Proper morphogenesis of neuronal dendritic spines is essential for the formation of functional synaptic networks. However, it is not known how spines are initiated. Here, we identify the inverse-BAR (I-BAR) protein MIM/MTSS1 as a nucleator of dendritic spines. MIM accumulated to future spine initiation sites in a PIP2-dependent manner and deformed the plasma membrane outward into a proto-protrusion via its I-BAR domain. Unexpectedly, the initial protrusion formation did not involve actin polymerization. However, PIP2-dependent activation of Arp2/3-mediated actin assembly was required for protrusion elongation. Overexpression of MIM increased the density of dendritic protrusions and suppressed spine maturation. In contrast, MIM deficiency led to decreased density of dendritic protrusions and larger spine heads. Moreover, MIM-deficient mice displayed altered glutamatergic synaptic transmission and compatible behavioral defects. Collectively, our data identify an important morphogenetic pathway, which initiates spine protrusions by coupling phosphoinositide signaling, direct membrane bending, and actin assembly to ensure proper synaptogenesis.
Selective insertion ⁄ removal of GluR-A (GluR1) subunit, containing a-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid (AMPA) receptors to ⁄ from the postsynaptic membrane, is a key early event in some experimental models of activity-dependent regulation of synaptic strength [1][2][3][4][5]. Rapid insertion of GluR-A to synaptic membrane is dependent on its cytoplasmic C-terminal domain of 80 amino acid residues [6]. Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser ⁄ Thr-x-/-COOH where / is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97 PDZ2 ). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97 PDZ2 variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 Å resolutions. SAP97 PDZ2 folds to a compact globular domain comprising six b-strands and two a-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel b-strand in the binding groove of SAP97 PDZ2 . The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The bA)bB loop moves away from the C-terminal end of aB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in bA)bB loop with Asp396 or Thr394 in bB. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.Abbreviations AMPA, a-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid; GluR-A, ionotropic glutamate receptor subunit A; GST, glutathione S-transferase; Maguk, membrane-associated guanylate kinase homolog; PDZ, postsynaptic density )95 ⁄ Discs large ⁄ zona occludens-1; PSD-93, postsynaptic density )93; PSD-95, postsynaptic density )95; SAP97, synapse-associated protein 97; SAP102, synapse-associated protein 102.
Synaptic delivery of GluR-A (GluR1) subunit-containing glutamate receptors depends on a C-terminal type I PDZ binding motif in GluR-A. Synapse-associated protein 97 (SAP97) is the only PDZ domain protein known to associate with GluR-A. We have used NMR spectroscopy and a biotinylated peptide binding assay to characterize the interaction between synthetic GluR-A C-terminal peptides and the PDZ2 domain of SAP97 (SAP97(PDZ2)), previously determined to be the dominant factor responsible for the interaction. The binding mode appeared to be strongly influenced by redox conditions. Chemical shift changes observed in NMR spectra indicate that under reducing conditions, the last four residues of GluR-A peptides bind to PDZ2 in a fashion typical of class I PDZ interactions. The binding is weak and relatively nonselective as it occurs similarly with a PDZ2 domain derived from PSD-95, a related protein not believed to directly interact with GluR-A. In the absence of reducing agents, conserved cysteine residues in SAP97(PDZ2) and the GluR-A C-terminus gave rise to an anomalous behavior in a microplate assay with a biotinylated GluR-A 18-mer peptide. A covalent disulfide-linked complex between SAP97(PDZ2) and the GluR-A peptide was seen in the binding assay and in the NMR experiments performed under oxidizing conditions. The results are consistent with a two-step binding mechanism consisting of an initial PDZ interaction followed by stabilization of the complex by a disulfide bond. The possible physiological relevance of redox regulation of SAP97-GluR-A interaction remains to be established.
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