Volume 110, Issue 3, Pages 623-634 (February 2016) Netrin-1-Regulated Distribution of UNC5B and DCC in Live Cells Revealed by TICCS  Angelica A. Gopal, Benjamin Rappaz, Vincent Rouger, Iain B. Martyn, Peter D. Dahlberg, Rachel J. Meland, Ian V. Beamish, Timothy E. Kennedy, Paul W. Wiseman  Biophysical Journal  Volume 110, Issue 3, Pages 623-634 (February 2016) DOI: 10.1016/j.bpj.2015.12.022 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 CLSM images of HEK293T cells expressing recombinant DCC-EGFP and UNC5B-mCherry before netrin-1 stimulation. (A) Channel 1: EGFP channel showing distribution of DCC. (B) Channel 2: mCherry channel showing UNC5B distribution. (C) Composite overlay image of (A) and (B). The yellow box shows a typical ROI selected for TICCS analysis. To see this figure in color, go online. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Representative TICCS correlation functions and fits to the 2D diffusion model (Eq. 3) calculated for the ROIs shown in Fig. 1. (A) Autocorrelation function and best fit for channel 1 DCC-EGFP. (B) Autocorrelation function and best fit for channel 2 UNC5B-mCherry. (C) Cross-correlation function and best fit for channel 1 × 2, with the inset showing a zoomed scale to show the decay and fit. R2 = 0.99 for the autocorrelation functions and 0.51 for the cross-correlation function. The correlation-function data are the discrete points and the solid lines are the best fits. Δt = 0.97 s. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Mean percentage changes in cluster density (CD), degree of aggregation (DA), and average intensity (Int.) of (A) transfected DCC-EGFP HEK293T cells and (B) transfected UNC5B-mCherry HEK293T cells in the control, blank, and netrin-1 stimulation conditions. Error bars indicate the mean ± SE; ∗p < 0.05 and ∗∗p < 0.001 compared to control and blank stimulation, evaluated by one-way analysis of variance (ANOVA); significance was evaluated at a 90% confidence level (CL) (∗) and a 95% CL (∗∗) via the two-tailed t-test. N = 5–9. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 TIRF microscopy images from a time series of HEK293T cells, expressing recombinant DCC-EGFP (A) Image frame 100 (50 s), before netrin-1 stimulation. (B) Image frame 600 (300 s), immediately post-netrin-1 stimulation at ∼550 frames. (C) Image frame 900 (450 s), showing cells well after netrin-1 stimulation. (D) Intensity traces taken from the yellow line at the different times shown from (A)–(C). The red trace displays the intensity from (A) before netrin-1 stimulation. The blue trace displays the intensity from (B) immediately after netrin-1 stimulation. The black trace shows the intensity from (C) following the redistribution well after netrin-1 stimulation. The single-frame time is 500 ms. To see this figure in color, go online. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 Mean percentage changes in (A) cluster density, (B) degree of aggregation, and (C) average intensity measured by TICCS from TIRF images of transfected DCC-EGFP HEK293T cells in the control and netrin-1 stimulation conditions. Error bars indicate the mean ± SE; ∗p < 0.05, ∗∗p < 0.001 compared to the control, evaluated by one-way ANOVA. Results marked + indicate significance at a 99% CL via a two-tailed t-test. N = 12–21. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 Mean percentage changes in cluster density (CD) and degree of aggregation (DA) for DCC-EGFP (A) and DCC-T-mCherry (B), in the average intensity (Int.) for both DCC variants (C), and in the interaction fractions (D) in HEK293T cells in the control, blank, and netrin-1 stimulation conditions. Error bars indicate the mean ± SE; ∗∗p < 0.001 compared to control and blank stimulation, evaluated by one-way ANOVA, and significance at a CL of 99% via a two-tailed t-test. N = 7–17. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 7 Mean percentage changes in cluster density (CD) and degree of aggregation (DA) for DCC-EGFP (A) and UNC5B-mCherry (B), in the average intensity (Int.) for DCC and UNC5B (C), and interaction fractions (D), in HEK293T cells in the control, blank, netrin-1, and netrin VI-V stimulation conditions. Error bars indicate the mean ± SE, ∗p < 0.05, ∗∗p < 0.001 compared to control and blank stimulation, evaluated by one-way ANOVA. All marked results are significant with a paired t-test at the 90% CL, whereas + indicates significance at the 99% CL in a two-tailed t-test. N = 15–18. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 8 Time profiles for cluster density (A) and degree of aggregation (B) of DCC-EGFP, and for the M1 interaction fraction (C) in HEK293T cells coexpressing DCC-EGFP and UNC5B-mCherry in a control experiment (dotted black line) and under netrin-1 stimulation (solid black line). Netrin-1 stimulation occurs at time frame 200, but due to the averaging nature of the analysis, the effect of netrin-1 is not exact in time. The single-frame time is 0.97 s. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 9 Mean percentage change in cluster density (CD) and degree of aggregation (DA) for DCC-EGFP (A) and UNC5B-mCherry (B), in the average intensity (Int.) for DCC and UNC5B (C), and in the interaction fractions (D) in HEK293T cells in control, PP2 control, PP3 control, and netrin-1 stimulation in PP2 and PP3. Error bars indicate the mean ± SE; ∗p < 0.05, ∗∗p < 0.001, evaluated by one-way ANOVA against the relevant control. Results marked + indicate significance with a two-tailed t-test at the 90% CL, whereas ++ indicates significance at the 95% CL. N = 8–12. Biophysical Journal 2016 110, 623-634DOI: (10.1016/j.bpj.2015.12.022) Copyright © 2016 Biophysical Society Terms and Conditions