Mammalian Drp1 is a dynamin-like GTPase required for mitochondrial fission. Although it exists primarily as a cytosolic homo-tetramer in vivo, it can also self-assemble into higher order structures on the mitochondrial outer membrane, where it is required for proper mitochondrial division. Functional studies and sequence comparisons have revealed four different structural domains in Drp1, comprising N-terminal GTP-binding, middle, insert B, and C-terminal GTPase effector (GED) domains. Here we describe an intramolecular interaction within Drp1 between the GED and the N-terminal GTP-binding and middle domains. A point mutation (K679A) within the C-terminal GED domain inhibits this intramolecular association, without affecting the formation of Drp1 tetramers or the intermolecular associations among isolated C-terminal domains. Mutant Drp1 K679A exhibits impaired GTPase activity, and when overexpressed in mammalian cells it decreases mitochondrial division. Sedimentation experiments indicate that the K679A mutation either increases Drp1 complex formation or, more likely, decreases complex disassembly as compared with wild-type Drp1. Taken together, these data suggest that the C-terminal GED domain is important for stimulation of GTPase activity, formation and stability of higher order complexes, and efficient mitochondrial division.

Excitatory synapses are located on actin-rich protrusions known as dendritic spines. alpha-Actinin is an actin binding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Because it also binds to NMDA receptors and other PSD components, alpha-actinin has been proposed to link NMDA receptors and the PSD to the underlying actin cytoskeleton of the dendritic spine. Although alpha-actinin has been implicated in modulation of NMDA receptor activity, the cell biological function of alpha-actinin in neurons is unknown. We report here that alpha-actinin is concentrated in spines. Both the actin binding domain and the spectrin repeat region (which interacts with NMDA receptors) of alpha-actinin2 are required for spine targeting. In live imaging experiments, Venus-tagged alpha-actinin2 in dendritic spines showed faster turnover than PSD-95, as determined by fluorescent recovery after photobleaching (FRAP), and individual spines often showed marked fluctuations in alpha-actinin content over a time-scale of minutes. Overexpression of alpha-actinin2 increased the length and density of dendritic protrusions in cultured hippocampal neurons, an effect that requires the actin binding domain and the spectrin repeats of alpha-actinin. These results suggest that alpha-actinin regulates spine morphology and density.

The efficacy of excitatory transmission in the brain depends to a large extent on synaptic AMPA receptors, hence the importance of understanding the delivery and recycling of the receptors at the synaptic sites. Here we report a novel regulation of the AMPA receptor transport by a PDZ (postsynaptic density-95/Drosophila disc large tumor suppressor zona occludens 1) and LIM (Lin11/rat Isl-1/Mec3) domain-containing protein, RIL (reversion-induced LIM protein). We show that RIL binds to the AMPA glutamate receptor subunit GluR-A C-terminal peptide via its LIM domain and to alpha-actinin via its PDZ domain. RIL is enriched in the postsynaptic density fraction isolated from rat forebrain, strongly localizes to dendritic spines in cultured neurons, and coprecipitates, together with alpha-actinin, in a protein complex isolated by immunoprecipitation of AMPA receptors from forebrain synaptosomes. Functionally, in heterologous cells, RIL links AMPA receptors to the alpha-actinin/actin cytoskeleton, an effect that appears to apply selectively to the endosomal surface-internalized population of the receptors. In cultured neurons, an overexpression of recombinant RIL increases the accumulation of AMPA receptors in dendritic spines, both at the total level, as assessed by immunodetection of endogenous GluR-A-containing receptors, and at the synaptic surface, as assessed by recording of miniature EPSCs. Our results thus indicate that RIL directs the transport of GluR-A-containing AMPA receptors to and/or within dendritic spines, in an alpha-actinin/actin-dependent manner, and that such trafficking function promotes the synaptic accumulation of the receptors.

PDZ domains are protein-interaction domains that are often found in multi-domain scaffolding proteins. PDZ-containing scaffolds assemble specific proteins into large molecular complexes at defined locations in the cell. In the postsynaptic density of neuronal excitatory synapses, PDZ proteins such as PSD-95 organize glutamate receptors and their associated signalling proteins and determine the size and strength of synapses. PDZ scaffolds also function in the dynamic trafficking of synaptic proteins by assembling cargo complexes for transport by molecular motors. As key organizers that control synaptic protein composition and structure, PDZ scaffolds are themselves highly regulated by synthesis and degradation, subcellular distribution and post-translational modification.

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(2+) sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn(2+) complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca(2+) and Mg(2+). In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM). The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn(2+) within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn(2+) stores.

The proper intracellular distribution of mitochondria is assumed to be critical for normal physiology of neuronal cells, but direct evidence for this idea is lacking. Extension or movement of mitochondria into dendritic protrusions correlates with the development and morphological plasticity of spines. Molecular manipulations of dynamin-like GTPases Drp1 and OPA1 that reduce dendritic mitochondria content lead to loss of synapses and dendritic spines, whereas increasing dendritic mitochondrial content or mitochondrial activity enhances the number and plasticity of spines and synapses. Thus, the dendritic distribution of mitochondria is essential and limiting for the support of synapses. Reciprocally, synaptic activity modulates the motility and fusion/fission balance of mitochondria and controls mitochondrial distribution in dendrites.

PDZ domains bind to short segments within target proteins in a sequence-specific fashion. Glutamate receptor-interacting protein (GRIP)/ABP family proteins contain six to seven PDZ domains and interact via the sixth PDZ domain (class II) with the C termini of various proteins including liprin-alpha. In addition the PDZ456 domain mediates the formation of homo- and heteromultimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class II PDZ domain and PDZ-mediated multimerization, we determined the crystal structures of the GRIP1 PDZ6 domain alone and in complex with a synthetic C-terminal octapeptide of human liprin-alpha at resolutions of 1.5 and 1.8 A, respectively. Remarkably, unlike other class II PDZ domains, Ile-736 at alphaB5 rather than conserved Leu-732 at alphaB1 makes a direct hydrophobic contact with the side chain of the Tyr at the -2 position of the ligand. Moreover, the peptide-bound structure of PDZ6 shows a slight reorientation of helix alphaB, indicating that the second hydrophobic pocket undergoes a conformational adaptation to accommodate the bulkiness of the Tyr side chain, and forms an antiparallel dimer through an interface located at a site distal to the peptide-binding groove. This configuration may enable formation of GRIP multimers and efficient clustering of GRIP-binding proteins.

PDZ domain proteins play critical roles in binding, clustering and subcellular targeting of membrane receptors and ion channels. PDZ domains in multi-PDZ proteins often are arranged in groups with highly conserved spacing and intervening sequences; however, the functional significance of such tandem arrangements of PDZs is unclear. We have solved the three-dimensional structure of the first two PDZ domains of postsynaptic density protein-95 (PSD-95 PDZ1 and PDZ2), which are closely linked to each other in the PSD-95 family of scaffold proteins. The two PDZs have limited freedom of rotation and their C-terminal peptide-binding grooves are aligned with each other with an orientation preference for binding to pairs of C termini extending in the same direction. Increasing the spacing between PDZ1 and PDZ2 resulted in decreased binding between PDZ12 and its dimeric targets. The same mutation impaired the functional ability of PSD-95 to cluster Kv1.4 potassium channels in heterologous cells. The data presented provide a molecular basis for preferential binding of PSD-95 to multimeric membrane proteins with appropriate C-terminal sequences.

The membrane-associated guanylate kinase PSD-95 scaffolds N-methyl-d-aspartate receptors to cytoplasmic signaling molecules, and associates with other glutamate receptors at central synapses. However, regulation of PSD-95 in vivo is poorly understood. We provide evidence of an activity-dependent redistribution of PSD-95 to dendrites in central visual neurons that is tied to eye opening. Six hours after eye opening, increased dendritic PSD-95 coimmunoprecipitates with the same proportions of stargazin, increased proportions of the N-methyl-d-aspartate receptor subunit NR2A, and decreased proportions of NR2B. Sustained high levels of PSD-95 in dendrites are dependent on continued pattern vision in juvenile but not mature animals, suggesting that the stabilization of PSD-95 at synapses may be involved in the control of developmental plasticity.