we present the realization of metasurface-based TIRF microscopy substrates consisting of periodic 2D arrays of asymmetric structures fabricated in titanium dioxide on borosilicate glass. These patterns, as small as 48nm, were optimized through rigorous coupled-wave analysis to couple 50-90% of the incoming normally incident light into the first diffraction order, which outputs at an angle that suffices total internal reflection in water and eliminates the requirement for high NA objectives or prisms to achieve TIRF. Being able to utilize lower-magnification air objectives and having a large evanescence field area provide unique TIRF conditions not accessible by traditional methods. Additionally, these structures are compatible with soft UV nanoimprint lithography, for cost-effective scale production, to give TIRF’s high contrast, low photodamage and low photobleaching capabilities to inexpensive wide-field microscopes.

Recent progress in the field of semiconductor nanocrystals or Quantum Dots (QDs) has seen them find wider acceptance as a tool in biomedical research labs. As produced, high quality QDs synthesized by high temperature organometallic synthesis, are coated with a hydrophobic ligand. Therefore, they must be further processed to be soluble in water and made biocompatible. A process to coat the QDs with silk fibroin, a fibrous protein derived from the Bombyx mori silk worm, is described. Following the coating process, the characterization of size, optical properties and biocompatibility profile of these particle systems is described. In addition, conjugation of the silk fibroin coated QDs to different labeling proteins such as phalloidin and streptavidin is described. Proteins on the surface of ovarian cancer cells (HeyA8) and of cytoskeletal components participating in the formation of focal adhesion complex (FAC), such as F-actin in endothelial cells (HUVECS) were labeled using the bio-conjugated QDs. Various imaging techniques such as epi-fluorescence, TIRF and AFM were used to study the QD labeled cells. Overall the project has produced luminescent nanoprobes that enable the study of FAC formation dynamics and potentially a better in vivo fluorescent marker tool.

In cell biology and chemistry, total internal reflection microscopy (TIRFM) has proven to be a useful technique that allows the probing of cellular processes with high-signal-to-noise ratio imaging. However, samples on solid substrates limit the accessibility to probe processes on extracellular membrane surface closest to the microscope objective. Colloidal crystals provide a porous alternative to the traditional solid substrates. Thin crystals exhibit optical properties similar to that of a fused silica coverslip allowing for TIRFM in the same manner as with a typical coverslip as demonstrated by the observance of Chinese hamster ovary cells with fluorescently labeled receptors on both types of substrates. Accessibility of the cell membrane closest to the substrate and the ability to probe fluorophore orientation information was observed by the binding of TIPP-cy5 to the human delta opioid receptor.

No description available.

Actin-based cell motility plays crucial roles throughout the lifetime of an organism. The dynamic rearrangement of the actin cytoskeleton triggers a plethora of cellular processes including cellular migration. Neural Wiskott Aldrich syndrome protein (N-WASP) is involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton. N-WASP activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3 complex, N-WASP binds actin filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures. We used nanofibers coated with N-WASP WWCA domains as model cell surfaces and single actin filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed end assembly. Individual barbed ends captured by WWCA domains of N-WASP grew at or below their diffusion limited assembly rate. At high filament densities, overlapping filaments formed buckles between their nanofiber tethers and myosin attachment points. These buckles grew 3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native poly-proline (PP) region showed similar rate enhancements. Increasing polycationic Mg2+ or Spermine to enhance filament bundling increased the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed end bundling. Our preliminary data shows that tethered N-WASP construct containing one WH2 domain does not generate processive bundles or filament loops leading us to believe that tandem WH2 is required for processivity. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion. Discovery of this bundle mediated unique pathway involved in invasion and metastasis will provide new targets for therapeutic development. / Ph. D.

Moderne Methoden für die Einzelzellanalyse werden dank der fortschreitenden Weiterentwicklung immer sensitiver. Dabei steigen jedoch auch die Anforderungen an das Probenmaterial. Viele Aufbereitungsprotokolle adhärenter Zellen beinhalten eine enzymatische Spaltung der Oberflächenproteine, um die Ablösung vom Zellkultursubstrat zu ermöglichen. Verschiedene Methoden, wie die Patch-Clamp-Technik oder eine auf der Markierung extrazellulärer Domänen von Membranproteinen basierende Durchflusszytometrie können dann nur noch eingeschränkt eingesetzt werden. Daher ist die Etablierung neuer Zellablösemethoden dringend notwendig. In der vorliegenden Arbeit werden erstmals PEG-basierte thermo-responsive Oberflächen erfolgreich für die Zellkultur eingesetzt. Dabei wird das zerstörungsfreie Ablösen verschiedener Zelllinien von den Oberflächen durch Temperatursenkung realisiert. Die Funktionalität der Oberflächen wird durch Variation der Polymerstruktur, sowie der Konzentration der Beschichtungslösung, durch Beschichtung der Oberflächen mit einem zelladhäsionsfördernden Protein (Fibronektin) und durch Adsorption zelladhäsionsvermittelnder Peptide (RGD) optimiert. Um den Zellablösungsprozess detaillierter zu untersuchen, wird hier zum ersten Mal der direkte Zellkontakt mit thermo-responsiven Oberflächen mittels oberflächensensitiver Mikroskopie (TIRAF) sichtbar gemacht. Mit dieser Technik sind die exakte Quantifizierung und die Analyse der Reduktion der Zelladhäsionsfläche während des Abkühlens möglich. Hierbei werden in Abhängigkeit von der Zelllinie Unterschiede im Zellverhalten während des Ablösens festgestellt: Zellen, wie eine Brustkrebszelllinie und eine Ovarzelllinie, die bekanntermaßen stärker mit ihrer Umgebung in Kontakt treten, vergrößern im Verlauf des Beobachtungszeitraumes den Abstand zwischen Zellmembran und Oberfläche, reduzieren jedoch ihre Zell-Substratkontaktfläche kaum. Mausfibroblasten hingegen verkleinern drastisch die Zelladhäsionsfläche. Der Ablösungsprozess wird vermutlich aktiv von den Zellen gesteuert. Diese Annahme wird durch zwei Beobachtungen gestützt: Erstens verläuft die Reduktion der Zelladhäsionsfläche bei Einschränkung des Zellmetabolismus durch eine Temperatursenkung auf 4 °C verzögert. Zweitens hinterlassen die Zellen Spuren, die nach dem Ablösen der Zellen auf den Oberflächen zurückbleiben. Mittels Kombination von TIRAF- und TIRF-Mikroskopie werden die Zelladhäsionsfläche und die Aktinstruktur gleichzeitig beobachtet. Die Verknüpfung beider Methoden stellt eine neue Möglichkeit dar, intrazelluläre Prozesse mit der Zellablösung von thermo-responsiven Oberflächen zu korrelieren. / Modern methods for single-cell analysis are becoming increasingly sensitive. At the same time, requirements for the sample material are on the rise. Today, sample preparation of adherent cells usually includes steps of enzymatic treatment to digest surface proteins thus, inducing cell detachment from culture substrates. This strongly limits the application of different techniques like patch clamp or labelling of extracellular domains of membrane proteins for flow cytometry. Therefore, a new cell detachment method is urgently required. In the present work, new PEG-based thermo-responsive polymers are used for cell culture for the first time. Here, non-destructive detachment of different cell lines from polymer-coated surfaces is realised by controlled temperature reduction. The surface functionality is systematically optimised by varying the concentration of the coating solutions, by artificial surface coating of a cell adhesion-mediating protein (fibronectin) and by co-adsorption of a cell adhesion-mediating peptide (RGD). For detailed analysis of the cell detachment process, TIRF microscopy is used to directly visualise the cell contacts on the thermo-responsive surfaces. Using this technique allows both the quantification and analysis of the reduction of the cell adhesion area during sample cooling. Furthermore, for several cell lines, different behaviours in cell detachment are observed. Cells that have close contact to their substrate like MCF-7 breast cancer cell line and CHO-K1 ovary cells increase the distance between cell membrane and surface, but there is only little decrease of cell-substrate adhesion area. In contrast, L929 fibroblasts reduce the cell adhesion area drastically. Furthermore, the hypothesis that the cell detachment is an active process is shown by lowering the cell metabolism by temperature reduction to 4 °C and by the cell traces that are left behind after rinsing the surfaces. A combination of TIRAF and TIRF enables visualising the cell adhesion area and actin structures. Measuring both parameters simultaneously opens up new possibilities to correlate intracellular and cell detachment processes on thermo-responsive surfaces.

Die G-Protein vermittelte Aktivierung der Phospholipase Cβ (PLCβ) stellt einen primären Mechanismus dar, um eine Vielzahl von physiologischen Ereignissen zu regulieren, z.B. die Kontraktion glatter Muskelzellen, Sekretion oder die Modulation der synaptischen Transmission. Sowohl Gαq- als auch Gβγ-Untereinheiten sind dafür bekannt mit PLCβ Enzymen zu interagieren und diese zu aktivieren. Über die Dynamik dieser Interaktion und den relative Beitrag der G-Protein Untereinheiten ist jedoch nur wenig bekannt. Unter Verwendung Fluoreszenz Resonanz Energie Transfer (FRET)- basierter Methoden in lebenden Zellen, wurde die Kinetik der Rezeptor-induzierten Interaktion zwischen Gβγ und Gαq Untereinheiten, die Interaktion von sowohl der Gαq als auch der Gβγ-Untereinheit mit der PLCβ3 und die Interaktion des regulator of G-Protein signaling 2 (RGS2) mit Gαq-Untereinheiten untersucht. Um die Untersuchung der Protein-Protein-Interaktion auf die Zellmembran zu beschränken, wurde die Total-Internal Reflection Fluorescence (TIRF) Mikroskopie angewandt. Zeitlich hoch auflösendes, ratiometrisches FRET-Imaging offenbarte eine deutlich schnellere Dissoziation von Gαq und PLCβ3 nach Entzug purinerger Agonisten verglichen mit der Deaktivierung von Gq Proteinen in der Abwesenheit der PLCβ3. Dieser offensichtliche Unterschied in der Kinetik kann durch die GTPase-aktivierende Eigenschaft der PLCβ3 in lebenden Zellen erklärt werden. Weiterhin zeigte es sich, dass PLCβ3 die Gq Protein Kinetik in einem ähnlich Ausmaß beeinflusst wie RGS2, welches in vitro deutlich effizienter darin ist, die intrinsische GTPase Aktivität der Gαq-Untereinheit zu beschleunigen. Als Antwort auf die Rezeptorstimulation wurde sowohl eine Interaktion von Gαq-Untereinheiten als auch von Gq-abstammende Gβγ-Untereinheiten mit der PLCβ3 beobachtet. Darüber hinaus zeigte sich auch eine Agonist-abhängige Interaktion von Gαq und RGS2. In Abwesenheit einer Rezeptorstimulation konnte kein spezifisches FRET-Signal zwischen Gq Proteinen und der PLCβ3 oder RGS2 detektiert werden. Zusammengefasst ermöglichte das ratiometrische FRET-Imaging in der TIRF Mikroskopie neue Einsichten in die Dynamik und Interaktionsmuster des Gq-Signalwegs. / G protein-mediated activation of phospholipase Cβ (PLCβ) represents a primary mechanism to regulate many physiological events such induce smooth muscle contraction, secretion and modulation of synaptic transmission. Both Gαq- and Gβγ-subunits are known to interact and activate PLCβ enzymes, however little is known about the dynamics of this interactions and the relative contribution of the G protein subunits in intact cells. Using fluorescence resonance energy transfer- (FRET-) based assays in single intact cells we studies kinetics of receptor-induced interactions between Gβγ- and Gαq-subunits, interactions of both Gαq and Gβγ with PLCβ3 as well as interactions of regulator of G proteins signalling 2 (RGS2) with Gαq- and Gβγ-subunits. In order to restrict the protein/protein interaction studies to the cell membrane we applied total internal reflection (TIRF) microscopy. High temporal resolution ratiometric FRET imaging uncovered a markedly faster dissociation of Gαq and PLC upon withdrawal of purinergic agonists compared to the deactivation of Gq proteins in the absence of PLCβ3. This apparent difference in kinetics could be contributed to the GTPase-activating property of PLCβ3 in living cells. Furthermore we found that PLCβ3 modulated Gq protein kinetics to a similar extent compared to RGS2, which in vitro is about 100 fold more efficient in activating Gq-GTPase activity. We observed that both Gαq subunits and Gq-derived Gβγ-subunits interact with PLCβ3 in response to receptor stimulation. In the absence of receptor stimulation we did neither detect any specific FRET signals between Gq protein subunits and PLCβ3 nor did we detect any interactions between RGS2 and Gαq subunits. Finally we could not detect agonist- dependent FRET between RGS2 and Gβγ-subunits. Taken together, ratiometric FRET-imaging under conditions of TIRF allowed new insights into dynamics and interaction patterns within the Gq signalling pathway.

Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.

Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.

Caveolae are a type of invaginated membrane domain that has been shown to be involved in several disease states, including lipodystrophy, muscular dystrophies and cancer. Several of these diseases are caused by the lack of caveolae or caveolae-related signaling deficiencies in the tissues in which the caveolar domain are abundant such as lung, adipose, muscle and their related endothelial cells. Caveolae are formed through the assembly of the membrane inserted protein caveolin, cholesterol and the recently described family of cavin proteins, which together form the caveolae coat. The work in this thesis focuses on understanding the protein components and mechanisms that control the biogenesis and dynamics of caveolae. We have found that the protein EHD2 is an important regulator and stabilizer of the caveolar domain at the cell membrane. EHD2 is a dimeric ATPase known to oligomerize into ring-like structures around lipid membranes to control their shape. We have characterized the domain interactions involved in the specific targeting and assembly of this protein at caveolae. We propose a stringent regulatory mechanism for the assembly of EHD2 involving ATP binding and switching of the EH domain position to release the N-terminus and facilitate oligomerization in the presence of membrane species. We show that loss of EHD2 in cells results in hyper- dynamic caveolae and that caveolae stability at the membrane can be restored by reintroducing EHD2 into these cells. In a study of the protein cavin-3, which is known to be an integral component of the caveolar coat, we showed that this protein is targeted to caveolae via direct binding to the caveolar core protein caveolin1. Furthermore, we show that cavin-3 is enriched at deeply invaginated caveolae and regulate the duration time of caveolae at the cell surface. In combination with a biochemical and cellbiological approach, the advanced fluorescence microscopy techniques, like Fluorescence Recovery After Photobleaching (FRAP), Total Internal Reflection microscopy (TIRF), combined with correlative Atomic Force Microscopy (AFM) have allowed us to characterize distinct caveolae-associated proteins and their respective functions at caveolae.