Correspondence to: David S. Bredt, University of California at San Francisco School of Medicine, 513 Parnassus Ave., San Francisco, CA 94143-0444. Tel:(415) 476-6310 Fax:(415) 476-4929 E-mail:bredt{at}itsa.ucsf.edu.
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Abstract |
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Postsynaptic density-95 (PSD-95/SAP-90) is a palmitoylated peripheral membrane protein that scaffolds ion channels at excitatory synapses. To elucidate mechanisms for postsynaptic ion channel clustering, we analyzed the cellular trafficking of PSD-95. We find that PSD-95 transiently associates with a perinuclear membranous compartment and traffics with vesiculotubular structures, which migrate in a microtubule-dependent manner. Trafficking of PSD-95 with these vesiculotubular structures requires dual palmitoylation, which is specified by five consecutive hydrophobic residues at the NH2 terminus. Mutations that disrupt dual palmitoylation of PSD-95 block both ion channel clustering by PSD-95 and its synaptic targeting. Replacing the palmitoylated NH2 terminus of PSD-95 with alternative palmitoylation motifs at either the NH2 or COOH termini restores ion channel clustering also induces postsynaptic targeting, respectively. In brain, we find that PSD-95 occurs not only at PSDs but also in association with intracellular smooth tubular structures in dendrites and spines. These data imply that PSD-95 is an itinerant vesicular protein; initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.
Key Words: PSD, palmitoylation, trafficking, MAGUK, clustering
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Introduction |
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Rapid and efficient neurotransmission requires proper synaptic assembly of signal transducing protein networks. At excitatory synapses, glutamate receptors are clustered at the postsynaptic density (PSD)1, a thickening of the cytoskeleton beneath the plasma membrane. Also associated with the PSD are cytosolic enzymes including calcium calmodulin-dependent protein kinase II and neuronal nitric oxide synthase that modulate glutamatergic stimuli (
Although recent studies have helped to define the composition of the PSD (
Because postsynaptic protein clustering critically participates in both synaptogenesis and plasticity, mechanistic understanding of postsynaptic protein trafficking is an important goal. PSD-95 itself is a valuable tool for studying this protein sorting problem because fluorescently tagged PSD-95-GFP fusion proteins are targeted appropriately to the PSD (
Some insight into mechanisms for protein sorting in neurons has been gained by comparison with polarized protein targeting in epithelial cells. Many transmembrane proteins that occur along the basolateral surface of epithelial cells have a somatodendritic or postsynaptic distribution in neurons whereas apical proteins of epithelial cells are typically found in neuronal axons (
To clarify mechanisms for postsynaptic sorting and assembly of postsynaptic proteins, we have evaluated subcellular trafficking of fluorescently labeled PSD-95 proteins. Strikingly, we find that PSD-95 is sorted with perinuclear vesicles in both heterologous cells and in developing neurons. When visualized in living cells, PSD-95 positive vesicles migrate to and from perinuclear vesicles and this movement requires intact microtubules. Site-directed mutagenesis demonstrates that perinuclear vesicular sorting and ion channel clustering of PSD-95 in heterologous cells and its postsynaptic targeting in neurons all rely on dual palmitoylation of cysteines 3 and 5, and that the yet unidentified palmitoyltransferase responsible for this modification recognizes a consensus of 5 consecutive hydrophobic amino acids. Replacing the palmitoylated NH2 terminus of PSD-95 with alternative palmitoylation motifs at either the NH2 or COOH terminus restores ion channel clustering and postsynaptic targeting. These data imply that PSD-95 is not merely a static cytoskeletal element but that PSD-95 is an itinerant vesicular protein and that initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.
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Materials and Methods |
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Antibodies
The following antibodies were used: rabbit polyclonal antibodies to Kv1.4 (
cDNA Cloning and Mutagenesis
Subcloning of wild-type cysteine mutant forms of PSD-95 as NH2-terminal fusions with GFP (PSD-95 GFP, C35S, C3S, and C5S), in pGW1 were previously described (
Cell Transfection and Metabolic Labeling
COS7, HEK-293 and MDCK cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, penicillin, and streptomycin. PC12 cells used the same media containing 5% fetal bovine serum and 10% horse serum and were differentiated for 3 d after transfection in media containing 50 ng/ml NGF. Cells were transfected using Lipofectamine reagent according to the manufacturer's protocol (Gibco). Stable MDCK cells were generated by cotransfecting a PSD-95 expression construct in a GW1 vector together with pcDNAIII and selecting for stable transformants with 1 mg/ml G418. To form polarized monolayers, MDCK cells were plated on 0.4 mm diameter TranswellsTM (Corning Costar) and grown for 23 d. For studies of palmitoylation, metabolic labeling of transfected COS7 cells was done as previously described (
Transfection of Primary Neuronal Cultures and Immunofluorescent Labeling
Hippocampal cultures were transfected as previously described (
Electron Microscopic Immunocytochemistry
Adult rats were fixed by transcardial perfusion with a phosphate-buffered aldehyde mixture, consisting of 4% paraformaldehyde and 3% acrolein or 2.5% glutaraldehyde. Sections were prepared (40 µm) and blocked in saline buffered with 0.01 M phosphate (PBS) containing 1% BSA. After an overnight incubation with affinity-purified anti-PSD-95 antibody (1 µg/ml), sections were processed for pre-embedding colloidal gold immunolabeling, using 1-nm gold conjugated anti-rabbit IgG as the label. A silver intensification kit (Amersham) was then used to enlarge the 1-nm gold particles to >5 nm for light and electron microscopic detection (
Imaging of PSD-95 GFP in Live Cells
5 h after transfection, COS7 cells on glass coverslips were placed into a Bioptechs culture dish heated to 37°C. Images were acquired (100-ms exposure) every 30 seconds for 30180 min with excitation at 470/40 nm and emission at 525/50 nm. Imaging was done using a Zeiss Axiovert S100 TV inverted microscope equipped with a C-Apochromat (NA = 1.2) 63x water objective, a Hamamatsu 12-bit ORCA interline CCD camera, a Sutter excitation and emission filter-wheel and a Universal Imaging Corporation MetaMorph Imaging system.
Supplemental Online Material
Video 1.
Visualization of transport intermediates of PSD-95.
Video 2.
PSD-95 is trafficked with vesiculotubular structures that accumulate in a perinuclear domain.
Video 3.
Nocodazole treatment paralyzes transport intermediates of PSD-95.
Video 4.
NH2-terminal palmitoylation is required for sorting of PSD-95 to the perinuclear vesicular domain.
Videos 57.
Ion channel clustering by PSD-95 appears to involve vesiculotubular trafficking. COS cells transfected with PSD-95 GFP and Kv1.4 were monitored over time for GFP.
All videos are available at http://www.jcb.org/cgi/content/full/148/1/159/DC1.
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Results |
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PSD-95 Is Sorted with Perinuclear Vesicles in Heterologous Cells and in Neurons
To define cellular processes that mediate postsynaptic ion channel clustering by PSD-95, we first analyzed the subcellular distribution of exogenous PSD-95 expressed in various cell lines. 12 h after transfection of COS cells, wild-type or tagged versions of PSD-95 occur diffusely throughout the cytoplasm but are also conspicuously concentrated at a perinuclear compartment (Figure 1 A, top). The accumulation of fluorescent PSD-95 at this perinuclear region steadily progresses from 58 h after transfection (see time lapse below). To determine whether this transient perinuclear localization of PSD-95 corresponds to a specific organelle, transfected cells were double-stained for PSD-95 and either the mannose-6-phosphate receptor, Golgi 58K, TGN38, or DND-99 (Lysotracker). The perinuclear localization of PSD-95 corresponds to that of the mannose 6-phosphate receptor (M6PR; Figure 1B and Figure C), a well-characterized marker of late endosomes (
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As found in COS cells, PSD-95 expressed in either HEK293 or PC12 cells also coincides with the M6PR positive compartment (Figure 1 A, bottom, and data not shown). Furthermore, in developing hippocampal neurons we find that transfected PSD-95 GFP (not shown) and endogenous PSD-95 (Figure 2) transiently colocalize with the M6PR during the first 2 d in culture. A similar perinuclear localization is observed for NMDA receptor subunits NR1 and NR2B (Figure 2). By contrast, the microtubule-associated protein, MAP2, occurs in developing neurites and is not concentrated in the perinuclear domain. As the neurons mature, PSD-95 becomes less concentrated in this perinuclear domain and begins to accumulate in small clusters along the processes (Figure 2).
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Accumulation of PSD-95 at the Perinuclear Domain Is Brefeldin A- and Nocodazole-sensitive
To help to determine whether sorting of PSD-95 to the perinuclear domain requires active vesicular trafficking, we treated PSD-95expressing heterologous cells with BFA. BFA has multiple and diverse effects on vesicular trafficking in cells including disassembly of the Golgi complex (
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PSD-95 GFP Is Trafficked through Pleomorphic Vesiculotubular Structures
We used video microscopy to monitor the dynamics of PSD-95-GFP trafficking in live COS cells starting 5 h after transfection. During the first hour, vesicles and tubular structures are constantly forming and moving towards the plasma membrane as well as to the perinuclear region (see video 1). 13 hours later, accumulation of PSD-95 GFP in the perinuclear region becomes obvious and the accumulated PSD-95 GFP persists for the remainder of the imaging period (videos 1 and 2). Pleomorphic tubular structures, rather than small vesicles, are the primary vehicles for the transport of PSD-95 GFP. Figure 3 D shows examples of the various vesicular and tubular structures containing PSD-95 GFP observed 6 h after transfection. These structures undergo dynamic shape changes as they move along tracks (possibly microtubules) to the cell periphery and to the perinuclear region. Some of these vesicular structures fuse with the plasma membrane. We also observed that tubular elements containing PSD-95 GFP typically undergo dramatic shape changes during their movement. These transport intermediates often bifurcate and show elastic properties, including extension and retraction during movement. Moving tubules undergo continuous budding, translocation, and fusion with other transport intermediates carrying PSD-95 GFP to the plasma membrane or to the perinuclear region. These properties are similar to the trafficking of Golgi-derived vesicular transport intermediates for transmembrane proteins (
PSD-95 Localizes to Intracellular Membranes and to the PSD in Cortical Neurons
To determine whether PSD-95 also associates with membranous intracellular structures of neurons within intact brain, we performed electron microscopic immunocytochemical staining of the rat cerebral cortex, using a well characterized antiPSD-95 antiserum (
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Vesicular Sorting of PSD-95 Requires a Short NH2-terminal Palmitoylated Motif
We next determined the region(s) of PSD-95 sufficient for its association with the perinuclear vesicular domain. We first transfected COS cells with a series of progressively larger deletion constructs encoding PSD-95 fused to GFP. This deletion analysis showed that a construct containing the first nine amino acids of PSD-95 fused to GFP occurs diffusely when expressed in COS cells, whereas constructs containing the first 13, 26, 46, or 64 amino acids all transiently accumulate at the perinuclear domain, just like full-length PSD-95 (Figure 3 B).
To determine whether association of PSD-95 with perinuclear vesicles requires NH2-terminal palmitoylation, we evaluated the distribution of palmitoylation-deficient mutants. Mutation of cysteine 3 or 5 of PSD-95 to serine prevents palmitoylation (
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Palmitoylation of PSD-95 Is Determined by a Hydrophobic Consensus Sequence
To analyze further palmitoylation of PSD-95, we determined the structural requirements for this protein lipidation. As shown in Figure 5 B, constructs containing at least the first 13 amino acids of PSD-95 are efficiently palmitoylated, whereas a construct containing only the first 9 amino acids is not detectably palmitoylated. These data indicate that the palmitoyltransferase that modifies PSD-95 recognizes a short sequence confined to the NH2 terminus. This short consensus need not occur at the extreme NH2 terminus, as insertion of 6 amino acids (VSKSGS) between the starter methionine and the palmitoylated cysteines does not influence the efficacy of palmitoylation (Figure 5 B).
We further dissected the sequence requirements within the first 13 amino acids. Mutating amino acids 1013 partially attenuates palmitoylation efficiency whereas mutating amino acids 69 almost completely abolishes palmitoylation (Figure 5B and Figure D). We next generated a series of constructs in which we individually mutated amino acids 29 of PSD-95 to alanine and evaluated [3H]palmitate labeling in transfected cells. Changing amino acids 4-Leu, 6-Ile or 7-Val to alanine significantly reduces or eliminates palmitoylation, but mutations of 2-Asp, 8-Thr, or 9-Thr has no effect (Figure 5 C, and data not shown). These data indicate that mutations of the hydrophobic amino acids disrupt palmitoylation, whereas mutations of hydrophilic amino acids do not. To investigate this further, we individually mutated amino acids 6-Ile and 7-Val to either serine, a hydrophilic amino acid, or to leucine, a hydrophobic amino acid. Both serine mutants are weakly palmitoylated (Figure 5 C) whereas the leucine mutants are robustly palmitoylated (Figure 5 C). Mutation of amino acid 4 from leucine to either aspartate or serine also disrupts palmitoylation, whereas mutation to the less hydrophilic amino acid tyrosine partially preserves palmitoylation (Figure 5 C).
Previous studies showed that mutating cysteine 3 or 5 to serine completely disrupts palmitoylation (Ser mutants are not palmitoylated might now be explained by a requirement of the palmitoyltransferase for a hydrophobic consensus. This interpretation would predict that mutating cysteine 3 or 5 (which themselves are hydrophobic) to another hydrophobic amino acid would preserve palmitoylation of the remaining cysteine. Indeed, we found that Cys3Leu and Cys5Leu mutants show residual palmitoylation that amount to ~20 and ~40% of the wild-type level, respectively (Figure 5 D). Taken together, these data imply that palmitoylation of PSD-95 is mediated by a short NH2-terminal consensus sequence that critically relies on five consecutive hydrophobic amino acids (Cys-Leu-Cys-Ile-Val).
To determine whether a similar hydrophobic consensus might determine NH2-terminal palmitoylation of other proteins, we evaluated GAP-43, whose site of palmitoylation also contains five consecutive hydrophobic amino acids (Met-Leu-Cys-Cys-Met) (
To evaluate further the role of palmitoylation in vesicular sorting of PSD-95, we monitored the trafficking of each NH2-terminal mutant of PSD-95 in transfected COS cells. We found that all mutations that dramatically reduce protein palmitoylation (Cys3,5Ser, Cys3Ser, Cys5Ser, Leu4Ser, Leu4Asp, and Ile6Ser, Val7Ser) also block accumulation of PSD-95 at the perinuclear compartment (Figure 3 C). Conversely, PSD-95 occurs normally at the perinuclear domain in transfections with mutants that preserve palmitoylation (Asp2Ala, Ile6Leu, Val7Leu, Thr8Ala, and Thr8,9Ala) (Figure 3 C). For mutants that have reduced efficiency of palmitoylation (Leu4Tyr, Leu4Ala, Cys3Leu, Cys5Leu, Ile6Ala, Val7Ala, and Val7Ser), the density of PSD-95 at the perinuclear domain decreases but remains apparent above the diffuse level in the cytosol (Figure 3 C).
We also examined the trafficking of a mutant form of PSD-95, Ile6Ser in live cells. This single amino acid change in PSD-95 dramatically reduces its palmitoylation and prevents its vesicular sorting. Imaging of live cells expressing this point mutant form of PSD-95 fails to show formation of any vesicular structures (video 4). Instead, diffuse cytoplasmic GFP signal is seen throughout the recording period.
Basolateral Sorting of PSD-95 in Polarized Epithelial Cells Requires Dual Palmitoylation
The cellular pathways for dendritic and for postsynaptic sorting of certain neuronal transmembrane proteins share features with protein targeting to the basolateral membrane of polarized epithelial cells (
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To determine the domains of PSD-95 that, together with the palmitoylated NH2 terminus, mediate basolateral localization, two stably transfected MDCK cell lines were generated that express increasingly longer NH2-terminal constructs of PSD-95. The first line, containing the NH2 terminus and the first PDZ domain (1-PDZ1) occurs diffusely in the cells whereas the second line, containing the NH2 terminus and the 3 PDZ domains (1-PDZ3) is polarized to the basolateral surface, just like full-length PSD-95 (Figure 6 B). These experiments indicate that basolateral membrane targeting requires not only the palmitoylated NH2 terminus but also the PDZ domains. On the other hand, the SH3 and guanylate kinase regions are not required. It is important to note that, in sub-confluent cultures, palmitoylated PSD-95 constructs colocalize with M6PR positive vesicles whereas mutants that disrupt palmitoylation are expressed diffusely in nonconfluent MDCK cells (data not shown). Taken together, these data indicate that basolateral membrane targeting of PSD-95 requires both palmitoylation and PDZ domain interactions.
Postsynaptic Targeting of PSD-95 Requires Dual Palmitoylation
To evaluate the role of dual palmitoylation in postsynaptic sorting of PSD-95, we evaluated targeting of a series of NH2-terminal mutants in primary hippocampal neurons (at DIV 12). We found that dually palmitoylated constructs (wild-type, Thr8,9Ala, Ile6Leu and Val7Leu) all target appropriately to postsynaptic dendritic clusters (Figure 7 A). By contrast, all nonpalmitoylated constructs occur diffusely in dendrites of transfected neurons (Cys3Ser, Leu4Ser, and Ile6Ser). Constructs that show reduced palmitoylation (Leu4Tyr, Leu4Ala, Cys3Leu, Cys5Leu, Ile6Ala, Val7Ala, and Val7Ser) are partially clustered in neurons (Figure 7 A). The lack of proper sorting of the Cys3Leu and Cys5Leu constructs, which are singly palmitoylated, suggests that there is a fundamental difference between single and dual palmitoylation and that postsynaptic targeting requires dual palmitoylation. Furthermore, adding the NH2-terminal dually palmitoylated motif of GAP-43 or the COOH-terminal prenylated and dually palmitoylated motif of paralemmin to PSD-95(C3,5S) significantly restore postsynaptic targeting. With the GAP-43 motif and the paralemmin motif PSD-95 clusters form in 46 and 81% of neurons, whereas only 22% of cells show clusters without these motifs (Figure 7 B).
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Ion Channel Clustering by PSD-95 Requires Dual Palmitoylation
Certain aspects of channel clustering by MAGUK proteins can be reproduced in a coclustering experiment in heterologous cells (
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To define the role for dual palmitoylation in ion channel clustering we analyzed a series of PSD-95 mutants. We found that mutants of PSD-95 that disrupt palmitoylation, including L4S, I6S, C3S, and C35S, do not form coclustered membrane patches when coexpressed with Kv1.4 (Figure 8 A, top and lower right panels and data not shown). In contrast, mutants of PSD-95 that do not disrupt palmitoylation of PSD-95 including I6L and V7L coclustered with Kv1.4 (Figure 8 A, and data not shown). Furthermore, the NH2-terminal palmitoylated motif of GAP-43 or the COOH-terminal palmitoylated motif of paralemmin can functionally substitute for the PSD-95 NH2 terminus to mediate ion channel clustering (Figure 8 B).
Finally, minimal constructs of PSD-95 were designed to define the absolute requirements for ion channel clustering. Kv1.4 clustering is induced by the expression of a construct containing only the first 26 amino acids of PSD-95 fused to PDZ domains 1 and 2. In addition, a construct containing GAP-43, fused to only PDZ domains 1&2 of PSD-95 is capable of clustering Kv1.4 (Figure 8 C). These experiments establish that ion channel clustering by PSD-95 requires (a) dual palmitoylation and (b) an appropriate PDZ domain.
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Discussion |
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This work demonstrates that dual palmitoylation of PSD-95 mediates a transient association with perinuclear vesicles and that this lipid modification is also essential for ion channel clustering and postsynaptic targeting. Dual palmitoylation of PSD-95 is determined by a consensus sequence of five consecutive NH2-terminal hydrophobic amino acids that include the two modified cysteines. Importantly, all mutations that disrupt dual palmitoylation block perinuclear trafficking, postsynaptic targeting, and ion channel clustering activity of PSD-95.
Synaptic Targeting and Ion Channel Clustering by PSD-95 Require Dual Palmitoylation
This work emphasizes the central role for NH2-terminal palmitoylation in protein sorting by PSD-95. Previous studies noted essential roles for cysteines 3 and 5 in both receptor clustering (
Five Consecutive Hydrophobic Amino Acids Specifies Palmitoylation of PSD-95
Our detailed mutagenesis of the NH2 terminus of PSD-95 demonstrates that a sequence of five consecutive hydrophobic amino acids (containing the two modified cysteines) is essential for palmitoylation. Conservative mutations of any of these five amino acids (including the cysteines) to other hydrophobic amino acids preserve palmitoylation whereas mutations to a hydrophilic amino acid prevent palmitoylation. Interestingly, palmitoylation of GAP-43 is also specified by five consecutive NH2-terminal hydrophobic amino acids that otherwise shares no similarity with the sequence of PSD-95.
Despite the central role for palmitoylation in diverse cellular processes, the identity of the putative palmitoyltransferase enzyme remains mysterious. Our identification of a strict consensus sequence in PSD-95 and GAP-43 for palmitoylation strongly suggests that modifications of these proteins are enzyme-mediated. Similar to what is found for protein kinases, the PAT appears to recognize a short consensus sequence. Because PSD-95 is such a potent substrate for PAT, the NH2 terminus of PSD-95 may be a useful tool for biochemical characterization and isolation of this important and elusive enzyme.
Comparison of Postsynaptic Clustering with Basolateral Membrane Sorting
That postsynaptic clustering in neurons and basolateral membrane targeting in epithelial cells both require palmitoylation of PSD-95 suggests interesting parallels between these pathways. Previous analyses of trafficking of transmembrane proteins in epithelial cells have identified critical protein and lipid modifications that mediate polarized protein sorting. Basolateral sorting of several transmembrane proteins is determined by short cytoplasmic tyrosine or dileucine based motifs (
Implications for Synaptic Plasticity Regulated by MAGUK Proteins
Mice with mutant PSD-95 protein have a dramatic shift in NMDA receptor-dependent plasticity in hippocampus such that long-term depression (LTD) is blocked and long-term potentiation (LTP) is enhanced (
How PSD-95 assembles these signaling complexes, however, remains uncertain. Previous models, based on the assumption that PSD-95 and other MAGUKs are static elements of the postsynaptic cytoskeleton suggested a passive role for these proteins in simply retaining receptor clusters and associated signaling enzymes at the synapse (
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: BFA, brefeldin A; dlg, discs large; GAP-43, growth associated protein-43; GFP, green fluorescent protein; MAGUK, membrane-associated guanylate kinase; NMDA, N-methyl-D-aspartate; PDZ, postsynaptic density-95, discs large, zonula occludens; PSD, postsynaptic density.
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Acknowledgements |
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We thank Alice Elste for excellent technical assistance with electron microscopy, Yorum Altschuler for help with confocal microscopy and Robert Edwards and Keith Mostov for critically reviewing the manuscript.
This work was supported by a pre-doctoral research grant from the National Science Foundation and an ARCS Foundation scholarship (to S.E. Craven), and postdoctoral grants from the Medical Research Council of Canada (to A.E. El-Husseini), the National Institute of Child Health and Development and Spinal Cord Research Foundation (to B.L. Firestein), and the Howard Hughes Medical Institute (to D.M. Chetkovich). This research was also supported by grants (to C. Aoki) from the National Institutes of Health (R01-EY08055), NSF (RCD 92-53750), NYU Research Challenge Fund, and Human Frontier's Science Program Award and by grants (to D.S. Bredt) from the National Institutes of Health (R01-NS36017), National Science Foundation, the National Association for Research on Schizophrenia and Depression, the EJLB, and the Culpeper and Beckman Foundations.
Submitted: 6 October 1999
Revised: 29 November 1999
Accepted: 1 December 1999
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References |
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