Volume 39, Issue 5, Pages 611-625 (December 2016) Basal Cell-Extracellular Matrix Adhesion Regulates Force Transmission during Tissue Morphogenesis  Katharine Goodwin, Stephanie J. Ellis, Emily Lostchuck, Teresa Zulueta-Coarasa, Rodrigo Fernandez-Gonzalez, Guy Tanentzapf  Developmental Cell  Volume 39, Issue 5, Pages 611-625 (December 2016) DOI: 10.1016/j.devcel.2016.11.003 Copyright © 2016 Elsevier Inc. Terms and Conditions

Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Focal Adhesion-like Structures in the Amnioserosa during DC (A and B) Dorsal view (A) and transverse cross-section (B) schematics of embryos undergoing DC. Arrows in (A) indicate direction of forces generated by AS, epidermis, and actin cable. (C) Zoomed-in view of region within black box in (B). Cell-cell adhesions at the apical part of the cell and cell-ECM adhesions on the basal surface are both coupled to cortical F-actin networks. (C′) Schematic of force transmission model in the AS. Active tension is generated apically and transmitted to neighboring cells via cell-cell adhesions, and to the substrate via apical-basal mechanical coupling and cell-ECM adhesion (“tethers”). (D and E) Integrin (green) and talin (red) colocalize in FALS (n = 68 FALS). Sqh-talin colocalization was used as a negative control. (F) FRAP was used to assess FALS turnover (n > 30). (G and H) Tracking and MSD curve of FALS labeled with TalinGFP, shown as trajectories (pink) with overlay of cell contours (gray). (I and J) FALS colocalize and track with F-actin in fixed tissue (I) and in real time (J). Arrowhead in (J) indicates a FALS moving with F-actin. (K–M) FALS are mechanosensitive and become more stable (K) and larger (M) in response to compression (n = 322–904 FALS). (N–P) FALS area and density change in response to altered actomyosin contractility by disruption of Rho1 activity (n = 6–12 embryos, 667–2127 FALS). Arrowheads indicate FALS. ∗∗∗p < 0.0001, ∗p < 0.05 based on two-sample t test. Scale bars represent 5 μm. Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 DC Defects Are Associated with Disruptions to FALS (A) Sample images of FALS in wild-type (WT), mys−/−, Talin(E1777A), and Rap1-CA mutants labeled with TalinGFP. Arrowheads indicate FALS. Scale bar represents 5 μm. (B and C) Mean FALS area (B) and density (C) in Talin(E1777A) and Rap1-CA embryos with wild-type rescue and wild-type controls, respectively. (D and E) FALS speed (D) and effective diffusion constant (E) as measured by slope of MSD in Talin(E1777A) and Rap1-CA embryos with respective controls (n = 3–8 embryos, 97–1,182 FALS). (F–H) Mean closure rate of AS area over time during DC in mys−/− (F), Talin(E1777A) (G), and Rap1-CA (H) embryos and controls (n = 5–9). ∗p < 0.05 based on two-sample t test. See also Figure S4. Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 AS Cell Displacement Correlates Inversely with Cell-ECM Adhesion (A) Wild-type AS cells labeled with DE-cadherin-GFP with average cell movement represented as centroid trajectories (pink). Scale bar represents 5 μm. (B) Sequential overlay of cell outlines and centroid trajectory of sample wild-type cell. (C) MSD plot of cell in (B) including fit. (D, G, and J) Temporal overlay of cell contours with centroid trajectories in mys−/− mutants and mys+/− controls (D), Talin(E1777A) and wild-type rescue controls (G), and Rap1-CA and wild-type controls (J). (E, H, and K) Mean instantaneous centroid speed. (F, I, and L) Mean range of cell movement measured as the average area described by the cell trajectory (n = 3–6 embryos, n = 21–66 cells). ∗∗∗p < 0.0001, ∗p < 0.05 based on two-sample t test. See also Figure S2. Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 FALS Displacements Exhibit Area-Dependent Correlation with Local Cell Membrane Movement and May Resist Apical Displacement of AS Cells (A) Sample images from time-lapse movies of two embryos at 0, 15, and 30 s, and overlay of cell outlines and FALS trajectories. Pink arrowheads indicate tracked FALS; blue arrowheads indicate closest point on cell membrane. (B and C) Average cross-correlation function (B) and distribution of correlation coefficients (C) for FALS and local membrane movement in smaller (area <0.23 μm2, n = 20) and larger (area >0.23 μm2, n = 29) FALS. (D) Time lag versus FALS area for each tracked FALS (n = 7 embryos, n = 49 FALS). (E) Sample images from embryos expressing DE-cadherin-mTomato and TalinGFP, zoomed-in views of region in white box, and overlay of membrane outlines showing local apical membrane movement. Pink arrowheads indicate FALS. (F) Membrane speed versus mean area of nearby FALS for each cell (n = 6 embryos, n = 25 cells). Slopes and R2 values are indicated on relevant plots. ∗p < 0.05 based on two-sample t test. Scale bars represent 5 μm. See also Figure S3. Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Cell-ECM Adhesion Regulates Recoil after Laser Ablation and Force Transmission in the AS (A–C) AS cell membranes before and after laser ablation and mean instantaneous recoil velocity of cell junctions after ablation in mys−/− and mys+/− control (A), Talin(E1777A) and wild-type rescue control (B), and Rap1-CA and wild-type control (C) embryos (n = 12–26 cuts). Arrowheads indicate tracked junctions; red lines indicate cut. (D) Example image of AS before cut showing cut location (red line) and first neighbor, second neighbor, and background junctions. (E–G) Mean recoil velocity of second neighbors (nbr) and background (Bkgd) junctions in controls and mys−/− (E), Talin(E1777A) (F), and Rap1-CA (G). (H–J) Extent of displacement of all junctions as a function of distance from the cut fit to an exponential for mys−/− (H, H′), Talin(E1777A) (I, I′), and Rap1-CA (J, J′) mutants and respective controls. Spatial decay rate (k) and R2 values indicated on relevant plots. (K–K″) Mean spatial decay rate in mys (K), Talin(E1777A) (K′), Rap1-CA (K″) and controls. ∗∗∗p < 0.0001, ∗p < 0.05 based on two-sample t test. Scale bars represent 5 μm. Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 FALS Exhibit Attenuated Response to Ablation and May Provide Resistance to Apical Junction Recoil (A) Sample images from two embryos before laser ablation and at 3 and 30 s after ablation, and trajectories of cell junctions and FALS for 60 s after laser ablation. Red lines indicate cut; blue arrowheads indicate junctions; pink arrowheads indicate a subset of tracked FALS. Scale bars represent 5 μm. (B) Mean cell junction and FALS displacement over time after ablation (n = 18 cuts, n = 126 FALS). (C) Mean recoil velocity of cell junctions and FALS. (D) Junction recoil versus mean FALS area in each embryo. (E) Mean maximum displacement of cell junctions and FALS. (F) Junction maximum displacement versus mean FALS area for each cut (n = 18 cuts). Slopes and R2 values indicated on relevant plots. ∗∗∗p < 0.0001 based on two-sample t test. Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Cell-Substrate Movement Correlation Requires Cell-ECM Adhesion (A–C′) Sample images of the AS in an embryo expressing Bsg-GFP and DE-Cadherin-mTomato. Maximum-intensity projection images taken from the dorsal side of the embryo are shown (A–C) alongside transverse side views reconstructed from z stacks (A′–C′). AP, anteroposterior; DV, dorsoventral. Scale bar represents 5 μm. (D) Sample cell outline at t = 0 and t = 10 s with arrows representing vector field determined by Bsg-GFP PIV. (E) Normalized cell centroid ML displacement and mean ML displacement of all Bsg-GFP PIV vectors within the cell. (F) Average cross-correlation function of cell-substrate movement in mys+/− controls and mys−/− embryos, including negative controls for each. (G) Distribution of correlation coefficients in control and mys−/− embryos (n = 6–10 embryos, n = 49–50 cells). ∗p < 0.05 based on Kolmogorov-Smirnov test. (H) Mean substrate speed in controls and mys−/− embryos (n = 6–10 embryos, n = 49–50 cells). n.s. indicates not significant based on two-sample t test. Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 8 Global Patterns of Cell Movement in the AS (A and B) Sample image of an embryo expressing nls-RFP undergoing DC with inner and outer cells indicated (A), and legend for inner and outer cells and coordinate system (B). AP, anteroposterior; ML, mediolateral. (C and D) Polarity and ML displacement of inner (C) and outer (D) cells over 20-min intervals during the first 40 min of AS contraction. Rose diagrams indicate angle relative to ML axis. (E) Density of FALS in inner and outer regions of the AS. (F) Recoil velocity after laser ablation in the inner and outer regions of the AS. (G–I) Inner and outer cell displacement over the first 20 min of AS contraction in mys−/− embryos and mys+/− controls (G), Talin(E1777A) and wild-type rescue controls (H), and Rap1-CA and wild-type controls (I). ∗∗∗p < 0.0001, ∗p < 0.05 based on two-sample t test. Developmental Cell 2016 39, 611-625DOI: (10.1016/j.devcel.2016.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions