Current Biology, Vol. 13, 510–515, March 18, 2003

Dynamin 3 Is a Component of the Postsynapse, Where it Interacts with mGluR5 and Homer

The dynamins comprise a large family of mechanoenzymes known to participate in membrane modeling events [1, 2]. All three conventional dynamin genes (Dyn1, Dyn2, Dyn3) are expressed in mammalian brain time and produce more than 27 different dynamin as a result of alternative splicing [3]. Past studies have suggested that Dyn1 participates in specialized neuronal functions such as rapid synaptic vesicle recycling [4], while Dyn2 may mediate the conventional clathrin-mediated uptake of surface receptors [5]. Currently, the distribution, expression, and function of Dyn3 in neurons, or in any other cell type, are completely undefined. Here, we demonstrate that Dyn1 and Dyn3 localize differentially in the synapse. Dyn1 concentrates within the presynaptic compartment, while Dyn3 localizes to dendritic spine tips. Within the postsynaptic density (PSD), we found Dyn3, but not Dyn1, to be part of a biochemically isolated complex comprised of Homer and metabotropic glutamate receptors. Finally, although dominant-negative Dyn3 did not seem to inhibit receptor endocytosis, overexpression of a specific Dyn3 spliced variant in mature neurons caused a marked remodeling of dendritic spines. These data suggest that Dyn3 is a postsynaptic dynamin and, like its binding partner Homer, plays a significant role in dendritic spine morphogenesis and remodeling.

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Figure 1. Dyn1 Is Enriched in Synaptic Boutons (A and A ) (A) Dyn1 antibodies stained the axons and axonal varicosities of mature neurons (see arrows). (A ) These varicosities were shown to be synaptic boutons, as demonstrated by colabeling with synaptotagmin (red, see arrows). (B and B ) Neurons transfected with Dyn1aa-GFP were labeled with antibodies against synapsin (red) and the dendrite-specific marker MAP2b (blue). (B) Dyn1aa-GFP trafficked into the axons and was sequestered in axonal varicosities. (B ) The addition of the synapsin channel confirmed that these varicosities were synaptic boutons, for the Dyn1aa-GFP and synapsin both label the same structures (see arrows). (C) Neurons transfected with Dyn1aa-GFP were challenged to take up FM4-64 in response to a depolarizing stimulus. Arrows indicate Dyn1aa- GFP-positive boutons that are also labeled with the dye (red). (D) Functional Dyn1 is necessary for synaptic vesicle recycling, for neurons transfected with a GTPase-deficient Dyn1 (Dyn1aaKA-GFP) were unable to take up FM4-64 following depolarization. Arrows indicate mutant Dyn1-positive boutons that are not labeled with the dye. The dye was actively endocytosed by surrounding nontransfected axons. The scale bar represents 5 m for (A)–(D). (E and E ) Two examples of Dyn1 antibody labeling in synaptosomes reveal a presynaptic localization for Dyn1 at the ultrastructural level. The white arrows indicate clusters of Dyn1 immunolabeling. Mit, mitochondria; DC, dense-core granule; PSD, postsynaptic density. The scale bar represents 100 nm.

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Figure 2. Dyn3 Resides at the Tips of Dendritic Spines (A) Neurons were colabeled with antibodies against Dyn3 (green) and MAP2b (red). Dyn3 puncta were observed along the dendrites but were adjacent to the dendritic shaft (arrows). (B) Neurons were triple labeled with Dyn3 (green) and MAP2b (blue) antibodies and rhodamine-phalloidin (red) to reveal Dyn3 puncta at the tips of actin-rich dendritic spines (arrows). (C) To confirm this postsynaptic localization, neurons were transfected with Dyn3aaa-GFP and were labeled for PSD-95 (red). The Dyn3aaa- GFP puncta labeled the tips of spines and colocalized with PSD-95 (arrows). (D) Neurons were transfected with Dyn3aaa-GFP and were colabeled for MAP2b (blue) and synapsin (red). The Dyn3 signal was distinct from the presynaptic marker synapsin (arrows). The scale bar represents 5 m for (A)–(D). (E and E ) Two examples of Dyn3 antibody labeling in synaptosomes reveal a predominantly postsynaptic localization at the ultrastructural level. The white arrows indicate clusters of Dyn3 immunolabeling. SVs, synaptic vesicles; DC, dense-core granule; PSD, postsynaptic density. The scale bar represents 100 nm.

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Figure 4. Dyn3 Does Not Mediate mGluR5 Internalization in Neurons, but Spliced Variants Regulate Dendritic Spine Morphogenesis (A–B ) Mature hippocampal neurons transfected with both (A) Dyn3aaa-GFP and (A ) mGluR5a-myc exhibit robust internalization of mGluR5 in a constitutive manner, similar to those neurons expressing both a (B) GTPase-deficient Dyn3 (Dyn3aaa(K44A)-GFP) and (B ) mGluR5a-myc. There was no difference in the amount of internalized mGluR5 puncta in the mutant Dyn3-expressing cells, and this finding suggests that Dyn3 may not play a role in mGluR5 receptor endocytosis. The scale bar represents 10 m. (C) Image analysis of transfected cells revealed no difference in the number of internalized puncta per cell area in Dyn3aaa, Dyn2aa, or Dyn1aa mutant-expressing cells. The error bars represent SEM. (D) Dyn3aaa-GFP does not have any effect on the morphogenesis of dendritic spines when expressed in neurons. (E) Although only differing by ten amino acids, expression of the Dyn3 spliced variant Dyn3baa-GFP in mature neurons results in an extensive increase in filopodial growth and a concomitant reduction in mushroom-shaped dendritic spines. The scale bar represents 10 um.