Automated quantitative analysis of single and double label autoradiographs

A method for the analysis of silver grain content in both single and double label autoradiographs is presented. The total grain area is calculated by counting the number of pixels at which the recorded light intensity in transmission dark field illumination exceeds a selected threshold. The calibration tests included autoradiographs with low (3H- thymidin) and high (3H-desoxyuridin) silver grain density. The results are proportional to the customary visual grain count. For the range of visibly countable grain densities in single labeled specimens, the correlation coefficient between the computed values and the visual grain counts is better than 0.96. In the first emulsion of the two emulsion layer autoradiographs of double labeled specimens (3H-14C- thymidin) the correlation coefficient is 0.919 and 0.906. The method provides a statistical correction for the background grains not due to the isotope. The possibility to record 14C tracks by shifting the focus through the second emulsion of the double labeled specimens is also demonstrated. The reported technique is essentially independent of size, shape and density of the grains

Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and Recycling.

SummaryClathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. Here, we show that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact μ-homology domain, and functional AP-2 heterotetramers. Our data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling

Probe-Configuration-Dependent Decoherence in an Aharonov-Bohm Ring

We have measured transport through mesoscopic Aharonov-Bohm (AB) rings with two different four-terminal configurations. While the amplitude and the phase of the AB oscillations are well explained within the framework of the Landaur-B\"uttiker formalism, it is found that the probe configuration strongly affects the coherence time of the electrons, i.e., the decoherence is much reduced in the configuration of so-called nonlocal resistance. This result should provide an important clue in clarifying the mechanism of quantum decoherence in solids.Comment: 4 pages, 4 figures, RevTe

Interplay between pulsations and mass loss in the blue supergiant 55 Cygnus = HD 198478

Blue supergiant stars are known to display photometric and spectroscopic variability that is suggested to be linked to stellar pulsations. Pulsational activity in massive stars strongly depends on the star's evolutionary stage and is assumed to be connected with mass-loss episodes, the appearance of macroturbulent line broadening, and the formation of clumps in the wind. To investigate a possible interplay between pulsations and mass-loss, we carried out an observational campaign of the supergiant 55 Cyg over a period of five years to search for photospheric activity and cyclic mass-loss variability in the stellar wind. We modeled the H, He I, Si II and Si III lines using the nonlocal thermal equilibrium atmosphere code FASTWIND and derived the photospheric and wind parameters. In addition, we searched for variability in the intensity and radial velocity of photospheric lines and performed a moment analysis of the line profiles to derive frequencies and amplitudes of the variations. The Halpha line varies with time in both intensity and shape, displaying various types of profiles: P Cygni, pure emission, almost complete absence, and double or multiple peaked. The star undergoes episodes of variable mass-loss rates that change by a factor of 1.7-2 on different timescales. We also observe changes in the ionization rate of Si II and determine a multiperiodic oscillation in the He I absorption lines, with periods ranging from a few hours to 22.5 days. We interpret the photospheric line variations in terms of oscillations in p-, g-, and strange modes. We suggest that these pulsations can lead to phases of enhanced mass loss. Furthermore, they can mislead the determination of the stellar rotation. We classify the star as a post-red supergiant, belonging to the group of alpha Cyg variables.Comment: 20 pages, 18 figures, 3 tables, accepted to Astronomy & Astrophysic

Wind properties of variable B supergiants. Evidence of pulsations connected with mass-loss episodes

Context. Variable B supergiants (BSGs) constitute a heterogeneous group of stars with complex photometric and spectroscopic behaviours. They exhibit mass-loss variations and experience different types of oscillation modes, and there is growing evidence that variable stellar winds and photospheric pulsations are closely related. Aims: To discuss the wind properties and variability of evolved B-type stars, we derive new stellar and wind parameters for a sample of 19 Galactic BSGs by fitting theoretical line profiles of H, He, and Si to the observed ones and compare them with previous determinations. Methods: The synthetic line profiles are computed with the non-local thermodynamic equilibrium (NLTE) atmosphere code FASTWIND, with a β-law for hydrodynamics. Results: The mass-loss rate of three stars has been obtained for the first time. The global properties of stellar winds of mid/late B supergiants are well represented by a β-law with β > 2. All stars follow the known empirical wind momentum-luminosity relationships, and the late BSGs show the trend of the mid BSGs. HD 75149 and HD 99953 display significant changes in the shape and intensity of the Hα line (from a pure absorption to a P Cygni profile, and vice versa). These stars have mass-loss variations of almost a factor of 2.8. A comparison among mass-loss rates from the literature reveals discrepancies of a factor of 1 to 7. This large variation is a consequence of the uncertainties in the determination of the stellar radius. Therefore, for a reliable comparison of these values we used the invariant parameter Qr. Based on this parameter, we find an empirical relationship that associates the amplitude of mass-loss variations with photometric/spectroscopic variability on timescales of tens of days. We find that stars located on the cool side of the bi-stability jump show a decrease in the ratio V∞/Vesc, while their corresponding mass-loss rates are similar to or lower than the values found for stars on the hot side. Particularly, for those variable stars a decrease in V∞/Vesc is accompanied by a decrease in Ṁ. Conclusions: Our results also suggest that radial pulsation modes with periods longer than 6 days might be responsible for the wind variability in the mid/late-type. These radial modes might be identified with strange modes, which are known to facilitate (enhanced) mass loss. On the other hand, we propose that the wind behaviour of stars on the cool side of the bi-stability jump could fit with predictions of the δ-slow hydrodynamics solution for radiation-driven winds with highly variable ionization.Based on observations taken with the J. Sahade Telescope at Complejo Astronómico El Leoncito (CASLEO), operated under an agreement between the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, the Secretaría de Ciencia y Tecnología de la Nación, and the National Universities of La Plata, Córdoba, and San Juan.Fil: Haucke, Maximiliano. Universidad Nacional de La Plata. Facultad de Ciencias Astronómicas y Geofísicas; ArgentinaFil: Cidale, Lydia Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Astrofísica La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Astronómicas y Geofísicas. Instituto de Astrofísica La Plata; ArgentinaFil: Venero, Roberto Oscar José. Universidad Nacional de La Plata. Facultad de Ciencias Astronómicas y Geofísicas; ArgentinaFil: Curé, M.. Universidad de Valparaíso - Chile; ChileFil: Kraus, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Astrofísica La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Astronómicas y Geofísicas. Instituto de Astrofísica La Plata; ArgentinaFil: Kanaan, S.. Universidad Nacional de La Plata. Facultad de Ciencias Astronómicas y Geofísicas; ArgentinaFil: Arcos, C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Astrofísica La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Astronómicas y Geofísicas. Instituto de Astrofísica La Plata; Argentin

Stabilization of GABAA Receptors at Endocytic Zones Is Mediated by an AP2 Binding Motif within the GABAA Receptor β3 Subunit

The strength of synaptic inhibition can be controlled by the stability and endocytosis of surface and synaptic GABAA receptors (GABAARs), but the surface receptor dynamics that underpin GABAAR recruitment to dendritic endocytic zones (EZs) have not been investigated. Stabilization of GABAARs at EZs is likely to be regulated by receptor interactions with the clathrin-adaptor AP2, but the molecular determinants of these associations remain poorly understood. Moreover, although surface GABAAR downmodulation plays a key role in pathological disinhibition in conditions such as ischemia and epilepsy, whether this occurs in an AP2-dependent manner also remains unclear. Here we report the characterization of a novel motif containing three arginine residues (405RRR407) within the GABAAR β3-subunit intracellular domain (ICD), responsible for the interaction with AP2 and GABAAR internalization. When this motif is disrupted, binding to AP2 is abolished in vitro and in rat brain. Using single-particle tracking, we reveal that surface β3-subunit-containing GABAARs exhibit highly confined behavior at EZs, which is dependent on AP2 interactions via this motif. Reduced stabilization of mutant GABAARs at EZs correlates with their reduced endocytosis and increased steady-state levels at synapses. By imaging wild-type or mutant super-ecliptic pHluorin-tagged GABAARs in neurons, we also show that, under conditions of oxygen–glucose deprivation to mimic cerebral ischemia, GABAARs are depleted from synapses in dendrites, depending on the 405RRR407 motif. Thus, AP2 binding to an RRR motif in the GABAAR β3-subunit ICD regulates GABAAR residency time at EZs, steady- state synaptic receptor levels, and pathological loss of GABAARs from synapses during simulated ischemia

Wind properties of variable B supergiants : Evidence of pulsations connected with mass-loss episodes

Context. Variable B supergiants (BSGs) constitute a heterogeneous group of stars with complex photometric and spectroscopic behaviours. They exhibit mass-loss variations and experience different types of oscillation modes, and there is growing evidence that variable stellar winds and photospheric pulsations are closely related. Aims: To discuss the wind properties and variability of evolved B-type stars, we derive new stellar and wind parameters for a sample of 19 Galactic BSGs by fitting theoretical line profiles of H, He, and Si to the observed ones and compare them with previous determinations. Methods: The synthetic line profiles are computed with the non-local thermodynamic equilibrium (NLTE) atmosphere code FASTWIND, with a β-law for hydrodynamics. Results: The mass-loss rate of three stars has been obtained for the first time. The global properties of stellar winds of mid/late B supergiants are well represented by a β-law with β > 2. All stars follow the known empirical wind momentum-luminosity relationships, and the late BSGs show the trend of the mid BSGs. HD 75149 and HD 99953 display significant changes in the shape and intensity of the Hα line (from a pure absorption to a P Cygni profile, and vice versa). These stars have mass-loss variations of almost a factor of 2.8. A comparison among mass-loss rates from the literature reveals discrepancies of a factor of 1 to 7. This large variation is a consequence of the uncertainties in the determination of the stellar radius. Therefore, for a reliable comparison of these values we used the invariant parameter Qr. Based on this parameter, we find an empirical relationship that associates the amplitude of mass-loss variations with photometric/spectroscopic variability on timescales of tens of days. We find that stars located on the cool side of the bi-stability jump show a decrease in the ratio V∞/Vesc, while their corresponding mass-loss rates are similar to or lower than the values found for stars on the hot side. Particularly, for those variable stars a decrease in V∞/Vesc is accompanied by a decrease in Ṁ. Conclusions: Our results also suggest that radial pulsation modes with periods longer than 6 days might be responsible for the wind variability in the mid/late-type. These radial modes might be identified with strange modes, which are known to facilitate (enhanced) mass loss. On the other hand, we propose that the wind behaviour of stars on the cool side of the bi-stability jump could fit with predictions of the δ-slow hydrodynamics solution for radiation-driven winds with highly variable ionization.Facultad de Ciencias Astronómicas y GeofísicasInstituto de Astrofísica de La Plat

Wind properties of variable B supergiants : Evidence of pulsations connected with mass-loss episodes

Context. Variable B supergiants (BSGs) constitute a heterogeneous group of stars with complex photometric and spectroscopic behaviours. They exhibit mass-loss variations and experience different types of oscillation modes, and there is growing evidence that variable stellar winds and photospheric pulsations are closely related. Aims: To discuss the wind properties and variability of evolved B-type stars, we derive new stellar and wind parameters for a sample of 19 Galactic BSGs by fitting theoretical line profiles of H, He, and Si to the observed ones and compare them with previous determinations. Methods: The synthetic line profiles are computed with the non-local thermodynamic equilibrium (NLTE) atmosphere code FASTWIND, with a β-law for hydrodynamics. Results: The mass-loss rate of three stars has been obtained for the first time. The global properties of stellar winds of mid/late B supergiants are well represented by a β-law with β > 2. All stars follow the known empirical wind momentum-luminosity relationships, and the late BSGs show the trend of the mid BSGs. HD 75149 and HD 99953 display significant changes in the shape and intensity of the Hα line (from a pure absorption to a P Cygni profile, and vice versa). These stars have mass-loss variations of almost a factor of 2.8. A comparison among mass-loss rates from the literature reveals discrepancies of a factor of 1 to 7. This large variation is a consequence of the uncertainties in the determination of the stellar radius. Therefore, for a reliable comparison of these values we used the invariant parameter Qr. Based on this parameter, we find an empirical relationship that associates the amplitude of mass-loss variations with photometric/spectroscopic variability on timescales of tens of days. We find that stars located on the cool side of the bi-stability jump show a decrease in the ratio V∞/Vesc, while their corresponding mass-loss rates are similar to or lower than the values found for stars on the hot side. Particularly, for those variable stars a decrease in V∞/Vesc is accompanied by a decrease in Ṁ. Conclusions: Our results also suggest that radial pulsation modes with periods longer than 6 days might be responsible for the wind variability in the mid/late-type. These radial modes might be identified with strange modes, which are known to facilitate (enhanced) mass loss. On the other hand, we propose that the wind behaviour of stars on the cool side of the bi-stability jump could fit with predictions of the δ-slow hydrodynamics solution for radiation-driven winds with highly variable ionization.Facultad de Ciencias Astronómicas y GeofísicasInstituto de Astrofísica de La Plat

The molecular organization of differentially curved caveolae indicates bendable structural units at the plasma membrane

Caveolae are small coated plasma membrane invaginations with diverse functions. Caveolae undergo curvature changes. Yet, it is unclear which proteins regulate this process. To address this gap, we develop a correlative stimulated emission depletion (STED) fluorescence and platinum replica electron microscopy imaging (CLEM) method to image proteins at single caveolae. Caveolins and cavins are found at all caveolae, independent of curvature. EHD2 is detected at both low and highly curved caveolae. Pacsin2 associates with low curved caveolae and EHBP1 with mostly highly curved caveolae. Dynamin is absent from caveolae. Cells lacking dynamin show no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by intermittent associations with pacsin2 and EHBP1. These coats can flatten and curve to enable lipid traffic, signaling, and changes to the surface area of the cell

Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2

The alpha,beta2,mu2,sigma2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu2Thr156-monophosphorylated AP2 mutated in their alphaPtdIns4,5P(2), mu2PtdIns4,5P(2), and mu2Yxxvarphi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxxvarphi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P(2), explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxxvarphi-containing membranes is two step: initial recruitment via the alphaPtdIns4,5P(2) site and then stabilization through the binding of mu2Yxxvarphi and mu2PtdIns4,5P(2) sites to their ligands. The second step is facilitated by a conformational change favored by mu2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxxvarphi binding and is not enhanced by mu2Thr156 phosphorylation