Correlating structure and dynamics of lamellipodia and determination of the F-and G-actin concentrations in lamellipodia

TITELBLATT: nur in PRINTAUSGABE! -- Eukaryotische Zellen bewegen sich in Phasen des Schiebens und Ziehens einer flachen Struktur an der Zell-Vorderseite, dem Lamellipodium. Die Vorwärtsbewegung entsteht durch die Polymerisation von Aktin-Monomeren (G-Aktin) in Aktin-Filamente (F-Aktin) am vorderen Rand des Lamellipodiums, wodurch die Membran nach vorne geschoben wird. Aktin-Filamente werden in Richtung zum hinteren Ende des Lamellipodiums abgebaut wodurch ein Vorrat an Aktin-Monomeren aufrecht erhalten wird. Im Laufe meiner Doktorarbeit habe ich mittels korrelativer Licht- und Negativ-Färbungs-Elektronenmikroskopie anhand von B16 Maus Melanomzellen, die mit GFP-Aktin und anderen Konstrukten, die Zellbewegungsproteine kodieren, den Zusammenhang zwischen den unterschiedlichen Aktivitäten des Lamellipodiums und seiner Ultrastruktur untersucht. Ich konnte zeigen, dass sich die Aktinfilamente während des Übergangs vom Schieben zum Ziehen neu anordnen um zusammen mit Myosin hinter dem Lamellipodium kontraktile Einheiten aus antiparallen Filamenten zu bilden. Die Ergebnisse stellen derzeitige Modelle für die Zellbewegung mittels Aktin infrage. Außerdem haben wir eine Methode entwickelt um die F- und G-Aktin-Konzentrationen im Lamellipodium von sich bewegenden Zellen zu bestimmen. Die Strategie war zunächst das Verhältnis von F- zu G-Aktin zu ermitteln. Dazu haben wir die Möglichkeiten von FRAP (Fluorescence Recovery After Photobleaching - das Wiedererlangen der Fluoreszenz nach Bleichen mit starkem Licht.) genutzt um die F- und G-Aktin-Komponenten der gesamten Fluoreszenzintensität räumlich zu trennen um dann die G-actin-Komponente durch Zell-Lyse zu erhalten. Die F-actin Konzentration wurde von Elektronenmikroskopiebildern ermittelt. Die Konzentrationsparameter können in mathematische Modelle inkorporiert werden und haben Auswirkungen auf die Art, wie Zellbewegung reguliert wird. Zusammenfassend haben die Studien neue Erkenntnisse über die Dynamik der Aktin-Filamente während der Zellbewegung geliefert.Eukaryotic cells move in phases of extension and retraction of a leaf-like structure, the lamellipodium, at the cell front. Protrusion occurs by the polymerization of monomeric (G) into polymeric (F) actin filaments at the tip of the lamellipodium, thereby pushing the membrane forward. Actin filaments are depolymerized towards the rear of the lamellipodium in a treadmilling process, thereby supplementing a G-actin pool for a new round of polymerization. During my thesis I used correlative light cell imaging and negative stain electron microscopy on B16 mouse melanoma cells transfected with GFP-actin and other constructs encoding cell motility proteins to determine the correlation between the protrusive activity and the ultra-structure of the lamellipodium. I show that during a shift from protrusion to retraction lamellipodial filaments rearrange to generate, together with myosin, contractile arrays of antiparallel filaments behind the lamellipodium. The results challenge current models of how actin filaments are used to drive protrusion. Moreover, I developed a technique to determine the F- and G-actin concentrations in lamellipodia of living cells. The strategy was to first determine the F- to G-actin ratio by exploiting the light microscopy technique FRAP (fluorescence recovery after photobleaching) to spatially segregate the F- and G-actin components of the total fluorescence intensity and specifically extract the G-actin component by cell lysis. The F-actin concentration was determined from electron micrographs. The concentration parameters can be incorporated into mathematical models and have implications on the way protrusion is regulated. In conclusion these studies have provided new information about the dynamics of actin filament turn-over during cell migration

Highly accelerated cardiac functional MRI in rodent hearts using compressed sensing and parallel imaging at 9.4T

Summary. Parallel Imaging and Compressed Sensing have individually been shown to speed up cardiac functional MRI in mice and rats at ultra-high magnetic fields whilst providing accurate measurement of the physiologically relevant parameters. This study demonstrates that the acquisition time for cine-MRI in rodent hearts can be significantly reduced further by combining both techniques

Patterned Surface Activation of Cyclo-Olefin Polymers for Biochip Applications

Two different surface activation methods (UV/ozone and oxygen plasma treatment) were applied for patterned surface activation of cyclo-olefin polymer (COP) surfaces combined with different masking techniques (metal shadow mask and protective tape). Surface properties were characterized by various methods such as contact angle measurement, ATR-IR, XPS and Surface enhanced ellipsometric contrast (SEEC) microscopy. UV/ozone and oxygen plasma allowed for patterned surface modification of COP leading to the formation of carboxylic and hydroxyl groups on the activated part of the surface. Stability against organic solvents was determined by rinsing the activated substrates with 2-propanol. For UV/ozone treatment it was found that a thin film of degradation products remains on the COP surface and is at least partly removed in the following washing or rinsing steps

Accelerating cine-MR Imaging in Mouse Hearts Using Compressed Sensing

PURPOSE: To combine global cardiac function imaging with compressed sensing (CS) in order to reduce scan time and to validate this technique in normal mouse hearts and in a murine model of chronic myocardial infarction. MATERIALS AND METHODS: To determine the maximally achievable acceleration factor, fully acquired cine data, obtained in sham and chronically infarcted (MI) mouse hearts were 2-4-fold undersampled retrospectively, followed by CS reconstruction and blinded image segmentation. Subsequently, dedicated CS sampling schemes were implemented at a preclinical 9.4 T magnetic resonance imaging (MRI) system, and 2- and 3-fold undersampled cine data were acquired in normal mouse hearts with high temporal and spatial resolution. RESULTS: The retrospective analysis demonstrated that an undersampling factor of three is feasible without impairing accuracy of cardiac functional parameters. Dedicated CS sampling schemes applied prospectively to normal mouse hearts yielded comparable left-ventricular functional parameters, and intra- and interobserver variability between fully and 3-fold undersampled data. CONCLUSION: This study introduces and validates an alternative means to speed up experimental cine-MRI without the need for expensive hardware

Synthesis and characterization of naphthalimide-functionalized polynorbornenes

ABSTRACT: Highly fluorescent and photostable (2-alkyl)-1H-benzo[de]isoquinoline-1,3(2H)-diones with a polymerizable norbornene scaffold have been synthesized and polymerized using ring-opening metathesis polymerization. The monomers presented herein could be polymerized in a living fashion, using different comonomers and different monomer ratios. All obtained materials showed good film-forming properties and bright fluorescence caused by the incorporated push–pull chromophores. Additionally, one of the monomers containing a methylpiperazine functionality showed protonation-dependent photoinduced electron transfer which opens up interesting applications for logic gates and sensing. GRAPHICAL ABSTRACT: [Image: see text

Embedded inertial sensor for tracking projectile impact on granular media

Due to the opacity of most granular materials, it is often desirable to have three dimensional (3D) particle tracking techniques beyond optical imaging to explore granular dynamics. Using inertial measurement units (IMU) embedded in a projectile, we obtain the trajectory of projectile impacting on a granular medium under microgravity using tri-axial acceleration and angular velocity data. In addition to the standard algorithm for reconstruction, we emphasize solutions to various sources of error to determine projectile trajectory accurately

Embedded inertial sensor for tracking projectile impact on granular media

Due to the opacity of most granular materials, it is often desirable to have three dimensional (3D) particle tracking techniques beyond optical imaging to explore granular dynamics. Using inertial measurement units (IMU) embedded in a projectile, we obtain the trajectory of projectile impacting on a granular medium under microgravity using tri-axial acceleration and angular velocity data. In addition to the standard algorithm for reconstruction, we emphasize solutions to various sources of error to determine projectile trajectory accurately

Electrokinetic investigation of polyelectrolyte adsorption and multilayer formation on a polymer surface

Self assembled polyelectrolyte layers of poly(sodium 4-styrenesulfonate) (PSS), and poly(diallyldimethylammonium chloride) (PDADMAC) were deposited on planar poly(ethylene terephthalate) (PET) substrates using the layer-by-layer technique. Charged functional groups were generated on the polymer substrates by means of a surface modification procedure prior to polyelectrolyte adsorption. The layers were characterised concerning their electrokinetic properties. The build-up of multilayer architectures could be followed by changes of the zeta-potential versus pH curves. An increase of coating density with increasing layer number was found. The electrokinetic properties of the PET substrates were not recognised anymore if more then four layers were applied. If PSS formed the outermost layer these assemblies were very stable against shear forces while if PDADMAC formed the outermost layer the films were partially destroyed by high shear forces