Subcellular distribution of NL1 and GluA1 surface presentation following global ER release. (A) The timing and location of surface trafficking for DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) are shown following global light-triggered ER release. Surface signal with a shorter latency of appearance is rendered in warmer colors. Scale bars, 20 µm. (B) Time courses of DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) surface delivery at different cellular domains following global ER release. Shown are the normalized intensities for surface signal at the soma (pink line) and proximal (peach line; up to 40 µm from the soma) or distal regions of the dendritic arbor (lavender line; 40–200 µm from the soma); mean ± SEM (NL1: n = 10 neurons/time point from four independent experiments; GluA1: n = 11 neurons/time point from two independent experiments). (C) Comparison of DHFR-GFP-NL1 (gray) and DHFR-GFP-GluA1 (black) surface accumulation (from B) at distal dendrites (40–200 µm from the soma) following ER release. The yellow shaded region denotes P < 0.05 (two-way ANOVA, Bonferroni’s multiple comparisons test). (D) Time to 10% of total surface accumulation is plotted for DHFR-GFP-NL1 (gray) and DHFR-GFP-GluA1 (black) in different cellular domains; mean ± SEM; **, P < 0.01 (Student's t test). (E) Representative images of DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) surface signal (magenta, Alexa647-anti-GFP) in spines 90 min following ER release. Solid arrowheads denote cargo-positive spines. Open arrowheads mark spines that lack detectable surface cargo. The outline of the cell (dashed line) was drawn based on the cell fill (green signal in merge). Scale bar, 2 µm. (F) Time course of the fraction of spines in proximal (circles) and distal (triangles) dendrites with detectable DHFR-GFP-NL1 (gray) or DHFR-GFP-GluA1 (black) signal following ER release. A comparison of the fraction of DHFR-GFP-NL1– and DHFR-GFP-GluA1–positive spines at 90 and 120 min is shown on the right; mean ± SEM; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (Student's t test; n = 10 neurons/time point for NL1 and GluA1). (G) Plotted is the ratio of the total spine surface signal to the total dendritic shaft surface signal for DHFR-GFP-NL1 (gray) or DHFR-GFP-GluA1 (black) in proximal and distal dendrites, 90 and 120 min following ER release; mean ± SEM; **, P < 0.01; ***, P < 0.001 (Student's t test; n = 8–10 neurons/time point [NL1], n = 7–9 neurons/time point [GluA1]). dist., distal; norm., normalized; prox., proximal.