Abstract
Dendritic spines have two major structural elements: postsynaptic densities (PSDs) and actin cytoskeletons. PSD proteins are proposed to regulate spine morphogenesis. However, other molecular mechanisms should govern spine morphogenesis, because the initiation of spine morphogenesis precedes the synaptic clustering of these proteins. Here, we show that synaptic clustering of drebrin, an actin-binding protein highly enriched in dendritic spines, governs spine morphogenesis. We immunocytochemically analyzed developing hippocampal neurons of low-density cultures. Filopodia-like dendritic protrusions were classified into two types: diffuse-type filopodia, which have diffuse distribution of drebrin, and cluster-type filopodia, which have drebrin clusters with filamentous actin (F-actin). Most cluster-type filopodia were synaptic filopodia. Postsynaptic drebrin clusters were found in both most synaptic filopodia and spines. Postsynaptic PSD-95 clusters, however, were found in only one-half of synaptic filopodia but in most spines. These data indicate that cluster-type filopodia are not mature spines but their precursors. Suppression of the upregulation of drebrin adult isoform (drebrin A) by antisense oligonucleotides against it attenuated synaptic clustering of PSD-95, as well as clustering of drebrin and F-actin. Furthermore, the restoration of drebrin A expression by injection of the expression vectors of drebrin A tagged with green fluorescent protein into the neurons treated with the antisense oligonucleotides induced synaptic reclustering of PSD-95 on clusters of the labeled drebrin A. These data indicated that the synaptic clustering of drebrin is necessary for that of PSD-95 in developing neurons. Together, these data suggest that synaptic clustering of drebrin is an essential step for spine morphogenesis.
