Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca 2 þ in membrane protein organization. We find that Ca 2 þ at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca 2 þ influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca 2 þ strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.

The spatial distribution of the target (t-)SNARE proteins (syntaxin and SNAP-25) on the plasma membrane has been extensively characterized. However, the protein conformations and interactions of the two t-SNAREs in situ remain poorly defined. By using super-resolution optical techniques and fluorescence lifetime imaging microscopy, we observed that within the t-SNARE clusters syntaxin and SNAP-25 molecules interact, forming two distinct conformations of the t-SNARE binary intermediate. These are spatially segregated on the plasma membrane with each cluster exhibiting predominantly one of the two conformations, representing the two- and three-helical forms previously observed in vitro. We sought to explain why these two t-SNARE intermediate conformations exist in spatially distinct clusters on the plasma membrane. By disrupting plasma membrane lipid order, we found that all of the t-SNARE clusters now adopted a single conformational state corresponding to the three helical t-SNARE intermediates. Together, our results define spatially distinct t-SNARE intermediate states on the plasma membrane and how the conformation adopted can be patterned by the underlying lipid environment.

SummaryInhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.

Fiuza et al. report that PICK1 localizes to clathrin-coated pits and makes direct, functional interactions with the endocytic adapter complex AP2 and dynamin. The PICK1–AP2 interaction is required for clustering AMPA receptors at endocytic sites and for consequent AMPA receptor endocytosis, defining PICK1 as a cargo-specific endocytic accessory protein.

SNAREs are a superfamily of small membrane proteins catalyzing membrane fusion (1,2). They share as a common feature a conserved stretch of 60-70 amino acids, called the SNARE-motif. Most SNARE-motifs are membrane anchored by a C-terminal transmembrane region (TMR), and a few SNAREs possess two SNAREmotifs linked by a region containing a palmitoylated cysteine cluster for membrane attachment.Each fusion step requires a specific set of three to four SNAREs providing four SNARE-motifs. For instance, neuronal exocytosis is driven by formation of a complex between SNAP25 (containing two SNARE-motifs) and syntaxin 1A at the plasma membrane and vesicle associated synaptobrevin 2.The fusion reaction results in a ternary cis-SNARE complex in which the four SNARE-motifs adopt alpha-helical conformation, forming a parallel, twisted bundle stabilized by 16 layers of interaction (3). All layers are hydrophobic with the exception of a highly conserved central ionic layer to which each of the SNARE-motifs contributes either one out of three glutamines (consecutively named Q a , Q b and Q c ) or an arginine (R). According to their contributed amino acids, the SNAREs have been classified as Q a -(syntaxin), Q b -and Q c -(N-and C-terminal SNARE-motifs of SNAP25, respectively) and R-SNARE (synaptobrevin 2) (4). Retrospectively, the neuronal SNARE complex laid the ground for the structural Q/R-SNARE classification of all SNAREs discovered in the animal kingdom, fungi or plants.Although the post-fusion SNARE complex is well characterized, it is still debated by what sequence of interactions it assembles (5). Because Q-SNAREs form complexes but the R-SNARE-motif does not interact with any of the individual Q-SNAREs [for exception see e.g. (6)], it is generally believed that initially a Q-SNARE complex forms, which then functions as acceptor for the R-SNARE.However, in vitro soluble Q-SNARE-motifs (lacking their membrane anchor) do not form an intuitively expected stable Q a Q b Q c or Q x Q y complex, but have the intrinsic property to assemble into several four-helix bundles [e.g. (Q a ) 2 Q b Q c (7), (Q a ) 2 (Q b ) 2 (8) and (Q a ) 4 (8,9); for a SNAP23 (Q b ) 4 bundle see (10)].More recently, several groups accomplished to prevent the stabilization of Q a Q b Q c complexes into (Q a ) 2 Q b Q c complexes by different approaches. For instance, a Q a Q b Q c complex forms when SNAP25 is immobilized on sepharose beads by anti-SNAP25 antibodies (11), a C-terminal synaptobrevin fragment is used for stabilization (12), SNAP25 is available in large excess over syntaxin (12), or complexes are prepared in supported lipid bilayers containing syntaxin at a very low protein to lipid ratio (13).The latter study even allowed the characterization of single complexes and found several states. In addition to the Q a Q b Q c complex, other complexes were also observed with one of the SNAP25 SNARE-motifs dissociated (in principle representing a Q a Q b or Q a Q c complex). 394 www.traffic.dk