Armin Lambacher

Fluorescence interference-contrast microscopy on oxidized silicon using a monomolecular dye layer

A silicon chip is covered by a monomolecular film of a fluorescence dye with silicon dioxide used as a spacer. The fluorescence depends on the distance of the dye from the silicon. The modulation of the intensity is described quantitatively by an optical theory which accounts for interference of the exciting light and of the emitted light. The effect is used to obtain a microscopic picture of the surface profile with a precision of a few Angströms. The perspectives for an application in wet systems such as neuron-silicon junctions and lipid membranes on silicon are pointed out.

Integrin-Linked Kinase Controls Microtubule Dynamics Required for Plasma Membrane Targeting of Caveolae

Summary Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we identify a critical role for adhesion-mediated signaling through β1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking β1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its effector mDia1 to locally stabilize MTs and to allow stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.

Luminescence of dye molecules on oxidized silicon and fluorescence interference contrast microscopy of biomembranes

The luminescence of dye molecules depends on their position in a layered optical system. Conversely, the luminescence can be applied to measure the position of dye molecules above an interface. We formulate the electromagnetic theory of stationary fluorescence in a layered optical system—of light absorption, light detection, and fluorescence lifetime—computing the angular dependence of dipole interaction with all plane waves by a classical Sommerfeld approach. The theory is checked by experiments with stained lipid membranes on silicon with 256 terraces of silicon dioxide. We apply the electromagnetic theory to fluorescence micrographs of living cells on oxidized silicon chips and evaluate distances between the cell membrane and the substrate in a range of 1–150 nm.

Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn2+. Despite compromised integrin activation and adhesion, Mn2+ enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner. DOI: http://dx.doi.org/10.7554/eLife.10130.001

Spectra of voltage-sensitive fluorescence of styryl-dye in neuron membrane

The voltage sensitivity of fluorescence of an aminobenzstyryl-pyridinium dye (di4-ANEPPS) is characterized in Retzius cells dissociated from the leech. The modulation of the complete spectra of excitation and emission is determined. The spectral changes induced by depolarization are described by a blue shift of the absorption spectrum, by a weaker blue shift and an enhanced width of the fluorescence spectrum and by a decrease of the yield of fluorescence. These changes are attributed tentatively to a superposition of electrochromism and of field-induced resolvation.

Axonal Transmission in the Retina Introduces a Small Dispersion of Relative Timing in the Ganglion Cell Population Response

Background Visual stimuli elicit action potentials in tens of different retinal ganglion cells. Each ganglion cell type responds with a different latency to a given stimulus, thus transforming the high-dimensional input into a temporal neural code. The timing of the first spikes between different retinal projection neurons cells may further change along axonal transmission. The purpose of this study is to investigate if intraretinal conduction velocity leads to a synchronization or dispersion of the population signal leaving the eye. Methodology/Principal Findings We ‘imaged’ the initiation and transmission of light-evoked action potentials along individual axons in the rabbit retina at micron-scale resolution using a high-density multi-transistor array. We measured unimodal conduction velocity distributions (1.3±0.3 m/sec, mean ± SD) for axonal populations at all retinal eccentricities with the exception of the central part that contains myelinated axons. The velocity variance within each piece of retina is caused by ganglion cell types that show narrower and slightly different average velocity tuning. Ganglion cells of the same type respond with similar latency to spatially homogenous stimuli and conduct with similar velocity. For ganglion cells of different type intraretinal conduction velocity and response latency to flashed stimuli are negatively correlated, indicating that differences in first spike timing increase (up to 10 msec). Similarly, the analysis of pair-wise correlated activity in response to white-noise stimuli reveals that conduction velocity and response latency are negatively correlated. Conclusion/Significance Intraretinal conduction does not change the relative spike timing between ganglion cells of the same type but increases spike timing differences among ganglion cells of different type. The fastest retinal ganglion cells therefore act as indicators of new stimuli for postsynaptic neurons. The intraretinal dispersion of the population activity will not be compensated by variability in extraretinal conduction times, estimated from data in the literature.

A neural tissue interfacing chip for in-vitro applications with 32k recording / stimulation channels on an active area of 2.6 mm 2

A neural tissue interfacing chip for in-vitro applications is presented based on an extended 0.5 μm, 5 V, standard CMOS process. The chip provides 256 × 128 bidirectional recording / stimulation sites on a total active area of 2.28 mm × 1.14 mm, and a fully digital interface. Full frame recording rate is 2.4 kHz. Selection of a subset of sites is possible and translates into higher bandwidth per site accordingly. Electrical and biological results are presented which reveal proper functionality.

Nascent Adhesions: From Fluctuations to a Hierarchical Organization

Integrins assemble a complex network of molecular interactions at cell-matrix adhesion sites. Fluorescence correlation microscopy has now shed light on the spatial, temporal and numerical distributions of protein complexes during assembly and stabilization of nascent adhesions.