Orchestrating vesicular and nonvesicular membrane dynamics by intrinsically disordered proteins

Compartmentalization by membranes is a common feature of eukaryotic cells and serves to spatiotemporally confine biochemical reactions to control physiology. Membrane‐bound organelles such as the endoplasmic reticulum (ER), the Golgi complex, endosomes and lysosomes, and the plasma membrane, continuously exchange material via vesicular carriers. In addition to vesicular trafficking entailing budding, fission, and fusion processes, organelles can form membrane contact sites (MCSs) that enable the nonvesicular exchange of lipids, ions, and metabolites, or the secretion of neurotransmitters via subsequent membrane fusion. Recent data suggest that biomolecule and information transfer via vesicular carriers and via MCSs share common organizational principles and are often mediated by proteins with intrinsically disordered regions (IDRs). Intrinsically disordered proteins (IDPs) can assemble via low‐affinity, multivalent interactions to facilitate membrane tethering, deformation, fission, or fusion. Here, we review our current understanding of how IDPs drive the formation of multivalent protein assemblies and protein condensates to orchestrate vesicular and nonvesicular transport with a special focus on presynaptic neurotransmission. We further discuss how dysfunction of IDPs causes disease and outline perspectives for future research

Super-resolution imaging of tight and adherens junctions: Challenges and open questions

The tight junction (TJ) and the adherens junction (AJ) bridge the paracellular cleft of epithelial and endothelial cells. In addition to their role as protective barriers against bacteria and their toxins they maintain ion homeostasis, cell polarity, and mechano-sensing. Their functional loss leads to pathological changes such as tissue inflammation, ion imbalance, and cancer. To better understand the consequences of such malfunctions, the junctional nanoarchitecture is of great importance since it remains so far largely unresolved, mainly because of major difficulties in dynamically imaging these structures at sufficient resolution and with molecular precision. The rapid development of super-resolution imaging techniques ranging from structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (SMLM) has now enabled molecular imaging of biological specimens from cells to tissues with nanometer resolution. Here we summarize these techniques and their application to the dissection of the nanoscale molecular architecture of TJs and AJs. We propose that super-resolution imaging together with advances in genome engineering and functional analyses approaches will create a leap in our understanding of the composition, assembly, and function of TJs and AJs at the nanoscale and, thereby, enable a mechanistic understanding of their dysfunction in disease

Optogenetics and electron microscopy reveal an ultrafast mode of synaptic vesicle recycling, adding a new twist to a 40-year-old controversy

Optogenetics and electron microscopy reveal an ultrafast mode of synaptic vesicle recycling, adding a new twist to a 40-year-old controversy. - See more at: http://elifesciences.org/content/2/e01233#sthash.K8kQedyo.dpu

Endocytosis in the adaptation to cellular stress

Cellular life is challenged by a multitude of stress conditions, triggered for example by alterations in osmolarity, oxygen or nutrient supply. Hence, cells have developed sophisticated stress responses to cope with these challenges. Some of these stress programs such as the heat shock response are understood in great detail, while other aspects remain largely elusive including potential stress-dependent adaptations of the plasma membrane proteome. The plasma membrane is not only the first point of encounter for many types of environmental stress, but given the diversity of receptor proteins and their associated molecules also represents the site at which many cellular signal cascades originate. Since these signaling pathways affect virtually all aspects of cellular life, changes in the plasma membrane proteome appear ideally suited to contribute to the cellular adaptation to stress. The most rapid means to alter the cell surface proteome in response to stress is by alterations in endocytosis. Changes in the overall endocytic flux or in the endocytic regulation of select proteins conceivably can help to counteract adverse environmental conditions. In this review we summarize recent data regarding stress-induced changes in endocytosis and discuss how these changes might contribute to the cellular adaptation to stress in different systems. Future studies will be needed to uncover the underlying mechanisms in detail and to arrive at a coherent picture

A phosphatidylinositol (4,5)-bisphosphate binding site within μ2-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. How 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]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within μ2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant μ2 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)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant μ2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to μ2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation

The Neural Cell Adhesion Molecule Promotes Maturation of the Presynaptic Endocytotic Machinery by Switching Synaptic Vesicle Recycling from Adaptor Protein 3 (AP-3)- to AP-2-Dependent Mechanisms

Newly formed synapses undergo maturation during ontogenetic development via mechanisms that remain poorly understood. We show that maturation of the presynaptic endocytotic machinery in CNS neurons requires substitution of the adaptor protein 3 (AP-3) with AP-2 at the presynaptic plasma membrane. In mature synapses, AP-2 associates with the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes binding of AP-2 over binding of AP-3 to presynaptic membranes, thus favoring the substitution of AP-3 for AP-2 during formation of mature synapses. The presynaptic endocytotic machinery remains immature in adult NCAM-deficient (NCAM−/−) mice accumulating AP-3 instead of AP-2 and its partner protein AP180 in synaptic membranes and vesicles. NCAM deficiency or disruption of the NCAM/AP-2 complex in wild-type (NCAM+/+) neurons by overexpression of AP-2 binding-defective mutant NCAM interferes with efficient retrieval of the synaptic vesicle v-SNARE synaptobrevin 2. Abnormalities in synaptic vesicle endocytosis and recycling may thus contribute to neurological disorders associated with mutations in NCAM

Generation of Coated Intermediates of Clathrin-Mediated Endocytosis on Protein-Free Liposomes

AbstractClathrin-coated buds and dynamin-coated tubules morphologically similar to corresponding structures observed in synaptic membranes can be generated on protein-free liposomes by incubation with cytosol, or with clathrin coat proteins and purified dynamin, respectively. Dynamin- and clathrin-coated intermediates may form independently of each other, despite the coupling between the two processes typically observed in synaptic membranes. Formation of both structures on liposomes can occur in the absence of nucleotides. These findings indicate that interfaces between lipids and cytosolic proteins are fully sufficient to deform lipids bilayers into buds and tubules. They suggest that a main function of membrane proteins is to act as positive and negative regulators of coat assembly, therefore controlling these processes in time and space

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

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