A phosphatidylinositol (4,5)-bisphosphate binding site within mu 2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. Flow precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)- bisphosphate (PI[4,5]P-2) in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant p,2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P-2- containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P-2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

Mechanisms that ensure robust long-term performance of synaptic transmission over a wide range of activity are crucial for the integrity of neuronal networks, for processing sensory information and for the ability to learn and store memories. Recent experiments have revealed that such robust performance requires a tight coupling between exocytic vesicle fusion at defined release sites and endocytic retrieval of synaptic vesicle membranes. Distinct presynaptic scaffolding proteins are essential for fulfilling this requirement, providing either ultrastructural coordination or acting as signalling hubs

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

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

Improved optical phenotyping of the grape berry surface using light-separation and automated RGB image analysis

Grape resilience towards Botrytis cinerea (B. cinerea) infections (Botrytis bunch rot) is an important concern of breeders and growers. Beside grape bunch architecture, berry surface characteristics like berry bloom (epicuticular wax) as well as thickness and permeability of the berry cuticle represent further promising physical barriers to increase resilience towards Botrytis bunch rot. In previous studies, two efficient sensor-based phenotyping methods were developed to evaluate both berry surface traits fast and objectively: (1) light-separated RGB (red-green-blue) image analysis to determine the distribution of epicuticular wax on the berry surface; and (2) electrical impedance characteristics of the grape berry cuticle based on point measurements. The present proof-of-concept study aiming at the evaluation of light-separated RGB images for both phenotyping applications, phenotyping wax distribution pattern and berry cuticle impedance values. Within the selected grapevine varieties like 'Riesling', 'Sauvignon Blanc' or 'Calardis Blanc' five contributions were achieved: (1) Both phenotyping approaches were fused into one prototypic unified phenotyping method achieving a wax detection accuracy of 98.6 % and a prediction of electrical impedance with an accuracy of 95 %. (2) Both traits are derived using only light-separated images of the grapevine berries. (3) The improved method allows the detection and quantification of additional surface traits of the grape berry surface such as lenticels (punctual lignification) and the berry stem that are also known as being able to affect the grape susceptibility towards Botrytis. (4) The improved image analysis tools are further integrated into a comprehensive workbench allowing end-users, like breeders to combine phenotyping experiments with transparent data management offering valuable services like visualizations, indexing, etc. (5) Annotation work is supported by a sophisticated annotation tool of the image analysis workbench. The usage of light-separated images enables fast and non-invasive phenotyping of different optical berry surface characteristics, which saves time-consuming labor and additionally allows the reuse of the berry samples for subsequent investigations, e.g. Botrytis infection studies

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

Fast neurotransmitter release regulated by the endocytic scaffold intersectin.

Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain

The cell adhesion protein CAR is a negative regulator of synaptic transmission

The Coxsackievirus and adenovirus receptor (CAR) is essential for normal electrical conductance in the heart, but its role in the postnatal brain is largely unknown. Using brain specific CAR knockout mice (KO), we discovered an unexpected role of CAR in neuronal communication. This includes increased basic synaptic transmission at hippocampal Schaffer collaterals, resistance to fatigue, and enhanced long-term potentiation. Spontaneous neurotransmitter release and speed of endocytosis are increased in KOs, accompanied by increased expression of the exocytosis associated calcium sensor synaptotagmin 2. Using proximity proteomics and binding studies, we link CAR to the exocytosis machinery as it associates with syntenin and synaptobrevin/VAMP2 at the synapse. Increased synaptic function does not cause adverse effects in KO mice, as behavior and learning are unaffected. Thus, unlike the connexin-dependent suppression of atrioventricular conduction in the cardiac knockout, communication in the CAR deficient brain is improved, suggesting a role for CAR in presynaptic processes

Crystal structure of the dynamin tetramer

The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane. Previous studies have described the architecture of dynamin dimers, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot-Marie-Tooth neuropathy and centronuclear myopathy, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction

Studies of h/e Aharonov-Bohm Photovoltaic Oscillations in Mesoscopic Au Rings

We have investigated a mesoscopic photovoltaic (PV) effect in micron-size Au rings in which a dc voltage Vdc is generated in response to microwave radiation. The effect is due to the lack of inversion symmetry in a disordered system. Aharonov-Bohm PV oscillations with flux period h/e have been observed at low microwave intensities for temperatures ranging from 1.4 to 13 K. For moderate microwave intensities the h/e PV oscillations are completely quenched providing evidence that the microwaves act to randomize the phase of the electrons. Studies of the temperature dependence of Vdc also provide evidence of the dephasing nature of the microwave field. A complete theoretical explanation of the observed behavior seems to require a theory for the PV effect in a ring geometry.Comment: 10 pages (RevTex twocolumn style), 8 figures-2 pages (one postscript file) To be published in Phys. Rev.