β-Pix modulates actin-mediated recruitment of synaptic vesicles to synapses

Abstract

Presynaptic compartments are formed through the recruitment of preassembled clusters of proteins to points of cell-cell contact, however, the molecular mechanism(s) underlying this process remains unclear. We demonstrate that clusters of polymerized actin can recruit and maintain synaptic vesicles to discrete sites along the axon, and that cadherin/β-catenin/scribble/β-pix complexes play an important role in this event. Previous work has demonstrated that β-catenin and scribble are important for the clustering of vesicles at synapses. We demonstrate that β-pix, a Rac/Cdc42 guanine nucleotide exchange factor (GEF), forms a complex with cadherin, β-catenin, and scribble at synapses and enhances localized actin polymerization in rat hippocampal neurons. In cells expressing β-pix siRNA or dominant-negative β-pix that lacks its GEF activity, actin polymerization at synapses is dramatically reduced, and synaptic vesicle localization is disrupted. This β-pix phenotype can be rescued by cortactin overexpression, suggesting that β-pix-mediated actin polymerization at synapses regulates vesicle localization.

Figure 1.

GFP-actin and Syn-RFP are localized to synapses. Confocal images of 10 DIV hippocampal neurons transfected with GFP-actin (A) or Syn-RFP (B) and immunolabeled with synaptic markers, PSD-95 and bassoon. Bassoon and PSD-95 immunopositive puncta that are not colocalized with GFP-actin or Syn-RFP may represent puncta on untransfected cells. Higher magnifications of the insets from A and B are shown in A' and B', respectively. GFP-actin and Syn-RFP were highly colocalized with PSD-95/bassoon (open arrowheads). Scale bars, 10 μm.

Figure 2.

The density and IntDen of GFP-actin and Syn-RFP clusters are increased following ALLN treatment. A, B, Confocal images of 8 DIV hippocampal neurons transfected with GFP-actin and Syn-RFP and immunolabeled with PSD-95. Neurons were treated either with 20 μm ALLN or with DMSO vehicle 24 h before fixation. Open arrowheads in A and B indicate synapses as defined by colocalized Syn-RFP/GFP-actin/PSD-95 clusters, whereas closed arrowheads indicate sites of colocalization between GFP-actin and Syn-RFP, but not PSD-95. ALLN treatment increased the density (A, B, open plus closed arrowheads; C) and IntDen (D) of GFP-actin clusters. C, D, The density and IntDen of Syn-RFP clusters that were associated with GFP-actin were specifically increased. Arrows indicate Syn-RFP clusters that were not associated with GFP-actin clusters. N = 23–33 cells per condition from 3 separate cultures. *p < 0.05, **p < 0.01; Student's t test. Scale bar, 10 μm. E, Synaptophysin protein levels were similar in control and ALLN-treated cells (α-tubulin was used as a loading control). N = 3 different blots from 3 separate cultures.

Figure 3.

The density and IntDen of GFP-actin, Syn-RFP, and UtrCH-RFP clusters are increased in neurons expressing cortactin. A, B, Confocal images of 8 DIV hippocampal neurons cotransfected with GFP-actin and Syn-RFP plus either empty vector or Cort-HA and immunolabeled for HA. GFP-actin, Syn-RFP, and Cort-HA were highly colocalized (B, open arrowheads). In neurons expressing Cort-HA, the density and IntDen of GFP-actin clusters and Syn-RFP clusters associated with GFP-actin clusters were increased (A, B, open arrowheads; C, G), whereas the density and IntDen of Syn-RFP clusters that were not associated with GFP-actin clusters were not significantly changed (A, B, closed arrowheads; C, G). N = 23–31 cells per condition from 3 separate cultures. D, The density of GFP-actin clusters associated with PSD-95 and gephyrin remained unchanged, whereas the number of GFF-actin clusters that were not colocalized with either marker was increased in cells expressing Cort-HA. N = 14–16 cells per condition from 2 separate cultures. E, F, Confocal images of 8 DIV hippocampal neurons cotransfected with GFP-actin and UtrCH-RFP plus either empty vector or Cort-HA and immunolabeled for HA. Utrophin-RFP clusters were completely colocalized with GFP-actin clusters (E, F, open arrowheads), and the IntDen of UtrCH-RFP clusters was significantly increased in cells expressing Cort-HA (H). N = 19 cells per condition from 3 separate cultures. H, The IntDen of UtrCH-RFP at synapses, defined by UtrCH-RFP clusters colocalized with PSD-95, was increased in cells expressing Cort-HA. N = 14–15 cells per condition from 2 separate cultures. I, J, Confocal images of 8 DIV hippocampal neurons cotransfected with Syn-GFP plus either empty vector or Cort-HA and immunolabeled for PSD-95 and HA. Open arrowheads indicate synapses, which are defined as sites of colocalization between Syn-GFP and PSD-95. A subset of Cort-HA clusters localized to synapses (J, open arrowheads). The IntDen of Syn-GFP puncta in these cells was significantly increased at synapses associated with Cort-HA clusters (+Cort-HA), but unaffected at synapses not associated with Cort-HA (−Cort-HA) (I, K). N = 19–24 cells per condition from 3 separate cultures. *p < 0.05; one-way ANOVA with Dunnett's post hoc. L, Time-lapse analysis demonstrated an increase in the density of stable Syn-RFP clusters in cells expressing Cort-HA. N = 16–17 cells per condition from 3 separate cultures. *p < 0.05, **p < 0.01, ***p < 0.001; Student's t test. Scale bars, 10 μm.

Figure 4.

β-Pix interacts with cadherin, β-catenin, and scribble at synapses. A, Confocal images of 10 DIV hippocampal cultures immunolabeled for β-pix and synaptic markers, PSD-95 and bassoon. β-Pix clusters are highly colocalized with PSD-95/bassoon in neurons. B, Synaptosomal fractions from adult brains were immunoprecipitated with antibodies against cadherin, β-pix, β-catenin, or scribble and separated by SDS-PAGE. β-Pix, scribble, β-catenin, and cadherin coimmunoprecipitated with one another, but not with the synaptic protein, synaptophysin. The input lane corresponds to the crude synaptosomal fraction, P2. Rabbit IgG was used as a control. N = 3 different blots from 2 separate preparations. C, D, Confocal images of 8 DIV hippocampal neurons transfected with GFP-β-pix plus either shRNA control or Scrib shRNAs and immunolabeled for PSD-95 and bassoon. The IntDen of synaptic GFP-β-pix clusters, defined by GFP-β-pix at sites of colocalization between PSD-95 and bassoon, was smaller in cells expressing Scrib shRNA (C, D, open arrowheads; E, N = 14–27 cells per condition from 3 separate cultures) or N-cadherin siRNA (F, N = 15–18 cells per condition from 2 separate cultures). *p < 0.05, **p < 0.01; one-way ANOVA with Tukey post hoc. ***p < 0.001; one-way ANOVA with Dunnett's post hoc. Scale bars, 10 μm.

Figure 5.

The IntDen of synaptic GFP-actin clusters is decreased in neurons with reduced β-pix activity. A, GFP-β-pix expression in HEK 293 cells is decreased in cells expressing β-pix siRNA. N = 2 different blots from 2 separate experiments. B, C, Confocal images of 8 DIV hippocampal neurons transfected with GFP-actin plus scrambled or β-pix siRNAs and immunolabeled for PSD-95 and bassoon. D, The density of GFP-actin clusters was unaltered in β-pix siRNAs expressing neurons. However, the IntDen of synaptic GFP-actin clusters (GFP-actin at sites of PSD-95 and bassoon colocalization) was decreased in cells expressing β-pix siRNAs (B, C, open arrowheads; E). N = 16–24 cells per condition from 2 separate cultures. *p < 0.05, ***p < 0.001; one-way ANOVA with Dunnett's post hoc. F, The IntDen of synaptic GFP-actin clusters was significantly attenuated in neurons expressing DN-β-pix. N = 11–15 cells per condition from 2 separate cultures. ***p < 0.001; one-way ANOVA with Tukey post hoc. G, H, Confocal images of 8 DIV hippocampal neurons transfected with UtrCH-RFP plus control or β-pix siRNA-1 and immunolabeled for PSD-95. I, The IntDen of synaptic UtrCH-RFP clusters (UtrCH-RFP clusters that were colocalized with PSD-95) was significantly decreased in neurons expressing β-pix siRNA-1. N = 12–16 cells per condition from 2 separate cultures. *p < 0.05; Student's t test. Scale bars, 10 μm.

Figure 6.

The localization of synaptic vesicles at synapses is disrupted in neurons with reduced β-pix activity. A–D, Confocal images of 9 DIV hippocampal neurons transfected with Syn-GFP plus the indicated β-pix construct and immunolabeled for PSD-95 and bassoon. In control neurons (A) and those expressing FL-β-pix (C), Syn-GFP exhibited a punctate distribution and colocalized with PSD-95 and bassoon (open arrowheads). In contrast, neurons expressing β-pix siRNA-1 (B) or DN-β-pix (D) exhibited a more diffuse pattern of Syn-GFP distribution, and the coverage of Syn-GFP (the sum of the length of Syn-GFP fluorescence signal per 10 μm axon length ± SE) was increased (B, D, E). A significant decrease in the IntDen of Syn-GFP fluorescence at synapses was observed in β-pix siRNA-expressing neurons and those expressing DN-β-pix (F). The area (G) and density (H) of PSD-95 and bassoon were similar in control and siRNA-expressing neurons. N = 18–29 cells per condition from 3 separate cultures. *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey post hoc. I, Neurons transfected with Syn-GFP plus either control or β-pix siRNA-1 were loaded with FM 4-64. The density of FM 4-64 puncta was reduced in cells expressing β-pix siRNA-1. N = 30–31 cells per condition from 3 separate cultures. ***p < 0.001; Student's t test. Scale bars, 10 μm.

Figure 7.

Cortactin expression rescues the disruption in synaptic vesicle localization in β-pix knockdown cells. A, B, The IntDen of synaptic GFP-actin clusters (A) and synaptic UtrCH-RFP clusters (B) was decreased in neurons expressing β-pix siRNA, but unchanged in neurons expressing β-pix siRNA plus Cort-HA compared with control. N = 12–20 cells per condition from 2 separate cultures. C–E, Confocal images of 9 DIV hippocampal neurons transfected with Syn-GFP and β-pix siRNA plus or minus Cort-HA and immunolabeled for PSD-95. D, F, β-Pix siRNA-1-expressing neurons exhibited a more diffuse localization of Syn-GFP (D) and Syn-GFP coverage was increased compared with control (F). In neurons expressing both β-pix siRNA-1 and Cort-HA, discrete Syn-GFP clusters were observed at PSD-95 sites (E, open arrowheads). Syn-GFP coverage in these neurons was significantly decreased compared with cells expressing β-pix siRNA-1 alone, however, Syn-GFP coverage was not completely rescued to control levels (F). G, The IntDen of Syn-GFP fluorescence (within masks of PSD-95 clusters) was significantly decreased in β-pix siRNA-1-expressing neurons, and unchanged in neurons expressing siRNA-1 plus Cort-HA compared with control. N = 13–20 cells per condition from 2 separate cultures. *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey post hoc. Scale bar, 10 μm.