1. α-Catenin Controls the Anisotropy of Force Distribution at Cell-Cell Junctions during Collective Cell Migration 
Kenji Matsuzawa, Takuya Himoto, Yuki Mochizuki, Junichi Ikenouchi 
Cell Reports 
Volume 23, Issue 12, Pages (June 2018)
DOI: /j.celrep
Copyright © 2018 The Author(s) Terms and Conditions

2. Cell Reports 2018 23, 3447-3456DOI: (10.1016/j.celrep.2018.05.070)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Cell-Density-Dependent α-Catenin Activation Determines Vinculin Accumulation at Cell Junctions in Migrating MDCKII Cells
(A) Phase contrast images of a migrating MDCKII cell monolayer.
(B) Quantification of cell densities at the periphery and center of the migrating MDCKII cell monolayer. N = 3 independent experiments with 5 image fields counted per condition per experiment; Student's t test.
(C) Migrating MDCKII cell monolayer was stained with the antibody for activated α-catenin (α18) and phalloidin for F-actin. The scale bar represents 60 μm. Insets show representative cells. The scale bar represents 10 μm. Intensity profiles of cell junction immunofluorescence in representative cells are shown in right panels. Signal intensity was normalized to the respective mean signal intensity of the entire image here and in all subsequent analyses unless otherwise specified.
(D) Migration direction of individual cells within the monolayer was determined by referencing Golgi positioning (GM130 staining) with respect to that of the nucleus. Golgi and the nucleus were bisected and the cell was divided into quadrants. The quadrant inclusive of the Golgi was defined as Q1, junction parallel to the Golgi stack orientation. Arrowhead shows migration direction.
(E) Alpha18 intensity profile at each quadrant normalized to that of Q1. Q1 and Q3 (parallel) and Q2 and Q4 (perpendicular) were considered together. N = 3 independent experiments with at least 10 cells counted per experiment. Student's t test.
(F) Migrating MDCKII cell monolayers were stained for vinculin and phalloidin. The scale bar represents 60 μm. Insets show representative cells. The scale bar represents 10 μm. Intensity profiles of representative cell junction immunofluorescence are shown in right panels.
(G) Peripheral cells of the migrating MDCKII monolayer stained for activated α-catenin (α18), vinculin, and phalloidin. The scale bar represents 60 μm. Inset shows a representative cell. The scale bar represents 10 μm.
(H) Signal intensity variabilities of α18 (left), vinculin (center), and phalloidin (right) were quantified as detailed in Experimental Procedures. Whiskers indicate the maximum and minimum values, and the box corresponds to the 75th percentile, mean, and 25th percentile values. N = 3 independent experiment with >100 cells counted per experiment; Student’s t test.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2
Generation and Characterization of MDCKII Cells Stably Expressing the α-Catenin Active Conformation Mutant
(A) Schematic of the M319G/R326E active conformation mutant of α-catenin (α-catenin-CA).
(B) FLAG-α-catenin immunoprecipitates were probed for GFP-vinculin. N = 3 independent experiments.
(C) Lysates of parental, α-catenin knockout, and stable rescue cells expressing GFP-α-catenin-WTres and -CAres were immunoblotted for α-catenin.
(D–H) Parental MDCKII and CAres cells were co-cultured and stained for α-catenin (D), α18 (E), vinculin (F), phalloidin (G), and myosin IIB (H). Maximum intensity projections of confocal images and representative X-Z orthogonal images are shown. The scale bars represent either 20 μm (D and E) or 10 μm (F–H).
Cell Reports  , DOI: ( /j.celrep )
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5. Figure 3
RhoA-Formin Signaling Mediates α-Catenin-Directed AJ Remodeling
(A) FLAG-α-catenin immunoprecipitates were probed for GFP-mDia2 (mDia2-WT).
(B) Active RhoA was pulled down from WTres and CAres cell lysates by GST-rhotekin.
(C) Quantification of (B). N = 3 independent experiments; Student’s t test.
(D) Sequential images of cell junction deformation following laser ablation.
(E and F) Vertex recoil (E) and initial recoil velocity (F) of cell junctions in -WTres and -CAres cells following laser ablation. N = 3 independent experiments with at least 10 junctions per condition per experiment; Student’s t test.
(G and I) Migrating MDCKII monolayer was treated with either vehicle (DMSO; upper panels) or a formin inhibitor (SMIFH2; 100 μM; lower panels) for 4 hr and then stained for α18 (G) or afadin (I) and phalloidin. The scale bar represents 60 μm. Insets show representative cells. The scale bar represents 10 μm. Intensity profiles of cell junction immunofluorescence are shown in right panels.
(H and J) Signal intensity variability of activated α-catenin (H) and afadin (J) were quantified as detailed in Experimental Procedures. Whiskers indicate the maximum and minimum values, and the box corresponds to the 75th percentile, mean, and 25th percentile values. N = 3 independent experiment with >100 cells counted per experiment; Student’s t test.
Cell Reports  , DOI: ( /j.celrep )
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6. Figure 4
AJ Remodeling Controls Cell-Cell Cooperation during Collective Cell Migration
(A) Representative phase-contrast images of migrating WTres and CAres cell monolayers at 1, 5, and 11 hr after migration was initiated. Images are representative of 10 independent experiments. The scale bar represents 100 μm.
(B) Quantification of the migration front velocity. N = 10 independent experiments with each data point representing means of at least 5 fields of view per experiment; Student’s t test.
(C) Schematic of the particle image velocimetry (PIV) analysis. In the heatmap, red indicates regions of high polar directional cooperativity and blue indicates regions of low polar directional cooperativity.
(D) Heatmap representation of the PIV analysis corresponding to the images shown in (A). The two-dimensional polar order parameter cosθ was calculated based on the velocity field as previously described (Petitjean et al., 2010).
(E) A representative kymograph of the PIV analysis of (D). Single-pixel-width line scans perpendicular to the initial migration front were taken across all time points and depicted as a kymograph.
(F) Theoretical model of molecular mechanisms of tension propagation through AJs in adjacent epithelial cells. We assumed that collective cell migration was enabled by the following two conditions. (1) Cells located at the front of the population form lamellipodia and tow following cells. (2) In the tensioned AJs, α-catenin undergoes structural change, which reinforces AJ binding to the circumferential actomyosin network through recruitment of actin-binding proteins, such as vinculin and afadin, and activation of the RhoA-mDia pathway. These qualitative and quantitative changes of AJs shift the balance of intrinsic centripetal contraction of circumferential actin cables. As a result, attractive force is exerted and maintained between the epithelial cells along the direction of cell movement. On the other hand, in the CAres cell line, cooperativity of cell motility is diminished because intercellular attraction works equally in every direction between adjacent epithelial cells.
(G) Heatmap representation of directional cell movement created from the mathematical simulation for the collective migratory behavior of WTres and CAres cell lines. Original simulations are shown in Videos S5 and S6.
(H) The kymograph of the analysis of (G).
(I) Statistical analyses of mean values of the two-dimensional polar order parameter cosθ during time steps 250–270 in the simulation of GFP-α-catenin-WTres and GFP-α-catenin-CAres cell lines; Student’s t test.
Cell Reports  , DOI: ( /j.celrep )
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