Coiled coils als molekulare Kraftsensoren für die extrazelluläre Matrix

Kraft spielt eine fundamentale Rolle bei der Regulation von biologischen Prozessen. Zellen messen mechanische Eigenschaften der extrazellulären Matrix und benutzen diese Information zur Regulierung ihrer Funktion. Dazu werden im Zytoskelett Kräfte generiert und auf extrazelluläre Rezeptor-Ligand Wechselwirkungen übertragen. Obwohl der grundlegende Einfluss von mechanischen Signalen für das Zellschicksal eindeutig belegt ist, sind die auf molekularer Ebene wirkenden Kräfte kaum bekannt. Zur Messung dieser Kräfte wurden verschiedene molekulare Kraftsensoren entwickelt, die ein mechanisches Inputsignal aufnehmen und in einen optischen Output (Fluoreszenz) umwandeln. Diese Arbeit etabliert einen neuen Kraftsensor-Baustein, der die mechanischen Eigenschaften der extrazellulären Matrix nachbildet. Dieser Baustein basiert auf natürlichen Matrixproteinen, sogenannten coiled coils (CCs), die α-helikale Strukturen im Zytoskelett und der Matrix formen. Eine Serie an CC-Heterodimeren wurde konzipiert und mittels Einzelmolekül-Kraftspektroskopie und Molekulardynamik-Simulationen charakterisiert. Es wurde gezeigt, dass eine anliegende Scherkraft die Entfaltung der helikalen Struktur induziert. Die mechanische Stabilität (Separation der CC Helices) wird von der CC Länge und der Zuggeschwindigkeit bestimmt. Im Folgenden wurden 2 CCs unterschiedlicher Länge als Kraftsensoren verwendet, um die Adhäsionskräfte von Fibroblasten und Endothelzellen zu untersuchen. Diese Kraftsensoren deuten an, dass diese Zelltypen unterschiedlich starke Kräften generieren und mittels Integrin-Rezeptoren auf einen extrazellulären Liganden (RGD-Peptid) übertragen. Dieses neue CC-basierte Sensordesign ist ein leistungsstarkes Werkzeug zur Betrachtung zellulärer Kraftwahrnehmungsprozesse auf molekularer Ebene, das neue Erkenntnisse über die involvierten Mechanismen und Kräfte an der Zell-Matrix-Schnittstelle ermöglicht. Darüber hinaus wird dieses Sensordesign auch Anwendung bei der Entwicklung mechanisch kontrollierter Biomaterialien finden. Dazu können mechanisch charakterisierte, und mit einem Fluoreszenzreporter versehene, CCs in Hydrogele eingefügt werden. Dies erlaubt die Untersuchung der Zusammenhänge zwischen molekularer und makroskopischer Mechanik und eröffnet neue Möglichkeiten zur Diskriminierung von lokalen und globalen Faktoren, die die zelluläre Antwort auf mechanische Signale bestimmen. Force plays a fundamental role in the regulation of biological processes. Cells can sense the mechanical properties of the extracellular matrix (ECM) by applying forces and transmitting mechanical signals. They further use mechanical information for regulating a wide range of cellular functions, including adhesion, migration, proliferation, as well as differentiation and apoptosis. Even though it is well understood that mechanical signals play a crucial role in directing cell fate, surprisingly little is known about the range of forces that define cell-ECM interactions at the molecular level. Recently, synthetic molecular force sensor (MFS) designs have been established for measuring the molecular forces acting at the cell-ECM interface. MFSs detect the traction forces generated by cells and convert this mechanical input into an optical readout. They are composed of calibrated mechanoresponsive building blocks and are usually equipped with a fluorescence reporter system. Up to date, many different MFS designs have been introduced and successfully used for measuring forces involved in the adhesion of mammalian cells. These MFSs utilize different molecular building blocks, such as double-stranded deoxyribonucleic acid (dsDNA) molecules, DNA hairpins and synthetic polymers like polyethylene glycol (PEG). These currently available MFS designs lack ECM mimicking properties. In this work, I introduce a new MFS building block for cell biology applications, derived from the natural ECM. It combines mechanical tunability with the ability to mimic the native cellular microenvironment. Inspired by structural ECM proteins with load bearing function, this new MFS design utilizes coiled coil (CC)-forming peptides. CCs are involved in structural and mechanical tasks in the cellular microenvironment and many of the key protein components of the cytoskeleton and the ECM contain CC structures. The well-known folding motif of CC structures, an easy synthesis via solid phase methods and the many roles CCs play in biological processes have inspired studies to use CCs as tunable model systems for protein design and assembly. All these properties make CCs ideal candidates as building blocks for MFSs. In this work, a series of heterodimeric CCs were designed, characterized and further used as molecular building blocks for establishing a novel, next-generation MFS prototype. A mechanistic molecular understanding of their structural response to mechanical load is essential for revealing the sequence-structure-mechanics relationships of CCs. Here, synthetic heterodimeric CCs of different length were loaded in shear geometry and their mechanical response was investigated using a combination of atomic force microscope (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations. SMFS showed that the rupture forces of short heterodimeric CCs (3-5 heptads) lie in the range of 20-50 pN, depending on CC length, pulling geometry and the applied loading rate (dF/dt). Upon shearing, an initial rise in the force, followed by a force plateau and ultimately strand separation was observed in SMD simulations. A detailed structural analysis revealed that CC response to shear load depends on the loading rate and involves helix uncoiling, uncoiling-assisted sliding in the direction of the applied force and uncoiling-assisted dissociation perpendicular to the force axis. The application potential of these mechanically characterized CCs as building blocks for MFSs has been tested in 2D cell culture applications with the goal of determining the threshold force for cell adhesion. Fully calibrated, 4- to 5-heptad long, CC motifs (CC-A4B4 and CC-A5B5) were used for functionalizing glass surfaces with MFSs. 3T3 fibroblasts and endothelial cells carrying mutations in a signaling pathway linked to cell adhesion and mechanotransduction processes were used as model systems for time-dependent adhesion experiments. A5B5-MFS efficiently supported cell attachment to the functionalized surfaces for both cell types, while A4B4-MFS failed to maintain attachment of 3T3 fibroblasts after the first 2 hours of initial cell adhesion. This difference in cell adhesion behavior demonstrates that the magnitude of cell-ECM forces varies depending on the cell type and further supports the application potential of CCs as mechanoresponsive and tunable molecular building blocks for the development of next-generation protein-based MFSs.This novel CC-based MFS design is expected to provide a powerful new tool for observing cellular mechanosensing processes at the molecular level and to deliver new insights into the mechanisms and forces involved. This MFS design, utilizing mechanically tunable CC building blocks, will not only allow for measuring the molecular forces acting at the cell-ECM interface, but also yield a new platform for the development of mechanically controlled materials for a large number of biological and medical applications

Molecular Force Sensors: From Fundamental Concepts toward Applications in Cell Biology

Mechanical signals are central for the regulation of developmental, physiological, and pathological processes within biological systems. Force transduction across the cell–extracellular matrix (ECM) interface is highly crucial for regulating cell fate via mechanosensing and mechanotransduction cascades. The key molecules involved in these highly sophisticated processes have been identified in recent years. But little is still known about their interactions and in particular the molecular forces that determine these interactions. This is due to the limited availability of techniques that allow for investigating force propagation and mechanobiochemical signal conversion at the molecular level in live cells. In this progress report, currently available tools for measuring the molecular forces involved in cellular mechanosensing and mechanotransduction are summarized, specifically highlighting recent advances in the development of molecular force sensors (MFSs). MFSs convert the applied force into a fluorescence signal, allowing for a direct readout of tension with optical microscopy techniques. Moving from molecular design principles to applications of MFSs, important results are summarized, highlighting the new mechanistic information that has been obtained about mechanobiochemical processes at the cell–ECM interface. This progress report finishes with a critical discussion of current promises and limitations, providing perspectives for future research in this quickly evolving field

Sub-bandgap absorption spectroscopy and minority carrier transport properties of hydrogenated microcrystalline silicon thin films

Hydrogenated microcrystalline silicon thin films have been prepared using HW-CVD and VHF-PECVD techniques with different silane concentrations. The steady-state photoconductivity, dual beam photoconductivity, photothermal deflection spectroscopy and steady-state photocarrier grating (SSPG) methods have been used to investigate the optical and electronic properties of the films. Two different sub-bandgap absorption methods have been applied and analyzed to obtain a better insight into the electronic states involved. For some films, differences existed in the optical absorption spectra when the measurements were carried out through the film side and through the substrate side. In addition, for some films, fringe patterns remained on the spectrum after the calculation of the fringe free absorption spectrum, which indicates that structural inhomogeneities were present throughout the film. Finally, minority carrier diffusion lengths deduced from the SSPG measurements were investigated as a function of the crystalline volume fraction (I-c(RS)) obtained from Raman spectroscopy. The longest diffusion lengths and lowest sub-bandgap absorption coefficients were obtained for films deposited in the region of the transition to the amorphous growth

Atmospheric methane sulfonate and non-sea salt sulfate records at the EPICA deep-drilling site in Dronning Maud Land, Antarctica

Abstract. During three summer campaigns in January/February 2000, 2001 and 2002 the ionic compo-sition of the aerosol at the EPICA deep-drilling site at Kohnen Station was measured in daily resolution. In 2000 and 2002 we observed mean (±std) non-sea salt sulfate (nss-SO42-) con-centrations of 353±100 ng m-3 and 320±250 ng m-3, as well as methane sulfonate (MS) con-centrations of 59±36 ng m-3 and 74±80 ng m-3, respectively. For the summer campaign in 2001, significantly lower nss-SO42- and MS levels of 164±150 ng m-3 and 19±12 ng m-3, respectively, were typical. The mean MS/nss-SO42- ratio ranged from about 0.1 to 0.2. MS and nss-SO42- concentrations and their variability were roughly comparable to coastal stations at summer. Supported by air mass back trajectory analyses this finding documented an effi-cient long-range transport to Kohnen via the free troposphere. MS/nss-SO42- ratios exhibited a strong dependence on the MS concentration with systematically higher ratios at higher MS concentrations, a peculiarity which is also evident in a firn core drilled at this site

Electronic transport properties of microcrystalline silicon thin films prepared by VHF-PECVD

Steady-state photocarrier grating (SSPG) and steady-state photoconductivity, sigma(ph), experiments have been carried out to investigate the electronic transport properties of undoped hydrogenated microcrystalline silicon (muc-Si: H) films prepared with very high frequency plasma enhanced chemical vapor deposition (VHF-PECVD). Material with different crystalline volume fractions was obtained by variation of the silane concentration (SC) in the process gas mixture. Pure amorphous silicon material was investigated for comparison. The ambipolar diffusion length, L-amb, which is dominated by the minority carrier properties, is obtained both from the best fit to the experimental photocurrents ratio, beta, versus grating period (Lambda), and from the "Balberg plot" for the generation rates between 10(19) and 10(21) cm(-3) s(-1). L-amb increases from 86 nm with increasing SC and peaks around 200 nm for the SC = 5.6% and decreases again for higher SCs. L-amb values obtained from the intercept of the Balberg plot result in a small difference of around 5% for most of the samples. Minority carrier mobility-lifetime (mutau)-products are much lower than those of majority carriers, however, both majority and minority carrier c-products in microcrystalline silicon are higher than those of undoped hydrogenated amorphous silicon. The grating quality factor (gamma(o)) changes from 0.70 to 1.0 indicating almost negligible surface roughness present in the samples. (C) 2004 Kluwer Academic Publishers

Photoconductivity spectroscopy in hydrogenated microcrystalline silicon thin films

Steady-state photoconductivity and sub-bandgap absorption measurements by the dual-beam photoconductivity (DBP) method were carried out on undoped hydrogenated microcrystalline silicon thin films prepared by VHF-PECVD and hot-wire chemical vapor deposition. The results are compared with those of the constant-photocurrent method (CPM) and photothermal deflection spectroscopy (PDS). It is found that DBP, CPM, and PDS provide complementary data on the optoelectronic processes in microcrystalline silicon. (C) 2003 Kluwer Academic Publishers