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A Septin-Dependent Diffusion Barrier at Dendritic Spine Necks.

Ewers H, Tada T, Petersen JD, Racz B, Sheng M, Choquet D - PLoS ONE (2014)

Bottom Line: We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane.We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck.Our findings indicate that Sept7 regulates membrane protein access to spines.

View Article: PubMed Central - PubMed

Affiliation: Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France.

ABSTRACT
Excitatory glutamatergic synapses at dendritic spines exchange and modulate their receptor content via lateral membrane diffusion. Several studies have shown that the thin spine neck impedes the access of membrane and solute molecules to the spine head. However, it is unclear whether the spine neck geometry alone restricts access to dendritic spines or if a physical barrier to the diffusion of molecules exists. Here, we investigated whether a complex of septin cytoskeletal GTPases localized at the base of the spine neck regulates diffusion across the spine neck. We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane. We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck. Finally, when Sept7 expression was suppressed by RNA interference, membrane molecules explored larger membrane areas. Our findings indicate that Sept7 regulates membrane protein access to spines.

No MeSH data available.


Related in: MedlinePlus

A membrane-aligned, stable Sept-positive structure locates to dendritic spine necks.(a) Fluorescence images of fixed primary hippocampal neurons at various days in vitro (DIV) expressing Sept7-GFP and mRFP for two days (+2). Scale bars are 20 µm. 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 µm. (b) Quantification of experiments as shown in (a). Over time, more protrusions bear Sept7-GFP at their neck (left) and Sept7-GFP spots are more likely to be localized to protrusion necks (right). Histograms show mean ± SEM; **p<0.01, in One-way ANOVA. n>30 neurons in >3 independent experiments. (c) Confocal images of fixed hippocampal neurons at DIV 21 after 2 weeks of Sept7-GFP (pseudocolored red) and mRFP (pseudocolored green) expression. Sept7-GFP staining is concentrated in arc-like structures aligning spine necks. (d) Quantification of fluorescence recovery after photobleaching experiments on Sept7-GFP complexes in DIV 7 neurons. Shown is the average recovery measured of a total of 345 spotlike Sept7-GFP structures in 31 cells in 4 cultures with SEM.
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pone-0113916-g001: A membrane-aligned, stable Sept-positive structure locates to dendritic spine necks.(a) Fluorescence images of fixed primary hippocampal neurons at various days in vitro (DIV) expressing Sept7-GFP and mRFP for two days (+2). Scale bars are 20 µm. 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 µm. (b) Quantification of experiments as shown in (a). Over time, more protrusions bear Sept7-GFP at their neck (left) and Sept7-GFP spots are more likely to be localized to protrusion necks (right). Histograms show mean ± SEM; **p<0.01, in One-way ANOVA. n>30 neurons in >3 independent experiments. (c) Confocal images of fixed hippocampal neurons at DIV 21 after 2 weeks of Sept7-GFP (pseudocolored red) and mRFP (pseudocolored green) expression. Sept7-GFP staining is concentrated in arc-like structures aligning spine necks. (d) Quantification of fluorescence recovery after photobleaching experiments on Sept7-GFP complexes in DIV 7 neurons. Shown is the average recovery measured of a total of 345 spotlike Sept7-GFP structures in 31 cells in 4 cultures with SEM.

Mentions: 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 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 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.


A Septin-Dependent Diffusion Barrier at Dendritic Spine Necks.

Ewers H, Tada T, Petersen JD, Racz B, Sheng M, Choquet D - PLoS ONE (2014)

A membrane-aligned, stable Sept-positive structure locates to dendritic spine necks.(a) Fluorescence images of fixed primary hippocampal neurons at various days in vitro (DIV) expressing Sept7-GFP and mRFP for two days (+2). Scale bars are 20 µm. 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 µm. (b) Quantification of experiments as shown in (a). Over time, more protrusions bear Sept7-GFP at their neck (left) and Sept7-GFP spots are more likely to be localized to protrusion necks (right). Histograms show mean ± SEM; **p<0.01, in One-way ANOVA. n>30 neurons in >3 independent experiments. (c) Confocal images of fixed hippocampal neurons at DIV 21 after 2 weeks of Sept7-GFP (pseudocolored red) and mRFP (pseudocolored green) expression. Sept7-GFP staining is concentrated in arc-like structures aligning spine necks. (d) Quantification of fluorescence recovery after photobleaching experiments on Sept7-GFP complexes in DIV 7 neurons. Shown is the average recovery measured of a total of 345 spotlike Sept7-GFP structures in 31 cells in 4 cultures with SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4262254&req=5

pone-0113916-g001: A membrane-aligned, stable Sept-positive structure locates to dendritic spine necks.(a) Fluorescence images of fixed primary hippocampal neurons at various days in vitro (DIV) expressing Sept7-GFP and mRFP for two days (+2). Scale bars are 20 µm. 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 µm. (b) Quantification of experiments as shown in (a). Over time, more protrusions bear Sept7-GFP at their neck (left) and Sept7-GFP spots are more likely to be localized to protrusion necks (right). Histograms show mean ± SEM; **p<0.01, in One-way ANOVA. n>30 neurons in >3 independent experiments. (c) Confocal images of fixed hippocampal neurons at DIV 21 after 2 weeks of Sept7-GFP (pseudocolored red) and mRFP (pseudocolored green) expression. Sept7-GFP staining is concentrated in arc-like structures aligning spine necks. (d) Quantification of fluorescence recovery after photobleaching experiments on Sept7-GFP complexes in DIV 7 neurons. Shown is the average recovery measured of a total of 345 spotlike Sept7-GFP structures in 31 cells in 4 cultures with SEM.
Mentions: 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 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 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.

Bottom Line: We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane.We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck.Our findings indicate that Sept7 regulates membrane protein access to spines.

View Article: PubMed Central - PubMed

Affiliation: Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France.

ABSTRACT
Excitatory glutamatergic synapses at dendritic spines exchange and modulate their receptor content via lateral membrane diffusion. Several studies have shown that the thin spine neck impedes the access of membrane and solute molecules to the spine head. However, it is unclear whether the spine neck geometry alone restricts access to dendritic spines or if a physical barrier to the diffusion of molecules exists. Here, we investigated whether a complex of septin cytoskeletal GTPases localized at the base of the spine neck regulates diffusion across the spine neck. We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane. We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck. Finally, when Sept7 expression was suppressed by RNA interference, membrane molecules explored larger membrane areas. Our findings indicate that Sept7 regulates membrane protein access to spines.

No MeSH data available.


Related in: MedlinePlus