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 Mn(2+). Despite compromised integrin activation and adhesion, Mn(2+) 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.
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.
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► Formation of plasma membrane caveolae in keratinocytes requires β1 integrin and ILK ► β1 integrin/ILK recruit IQGAP1/mDia to nascent focal adhesions at the cell cortex ► IQGAP1/mDia stabilizes MTs and promotes caveolin trafficking to the plasma membrane ► Thus, the ILK/mDia/MT pathway drives caveolin availability for caveolae formation
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Total internal reflection fluorescence (TIRF) microscopy is a commonly used method for studying fluorescently labeled molecules in close proximity to a surface. Usually, the TIRF axial excitation ...profile is assumed to be single-exponential with a characteristic penetration depth, governed by the incident angle of the excitation laser beam towards the optical axis. However, in practice, the excitation profile does not only comprise the theoretically predicted single-exponential evanescent field, but also an additional non-evanescent contribution, supposedly caused by scattering within the optical path or optical aberrations. We developed a calibration slide to directly characterize the TIRF excitation field. Our slide features ten height steps ranging from 25 to 550 nanometers, fabricated from a polymer with a refractive index matching that of water. Fluorophores in aqueous solution above the polymer step layers sample the excitation profile at different heights. The obtained excitation profiles confirm the theoretically predicted exponential decay over increasing step heights as well as the presence of a non-evanescent contribution.
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.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A novel class of amphiphilic hemicyanine dyes is described where electron-pushing aniline and electron-pulling pyridinium are joined by anellated benzene rings. Enhancing the solvent polarity, the ...absorption band of these ANNINE dyes is shifted to the blue and the fluorescence band is shifted to the red at an invariant 00 energy. The increasing Stokes shift spans the whole visible spectrum. The divergent symmetrical solvatochromism of the positively charged chromophores is parametrized by a monopole−dipole model using a Born−Onsager−Marcus approach. From the intramolecular charge shift that is induced by electronic excitation, the linear Stark effect that is expected when the ANNINE dyes are used as voltage-sensitive probes in a biomembrane is estimated.
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IJS, KILJ, NUK, PNG, UL, UM
Amphiphilic hemicyanine dyes are fluorescent probes for voltage transients in nerve cells. Their sensitivity is assumed to be related with an intramolecular charge shift along the oriented ...chromophore that interacts with the electrical field across the cell membrane. Here we report on a measurement of the molecular orientation of the hemicyanine dye Di8ANEPPS in a lecithin membrane. We took advantage of the features of dipole radiation in front of a mirror. The fluorescence intensity of a stained membrane on oxidized silicon was measured as a function of the thickness of silicon dioxide up to 1000 nm and fitted with an electromagnetic theory accounting for the interference of the exciting light, for the interference of the emitted light and for the change of fluorescence lifetime. We found an angle of 37.8 ± 1.6° between the transition dipole moment and the membrane normal for an uniaxial cone model of the angular distribution function, with an order parameter 〈P 2〉 = 0.44 similar to the hydrocarbon chains of the lipid matrix.
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IJS, KILJ, NUK, PNG, UL, UM
The voltage sensitivity of fluorescence of an aminnobezystyryl-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.
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