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1.
PLoS One ; 6(3): e16998, 2011 Mar 07.
Article in English | MEDLINE | ID: mdl-21408225

ABSTRACT

Dendritic filopodia are dynamic protrusions that are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between filopodia formation and synaptogenesis. We investigated the role of filopodia in synapse formation by contrasting the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, increased the rate at which filopodia transformed into spines by stabilization of the initial contact with axons. Taken together, these results suggest that increased filopodia stability and not density, may be the rate-limiting step for synapse formation.


Subject(s)
Axons/metabolism , Dendrites/metabolism , Nerve Tissue Proteins/metabolism , Pseudopodia/metabolism , Amino Acid Motifs , Animals , Models, Biological , Nerve Tissue Proteins/chemistry , Rats , Synapses/metabolism
2.
Mol Biol Cell ; 19(5): 2026-38, 2008 May.
Article in English | MEDLINE | ID: mdl-18287537

ABSTRACT

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.


Subject(s)
Dendritic Spines/metabolism , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Pseudopodia/metabolism , Alternative Splicing/genetics , Animals , COS Cells , Cell Membrane/metabolism , Chlorocebus aethiops , Lipoylation , Mice , Protein Transport , Rats , Receptors, AMPA/metabolism , Time Factors
4.
Neuron ; 44(6): 977-86, 2004 Dec 16.
Article in English | MEDLINE | ID: mdl-15603740

ABSTRACT

In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.


Subject(s)
Carnitine O-Palmitoyltransferase/physiology , Carrier Proteins/physiology , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/physiology , Palmitic Acid/metabolism , Acyltransferases , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , COS Cells , Carnitine O-Palmitoyltransferase/genetics , Carnitine O-Palmitoyltransferase/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cells, Cultured , Chlorocebus aethiops , Humans , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Protein Transport/physiology , Substrate Specificity
5.
Mol Biol Cell ; 15(5): 2205-17, 2004 May.
Article in English | MEDLINE | ID: mdl-14978216

ABSTRACT

Although neuronal axons and dendrites with their associated filopodia and spines exhibit a profound cell polarity, the mechanism by which they develop is largely unknown. Here, we demonstrate that specific palmitoylated protein motifs, characterized by two adjacent cysteines and nearby basic residues, are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and the branching of dendrites and axons in neurons. Such motifs are present at the N-terminus of GAP-43 and the C-terminus of paralemmin, two neuronal proteins implicated in cytoskeletal organization and filopodial outgrowth. Filopodia induction is blocked by mutations of the palmitoylated sites or by treatment with 2-bromopalmitate, an agent that inhibits protein palmitoylation. Moreover, overexpression of a constitutively active form of ARF6, a GTPase that regulates membrane cycling and dendritic branching reversed the effects of the acylated protein motifs. Filopodia induction by the specific palmitoylated motifs was also reduced upon overexpression of a dominant negative form of the GTPase cdc42. These results demonstrate that select dually lipidated protein motifs trigger changes in the development and growth of neuronal processes.


Subject(s)
Dendrites/ultrastructure , Hippocampus/cytology , Nerve Tissue Proteins/chemistry , Neurons/cytology , Pseudopodia/ultrastructure , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/physiology , Acylation , Amino Acid Motifs/genetics , Amino Acid Motifs/physiology , Amino Acid Sequence , Amino Acids, Basic/genetics , Animals , COS Cells , Chlorocebus aethiops , Cysteine/genetics , Dendrites/physiology , GAP-43 Protein/genetics , GAP-43 Protein/physiology , Membrane Proteins/genetics , Membrane Proteins/physiology , Molecular Sequence Data , Mutagenesis , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Neurons/physiology , Palmitates/pharmacology , Phosphoproteins , Pseudopodia/drug effects , Pseudopodia/physiology , Rats , Sequence Alignment , Transfection , cdc42 GTP-Binding Protein/physiology
6.
Cell ; 108(6): 849-63, 2002 Mar 22.
Article in English | MEDLINE | ID: mdl-11955437

ABSTRACT

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.


Subject(s)
Nerve Tissue Proteins/metabolism , Palmitates/metabolism , Synapses/metabolism , Animals , Calcium Channels/genetics , Calcium Channels/metabolism , Cell Membrane/metabolism , Cells, Cultured , Cerebral Cortex/cytology , Disks Large Homolog 4 Protein , Green Fluorescent Proteins , Hippocampus/cytology , Hypoglycemic Agents/pharmacology , Indicators and Reagents/metabolism , Intracellular Signaling Peptides and Proteins , Luminescent Proteins/genetics , Membrane Proteins , Nerve Tissue Proteins/genetics , Neuronal Plasticity/physiology , Neurons/cytology , Neurons/physiology , Palmitates/pharmacology , Patch-Clamp Techniques , Rats , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Synaptic Transmission/drug effects , Synaptic Transmission/physiology
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