Figure 6. Disrupting Stx4 Inhibits Activity-Triggered Postsynaptic Exocytosis.
(A) Stx4 knock down blocks TfR-SEP insertion following stimulation with Bic/Gly. The fractional increase in TfR-SEP fluorescence (ΔF/F0) was monitored in hippocampal neurons expressing two different shRNAs against Stx4 (shRNA#1, shRNA#3) or a scrambled control shRNA (Scr) along with TfR-SEP. Bic/Gly treatment is indicated by the horizontal bar. Error bars represent SEM. n = 6, 9, 10 for shRNA#1, shRNA#3, and Scr, respectively.
(B) Soluble Stx4 blocks TfR-SEP insertion following stimulation with Bic/Gly. Total TfR-SEP fluorescence was monitored in hippocampal neurons expressing mCh or mCh-Stx4ΔTM. Bic/Gly treatment is indicated by the horizontal bar. Error bars represent SEM. n = 9 cells for each condition.
(C) Postsynaptic exocytosis is blocked by Stx4ΔTM. TfR-SEP insertion was measured on the dendrites of cells expressing mCh, mCh-Stx1ΔTM, mCh-Stx3ΔTM, or mCh-Stx4ΔTM five minutes following stimulation with Bic/Gly. Error bars represent SEM. n = 6, 8, 9, 9 cells respectively, * p < 0.05 Student's t-test.
(D) Acute disruption of Stx4 blocks postsynaptic exocytosis. Hippocampal neurons expressing Stx4-HA were incubated with either mouse anti-HA or control anti-Myc antibodies, followed by anti-mouse IgG to acutely crosslink and aggregate Stx4-HA. Error bars represent SEM. n = 7, 6, 9 cells respectively; * p<0.05, Student's t-test.
(E) Stx4 mediates exocytosis in dendritic spines. The total number of TfR-SEP exocytic events in dendritic spines was quantified in hippocampal neurons expressing mCh, mCh-Stx1ΔTM, mCh-Stx3ΔTM, mCh-Stx4ΔTM, Scr shRNA control, Stx4 shRNA#1 or Stx4 shRNA#3 during and 10 min following exposure to Bic/Gly. The total number of spine exocytic events observed during this time period was divided by the total number of spines in the imaged field for each cell. Error bars represent SEM. * p<0.05, Student's t-test, n = 6, 5, 5, 6, 8, 6, 6 cells respectively.
(F) Blocking Stx4 function leads to accumulation of REs in spines. GFP (top panel) or GFP-Stx4ΔTM (bottom panel) was expressed for 48 h along with TfR-mCh to mark REs in hippocampal neurons. Arrowheads designate RE-positive spines. Asterisks denote endosome-negative spines. Scale bar, 5 μm; inset, 1 μm.
(G) Quantification of data from (F). Data represent means ± SEM of the percent spines containing a TfR-positive RE in cells expressing GFP, Stx1ΔTM, Stx3ΔTM, Stx4ΔTM, scr shRNA, Stx4 shRNA#1 or Stx4 shRNA#3. *p<0.05, Student's t-test, n = 9, 6, 6, 9, 6, 6, 6 cells respectively. See also Figure S4 and Movie S5.