We found that Sept7-GFP is part of a stable, membrane-aligned structure that becomes localized with high fidelity to dendritic spine necks during development

We found that Sept7-GFP is part of a stable, membrane-aligned structure that becomes localized with high fidelity to dendritic spine necks during development. some forms of synaptic plasticity. Diffusion also contributes to the turnover of desensitized receptors during synaptic transmission[1],[2]and the redistribution of components during the formation and breakdown of synapses[3][5]. Most excitatory synapses in mature neurons are located on small protrusions from the dendrite shaft termed spines. Typically the Lifitegrast bulbous spine head bearing the postsynaptic density is separated from the dendrite shaft by an elongated neck. This neck is thought to impede the flow of solute and membrane molecules from the dendrite into the spine head and vice-versa. However, while neck width and length seem to play a role in modulating molecular exchange[3],[6],[7], the mechanism for this restriction is not clear. Recently, a novel protein complex was described that localizes to dendritic branching points and spine necks and is required for dendritic arborization[8],[9]. This complex is comprised of at least three members of the septin family of GTPases, namely septin 5, septin 7 and septin 11[9], and biochemically associates with membranes[8]. The crystal structure of a mammalian septin complex has been solved[10], in which hetero-hexameric complexes assemble end-over-end into filaments Lifitegrast and rings. Such structures have been reported for the evolutionarily conserved septins in many systems, most prominently for budding yeast, where a septin ring surrounding the cleavage furrow forms a diffusion barrier between the two separating cells[11],[12]. Septin-dependent membrane partitioning seems to be ubiquitous in biology[13], raising the possibility that septins may form a diffusion barrier at the spine neck as well[14]. Here we investigated the localization, structure and stability of the spine-localized septin complex in cultured hippocampal neurons by fluorescence and electron microscopy and asked whether the presence of Sept7 at spine necks influences membrane protein mobility across the spine neck. In particular, we tested if the presence of septin at the spine neck alters the diffusion of AMPA-type glutamate receptors (AMPARs), the diffusion of which into and out of synapses regulates the strength of synaptic transmission and short-term synaptic plasticity[1]. We found that Sept7-GFP is part of a stable, membrane-aligned structure that becomes localized with high fidelity to dendritic spine necks during development. Single particle tracking showed that GluA2 subunit-containing AMPARs move slower and dwell longer in Sept7-positive spines. Furthermore, in IL1R2 antibody fluorescence recovery after photobleaching (FRAP) experiments, transmembrane molecules and molecules attached to the inner membrane leaflet were restricted in their flow into Sept7-positive spines, while outer leaflet associated GPI-anchored molecules and soluble molecules were not detectably restricted. Finally, we found that RNAi-mediated reduction of Sept7 expression led to increased spine surface exploration of transmembrane proteins. We conclude that Sept7 at dendritic spine necks restricts membrane protein but not cytoplasmic flow across spine necks. == Results == Sept7-containing complexes localize to dendritic branching points and to the neck of dendritic filopodia and spines[8],[9]To further examine the generation of this pattern during Lifitegrast the morphological development of neurons, we expressed Sept7-GFP in cultured neurons at different time points (Fig. 1a). Expression of Sept7-GFP resulted in a characteristic pattern of discrete spots that exhibited the same distribution Lifitegrast as immunofluorescence staining of endogenous Sept7 (Fig. 1a, bottom row), suggesting that Sept7-GFP localizes and assembles into discrete structures that are indistinguishable from endogenous Sept7. == Figure 1. A membrane-aligned, stable Sept-positive structure locates to dendritic spine necks. == (a) Fluorescence images of fixed primary hippocampal neurons at various days Lifitegrast in vitro (DIV) expressing Sept7-GFP and mRFP for two days (+2). Scale bars are 20 m. Below are magnified regions to clarify the localization of Sept7-GFP (first row) and immunostained endogenous Sept7 (second row) respectively, these are also shown as single channel mages at the bottom to emphasize the discrete localization of septins at the base of protrusions. Scale bars are 5 m. (b) Quantification of.