Figure 4. The AIS is a surface... | Rockefeller University Press

Figure 4.

$The AIS is a surface trafficking hotspot for specific cargoes. (A) Representative images of DHFR-GFP-NL1 (left) and DHFR-GFP-GluA1 (right) expressing neurons expressing the AIS marker ankyrinG-mCh (not shown) and a cell fill (green). The axon is indicated by the white arrowhead. Magnified images to the right (taken from the yellow boxes) display surface signal at the AIS for DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) before and 60 min after ER release. Note that Alexa647-anti-GFP was present in the extracellular solution following ER release to continuously trap and label proteins as they surfaced. Red arrowheads denote accumulated cargo at the surface of the AIS. Scale bars, 20 µm; inset, 10 µm. (B) AIS surface signal (expressed as a fraction of total surface signal throughout the entire cell) for DHFR-GFP-NL1 (orange) or DHFR-GFP-GluA1 (blue) 60 min after ER release; mean ± SEM; ****, P < 0.0001 (Student's t test, n = 8 from two independent experiments for NL1 and GluA1). (C) Surface trafficking at the AIS occurs early following ER release. Confocal image (left) and heatmap displaying the timing and location of DHFR-GFP-NL1 surface appearance (right). Insets show the AIS and proximal and distal dendrites. Scale bars, 20 µm. Inset scale bars, 5 µm. (D) Shown are mean NL1 surface intensities (normalized to their maximum values) at the AIS (teal line), soma (pink line), proximal dendrites (5–40 µm from the soma; peach line) or distal dendrites (40–200 µm from the soma; lavender line); mean ± SEM (n = 10 neurons/time point from two independent experiments). (E) Time to reach 10% of maximum DHFR-GFP-NL1 surface signal is plotted for each subcellular compartment; mean ± SEM; *, P < 0.05; ****, P < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test; n = 10 neurons from two independent experiments). (F) ER release can be titrated by decreasing photoexcitation power. DHFR-GFP-NL1 accumulation in the GA is plotted following illumination with decreasing 405-nm light intensities (912 µW, 194 µW, and 97 µW); mean ± SEM (n = 7–14 neurons/condition from at least two independent experiments). (G) DHFR-GFP-NL1 appears at the surface of the AIS even when decreasing amounts are released from the ER. GluA1 surface signal at the AIS following exposure to a saturating light intensity (912 µW) is shown for comparison; mean ± SEM; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test; n = 5–8 neurons/condition from two independent experiments). (H) DHFR-GFP-NL1 traffics to the surface of the AIS when network activity is elevated (Bic) or suppressed (TTX). Student's t test; n = 7 neurons/condition from three independent experiments. norm., normalized; prox., proximal.$

The AIS is a surface trafficking hotspot for specific cargoes. (A) Representative images of DHFR-GFP-NL1 (left) and DHFR-GFP-GluA1 (right) expressing neurons expressing the AIS marker ankyrinG-mCh (not shown) and a cell fill (green). The axon is indicated by the white arrowhead. Magnified images to the right (taken from the yellow boxes) display surface signal at the AIS for DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) before and 60 min after ER release. Note that Alexa647-anti-GFP was present in the extracellular solution following ER release to continuously trap and label proteins as they surfaced. Red arrowheads denote accumulated cargo at the surface of the AIS. Scale bars, 20 µm; inset, 10 µm. (B) AIS surface signal (expressed as a fraction of total surface signal throughout the entire cell) for DHFR-GFP-NL1 (orange) or DHFR-GFP-GluA1 (blue) 60 min after ER release; mean ± SEM; ****, P < 0.0001 (Student's t test, n = 8 from two independent experiments for NL1 and GluA1). (C) Surface trafficking at the AIS occurs early following ER release. Confocal image (left) and heatmap displaying the timing and location of DHFR-GFP-NL1 surface appearance (right). Insets show the AIS and proximal and distal dendrites. Scale bars, 20 µm. Inset scale bars, 5 µm. (D) Shown are mean NL1 surface intensities (normalized to their maximum values) at the AIS (teal line), soma (pink line), proximal dendrites (5–40 µm from the soma; peach line) or distal dendrites (40–200 µm from the soma; lavender line); mean ± SEM (n = 10 neurons/time point from two independent experiments). (E) Time to reach 10% of maximum DHFR-GFP-NL1 surface signal is plotted for each subcellular compartment; mean ± SEM; *, P < 0.05; ****, P < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test; n = 10 neurons from two independent experiments). (F) ER release can be titrated by decreasing photoexcitation power. DHFR-GFP-NL1 accumulation in the GA is plotted following illumination with decreasing 405-nm light intensities (912 µW, 194 µW, and 97 µW); mean ± SEM (n = 7–14 neurons/condition from at least two independent experiments). (G) DHFR-GFP-NL1 appears at the surface of the AIS even when decreasing amounts are released from the ER. GluA1 surface signal at the AIS following exposure to a saturating light intensity (912 µW) is shown for comparison; mean ± SEM; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test; n = 5–8 neurons/condition from two independent experiments). (H) DHFR-GFP-NL1 traffics to the surface of the AIS when network activity is elevated (Bic) or suppressed (TTX). Student's t test; n = 7 neurons/condition from three independent experiments. norm., normalized; prox., proximal.