Arndt Pechstein

Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2μ–cargo complexes

Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is an important factor for a variety of cellular functions ranging from cell signaling to actin cytoskeletal dynamics and endocytic membrane traffic. Here, we have identified the clathrin adaptor complex AP-2 as a regulator of phosphatidylinositol 4-phosphate 5-kinase (PIPK)-mediated PI(4,5)P2 synthesis. AP-2 directly interacts with the kinase core domain of type I PIPK isozymes via its μ2-subunit in vitro and in native protein extracts. Endocytic cargo protein binding to μ2 leads to a potent stimulation of PIPK activity. These data thus identify a positive feedback loop consisting of endocytic cargo proteins, AP-2μ, and PIPK type I which may provide a specific pool of PI(4,5)P2 dedicated to clathrin/AP-2-dependent receptor internalization.

Molecular basis for SH3 domain regulation of F-BAR–mediated membrane deformation

Members of the Bin/amphiphysin/Rvs (BAR) domain protein superfamily are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicle and T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, α-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function.

Clathrin/AP-2 Mediate Synaptic Vesicle Reformation from Endosome-like Vacuoles but Are Not Essential for Membrane Retrieval at Central Synapses

Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.

Molecular determinants for the interaction between AMPA receptors and the clathrin adaptor complex AP-2

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. We report here that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with μ2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2μ and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre- and postsynaptic membrane proteins.

How synapsin I may cluster synaptic vesicles.

Synapsin I is the most abundant brain phosphoprotein present in conventional synapses of the CNS. Knockout and rescue experiments have demonstrated that synapsin is essential for clustering of synaptic vesicles (SVs) at active zones and the organization of the reserve pool of SVs. However, in spite of intense efforts it remains largely unknown how exactly synapsin I performs this function. It has been proposed that synapsin I in its dephosphorylated state may tether SVs to actin filaments within the cluster from where SVs are released in response to activity-induced synapsin phosphorylation. Recent studies, however, have failed to detect actin filaments inside the vesicle cluster at resting central synapses. Instead, proteins with established functional roles in SV recycling have been found within this presynaptic compartment. Here we discuss potential alternative mechanisms of synapsin I-dependent SV clustering in the reserve pool.

Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P2]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domain-containing accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the “side sites” of the AP2 α- and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrin-mediated SV recycling in synapses.

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.

Synapsin I Senses Membrane Curvature by an Amphipathic Lipid Packing Sensor Motif

Sustained neurotransmitter release at synapses during high-frequency synaptic activity involves the mobilization of synaptic vesicles (SVs) from the tightly clustered reserve pool (RP). Synapsin I (Syn I), a brain-specific peripheral membrane protein that undergoes activity-dependent cycles of SV association and dissociation, is implicated in RP organization via its ability to cluster SVs. Although Syn I has affinity for phospholipids, the mechanism for the reversible association of synapsin with SV membranes remains enigmatic. Here, we show that rat Syn I is able to sense membrane curvature via an evolutionary conserved amphipathic lipid packing sensor motif (ALPS). Deletion or mutational inactivation of the ALPS impairs the ability of Syn I to associate with highly curved membranes and with SVs. Furthermore, a Syn I mutant lacking ALPS displays defects in its ability to undergo activity-induced cycles of dispersion and reclustering in neurons and fails to induce vesicle clustering in vitro. Our data suggest a crucial role for ALPS-mediated sensing of membrane curvature in regulating synapsin function.

Taking a Back Seat: Synaptic Vesicle Clustering in Presynaptic Terminals

Central inter-neuronal synapses employ various molecular mechanisms to sustain neurotransmitter release during phases of high-frequency synaptic activity. One of the features ensuring this property is the presence of a pool of synaptic vesicles (SVs) in the presynaptic terminal. At rest and low rates of stimulation, most of the vesicles composing this pool remain in a tight cluster. They are actively utilized when neurons fire action potentials at higher rates and the capability of the recycling machinery is limited. In addition, SV clusters are capable of migrating between release sites and reassemble into clusters at neighboring active zones (AZs). Within the cluster, thin “tethers” interconnect SVs. These dynamic filamentous structures are reorganized during stimulation thereby releasing SVs from the cluster. So far, one protein family, the synapsins, which bind actin filaments and vesicles in a phosphorylation-dependent manner, has been implicated in SV clustering in vertebrate synapses. As evident from recent studies, many endocytic proteins reside in the SV cluster in addition to synapsin. Here we discuss alternative possible mechanisms involved in the organization of this population of SVs. We propose a model in which synapsins together with other synaptic proteins, a large proportion of which is involved in SV recycling, form a dynamic proteinaceous “matrix” which limits the mobility of SVs. Actin filaments, however, do not seem to contribute to SV crosslinking within the SV cluster, but instead they are present peripherally to it, at sites of neurotransmitter release, and at sites of SV recycling.

Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses.

Neurotransmission involves the exo‐endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin‐mediated SV reformation require curvature‐sensing and membrane‐bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi‐domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin‐coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non‐canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin‐binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1‐3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin‐mediated SV recycling, thereby facilitating vesicle uncoating.