Volume 27, Issue 24, Pages 3783-3795.e8 (December 2017) Tension-Dependent Stretching Activates ZO-1 to Control the Junctional Localization of Its Interactors  Domenica Spadaro, Shimin Le, Thierry Laroche, Isabelle Mean, Lionel Jond, Jie Yan, Sandra Citi  Current Biology  Volume 27, Issue 24, Pages 3783-3795.e8 (December 2017) DOI: 10.1016/j.cub.2017.11.014 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 3783-3795.e8DOI: (10.1016/j.cub.2017.11.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Stretched and Folded and/or Disordered ZO-1 Conformations Are Detected by SIM and PLA (A) Scheme of myc-ZO-1-HA expressed in ZO-1-KO Eph4 cells. (B–G) SIM images of cells expressing exogenous myc-ZO-1-HA, and treated either with control siRNA either in the absence (B, F, and G) or in the presence (C) of blebbistatin, or with si-ZO-2 either in the absence (D and F’) or in the presence (E and F”) of blebbistatin. Red (Cy3) and green (Alexa 488) fluorophores (insets/arrows) label N- and C-terminal ends of ZO-1. Yellow arrows indicate overlapped signal. Experimental treatments (siRNA and blebbistatin) are indicated on the left of images. (F) si-control, (F’) si-ZO-2, and (F”) si-ZO2+blebbistatin show calibration of chromatic shift, where junctions are imaged next to TetraSpeck Fluorescent microspheres (b, beads; merge image in bottom right panel). (G) The junction between two cells expressing tagged ZO-1. (H) Distribution of fluorophores/tags signal intensities as a function of distance. (I) Distance between intensity peaks. Error bars indicate SD. (J and K) PLA analysis on cells expressing myc-ZO-1HA, with the indicated treatments. Arrows and arrowheads indicate strong or weak PLA signal, respectively. The scale bars represent 5 μm (B–E) and 30 μm (J and K). Related Figure S1 shows experimental system, control experiments, and additional measures and quantifications. Related Figure S7 shows schemes of ZO-1 in stretched and folded conformations under the different experimental conditions. Current Biology 2017 27, 3783-3795.e8DOI: (10.1016/j.cub.2017.11.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Mechanical Stretching of Single Full-Length ZO-1 Molecules In Vitro (A) Scheme of experimental setup. (B) Force-extension curves of ZO-1 unfolding (colored) and refolding (gray). (C) Zoom-in of refolding events during force-decrease scans (loading rate = −0.1 pN/s) from (B). (D) Distribution of the ZO-1 domains unfolding forces (loading rate = 1 pN/s). (E) Distribution of number of residues involved in the individual unfolding events (N, number of unfolding events). The inset shows the distribution of total number of residues involved in unfolding in individual force-increase scans (N, number of scans). (F) Theoretical calculation of the force-extension curves of full-length ZO-1. Numbers of residues assumed to be non-structured are indicated. Related Figure S2 shows additional extension/refolding curves and step size measurements. Current Biology 2017 27, 3783-3795.e8DOI: (10.1016/j.cub.2017.11.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 The Junctional Accumulation and Expression of DbpA Are Regulated by Force in a ZO-Dependent Manner (A, B, E, and F) Immunofluorescent localization of DbpA, ZO-2, PLEKHA7 (internal reference for junctions), and ZO-1 either in WT (A and B) or mixed WT+ZO-1-KO (E and F) cells following treatment with either si-control or si-ZO-2, with the indicated drugs treatment. The scale bar represents 5 μm. The localization of occludin is shown in related Figure S3. Arrows and arrowheads indicate normal or decreased/undetected junctional labeling. (n) in (F) (BL/MG) indicates nuclear labeling for DbpA. Merge images show nuclei in blue (DAPI). (C and D) Quantification of junctional labeling of ZO-2 and DbpA (C) and immunoblotting analysis of the indicated proteins following experimental treatments (D). (G and H) Immunoblot of the indicated junctional proteins (red and green boxes indicate decreased and rescued DbpA; G) and DbpA levels quantification following indicated experimental treatments (H). Asterisks indicate depleted/KO cells in immunofluorescence and statistical significance in plots. Error bars indicate SD. Current Biology 2017 27, 3783-3795.e8DOI: (10.1016/j.cub.2017.11.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Tension Controls Cell Proliferation and Paracellular Barrier Function through ZO Proteins (A) Analysis of proliferation (% Ki67 positivity) of cells grown in 2D, with the indicated treatments. NT, not treated. (B and B’) Immunoblotting analysis (B) and quantification of DbpA levels (B’). (C and D) mRNA levels (relative expression by qRT-PCR, 100% = synchronized proliferating cells in G1) of DbpA target genes Cyclin D1 and PCNA (C) and ErbB2 (D) following the indicated experimental treatments. (E and F) Transepithelial electrical resistance (TEER) (E) and dextran permeability (Papp) in WT and ZO-1-KO cells (F), with the indicated treatments. Asterisks indicate statistical significance. Error bars indicate SD. Related Figure S7 shows schematically the different fates of DbpA and its activity on target genes, depending on the experimental conditions. Current Biology 2017 27, 3783-3795.e8DOI: (10.1016/j.cub.2017.11.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 ZO Proteins and Actomyosin Contractility Control Cell Proliferation, Cyst Growth, and DbpA Localization in Eph4 Cells Grown in Matrigel (A–D) Brightfield microscopy images of Eph4 cysts (A and B, WT; C and D, ZO-1-KO) at different days (d) of culture in Matrigel, without (A and C) or with (B and D) dATP treatment. Asterisks at the 21d time point indicate cyst lumens. Scale bars represent 10 μm. (E) Analysis of cyst size (diameter). Cell proliferation/number in cysts is shown in related Figure S4. Error bars indicate SD. (F) Immunoblotting analysis of levels of the indicated proteins in lysates of cysts at 21d. Red/green box indicates decreased/rescued DbpA levels. (G–J) Immunofluorescent localization of DbpA and E-cadherin (to label junctions) in untreated or dATP-treated (dATP) WT or ZO-1-KO cysts at days 4 (G and I) and 21 (H and J). Arrows and arrowheads indicate normal and reduced/undetected staining, respectively. Current Biology 2017 27, 3783-3795.e8DOI: (10.1016/j.cub.2017.11.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Intra-molecular Interactions between ZPSG and C-Terminal Domains of ZO Proteins Prevent Binding of DbpA (A and C) Schemes of ZO-1 (A) and ZO-2 (C), with the indicated domains, and prey (GFP-tag/green) and bait (GST-tag/red) constructs for pull-down experiments. (B, B’, and D) Interaction between ZPSG and C-terminal regions of ZO-1 (B; quantification in B’, with error bars indicating SD), and ZO-2 (D). (E–J) Competition between Cter of ZO-1 (1,619–1,748) and either DbpA (E–G) or occludin (H–J) for binding to ZPSG-1. (K and L) ZO-2 promotes ZO-1 interaction with DbpA. Immunoblot analysis of GST pull-downs using GST-DbpA as a bait, and full-length ZO-1 as a prey, in the presence of increasing amounts (μL of lysate indicated below each lane) of either vsv-tagged ZO-2 (K), or vsv-tagged p114-RhoGEF (L). Immunoblots (B, D, E, F, and H–M) show normalized loadings of preys (INPUT-prey, green), competing prey proteins (INPUT, blue), and GST pull-downs (preys in green) without or with competing proteins (blue). GST (negative control) and GST-fusion baits are in red, and their levels are shown in PonceauS-stained blots below immunoblots. Numbers indicate amino acid residues spanned in constructs. Preys were tagged either with GFP, HA, or vsv. FL-ZO-1 had no tag. Quantification of prey binding to GST-ZPSG- is shown in (B’) and (G). Binding of GFP-tagged ZPSG domains of ZO-1, ZO-2, and ZO-3 to either GST-DbpA or GST-occludin are shown in related Figure S5. Binding of Cter and DbpA to ZPSG-1 subdomains, competition experiments for binding to ZPSG-2, and determination of affinities of interaction of ZPSG-1 for either Cter or DbpA are shown in related Figure S6. Current Biology 2017 27, 3783-3795.e8DOI: (10.1016/j.cub.2017.11.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Force-Dependent Interaction of ZPSG and Cter In Vitro (A) Scheme of experimental setup. (B) Force-extension curves of rupturing/unfolding of ZPSG-FH1-Cter construct during multiple force-increase scans (1 pN/s). (C) Distributions of number of residues (top) and contour length (bottom) involved in the rupturing/unfolding events. (D) Force distributions for rupturing (>300 aa, top) and unfolding (<300 aa, bottom) of the ZPSG-Cter complex. (E) Typical force-extension curves of unfolding of ZPSG domain (loading rate of 1 pN/s). Left and right dashed boxes indicate regions where ZPSG/I27 domains unfolding occur, respectively. (F) Unfolding force distributions of ZPSG domain. N, number of events. Current Biology 2017 27, 3783-3795.e8DOI: (10.1016/j.cub.2017.11.014) Copyright © 2017 Elsevier Ltd Terms and Conditions