Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6

Abstract

Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell-cell junctions. Here we show that collective invasion by cancer cells depends on decreasing actomyosin contractility at sites of cell-cell contact. When actomyosin is not downregulated at cell-cell contacts, migrating cells lose cohesion. We provide a molecular mechanism for this downregulation. Depletion of discoidin domain receptor 1 (DDR1) blocks collective cancer-cell invasion in a range of two-dimensional, three-dimensional and 'organotypic' models. DDR1 coordinates the Par3/Par6 cell-polarity complex through its carboxy terminus, binding PDZ domains in Par3 and Par6. The DDR1-Par3/Par6 complex controls the localization of RhoE to cell-cell contacts, where it antagonizes ROCK-driven actomyosin contractility. Depletion of DDR1, Par3, Par6 or RhoE leads to increased actomyosin contactility at cell-cell contacts, a loss of cell-cell cohesion and defective collective cell invasion.

Figure 1. DDR1 is required for collective cell migration

(a) Top-left panel shows A431 SCC cells collectively invading a 3D matrix (F-actin – red, reflectance – cyan). Other left-hand panels show higher magnification of the indicated area. β-catenin – blue, F-actin – red, GFP-MLC – green. Right-hand panels show A431 cells on a 2D substrate, colours as in left-hand panels. Scale bars are 10 μm. (b) DDR1 western blots showing efficacy of siRNA (upper panel) and shRNA (lower panel); tubulin is used as loading control. (c) Representative images of F-actin organisation, pS19-MLC, myosin IIa or E-cadherin in A431 cells transfected with control siRNA (left) or with two different siRNA oligonucleotides against DDR1: #3 (middle) and #4 (right). Scale bars are 20 μm. (d) i) Upper panels show snap shots from phase contrast movies of control and DDR1 shRNA-transfected A431 cells moving on collagen gels. Scale bars are 20μm. Blue shading shows the position of cell groups at t=0 and red shading shows the position of the same cell groups at t=6 hours. ii) and iii) show the cell dispersion index and the average speed of cells within groups, respectively. Data calculated from tracking multiple cell groups from multiple experiments (analysis of 7-10 colonies from three independent experiments). * indicates p<0.01 student’s t-test. (e) Panels show 3D reconstruction of ‘spheroid’ SCC invasion assays in control and DDR1 depleted A431 cells. Grid spacing is 50μm. (f) Panels show H&E sections of control or DDR1 siRNA transfected SCC cells in an organotypic assay. The number in the bottom-left corner shows the invasion index of three independent experiments as percentage of the non-transfected cells.

Figure 2. DDR1 does not require kinase activity or collagen binding to regulate acto-myosin at cell contacts

(a) Representative pictures where DDR1 resistant to siRNA oligonucletides #3 was expressed in two DDR1 stable knock down clones (shDDR1#3 and shDDR1#4) or control cells (shCtr). F-actin is shown in red and DDR1 in green. The bottom panels show shDDR1#3 cells transfected with cherry as a control; scale bars are 10 μm. (b) The upper graph shows the quantification of recovery of normal actin organisation in two DDR1 stable knock down clones (shDDR1#3 and shDDR1#4) or control cells (shCtr) transfected with cherry as a control or with a DDR1 construct resistant to siRNA oligonucleotides #3. Left panels show when DDR1 (or cherry) is expressed only in one of the two cells in contact, while right panels show when DDR1 (or cherry) is expressed in both two cells in contact with one another. In the bottom graph is represented the percentage of cells with normal actin organisation in the cell-cell contacts in shDDR1#3 and shDDR1#4 cells transfected with cherry as a control or with different DDR1 constructs resistant to siRNA oligonucleotides #3 (wt, K618A, R105A or ΔDS1). Average of three experiments is shown: 20-30 cells were counted per experiment. (c) F-actin (left) and pS19-MLC organisation in A431 cells transfected with ECTM-GFP (wt in top panels and R105A in bottom panels). In the top panels, cells on the left are non-transfected cells. Scale bars are 20 μm. (d) example of DDR1 staining (green) in apical (top panel) and basal (bottom panel) membranes of A431 cells. Scale bars are 20 μm. (e) F-actin and DDR1 staining is shown in A431 cells transfected with control or E-cadherin siRNA. Scale bar is 20μm. (f) Western blot showing E-cadherin and DDR1 levels in A431 cells transfected with control or E-cadherin siRNA.

Figure 3. DR1 interacts with Par3 and Par 6

(a) Comparison of the last C-terminal aminoacids of DDR1 in human, rat, mouse, claudin 4, 5 and 6 and DDR2. (b) Negative staining of GST pulldown using GST alone as control (left), DDR1 amino acids 801-876 (middle) and DDR1 amino acids 801-875 (right). Asterisks show bands present only in the 801-876 lane. Bottom panel: Par3 western blot showing Par3 pulled down only by DDR1 801-876. (c) i) Non-transfected (first lane), GFP-transfected (second lane) or Par3-GFP-transfected (third lane) A431 cell lysates were incubated with anti-GFP antibody and the amount of endogenous DDR1 bound was determined by western blot. The Par3 western blot is shown on the middle. The amount of DDR1 in the starting lysates is shown on the bottom. ii) Non-transfected (first lane), empty PRK5.1-transfected (second lane) or Par6-flag-transfected (third lane) A431 cell lysates were incubated with anti-Flag antibody and the amount of endogenous DDR1 bound was determined by western blot. The amount of DDR1 in the starting lysates is shown on the bottom. (d) Flag-tagged PDZ domains of Par3 (third to fifth lanes) and Par6 (sixth lane) were immunoprecipitated from 293 cells also expressing DDR1. Non-transfected cells (first lane) and DDR1 alone (second lane) were used as controls. (e) Representative pictures showing Par3, DDR1 and F-actin staining (blue in merge) in A431 cells in apical membranes. Scale bar is 10 μm. (f) representative pictures showing actin (red) and Par3 (green) localisation in two clones stably knocked down of DDR1 (shDDR1#3 or shDDR1#4) or control A431 cells (shCtr) Scale bars are 10μm. (g) Representative pictures of Par3 (top) and DDR1 (bottom) in A431 cells transfected with control siRNA (left panels) or siRNA against Par3 (right panels). Scale bar is 20 μm.

Figure 4. Par3 and Par6 are required for efficient collective invasion

(a) i) Representative images showing pS19-MLC (green) and F-actin (red) localisation in siRNA control, Par3 or Par6 transfections with two different siRNA oligonucleotides. Scale bars are 10 μm. ii) Quantification of F-actin organization at cell-cell contacts. A431 cells were transfected with the indicated siRNA and 60 hours after transfection were fixed and stained for F-actin. The proportion of cells with tight F-actin at cell-cell contacts was scored (n=2, >200 cells for each data point). (b) Left-hand panels show Par3 and DDR1 levels in A431 cells transfected with control or Par3 siRNA. β-tubulin is shown as a loading control. Right-hand panels show QRT-PCR confirmation of Par6 depletion by two siRNA oligonucletides in A431 cells (normalised to GAPDH). (c) Dispersion index of cells moving on collagen gels in control, Par3 or Par6 siRNA transfected A431 cells. Analysis of 7-10 colonies from three independent experiments is shown. * indicates p<0.01 student's t-test. (d) Representative images of H&E sections of control or Par3 or Par6 siRNA transfected SCC cells in an organotypic assay. The number in the right-bottom corner represents the invasion index after quantification of three independent experiments as percentage of the control cells.

Figure 5. DDR1 controls RhoE localization at cell-cell contacts

(a) Representative images of F-actin organisation or pS19-MLC in A431 cells transfected with control siRNA or with two different siRNA oligonucleotides against RhoE or p190RhoGAP. Scale bars are 10 μm. (b) RhoE (upper) and p190RhoGAP (lower) western blots showing efficacy of siRNA; β-tubulin is used as loading control. (c) Dispersion index of cells moving on collagen gels in control or RhoE siRNA transfected A431 cells. Analysis of 7-10 colonies from two independent experiments is shown. * indicates p<0.01 student’s t-test.. (d) Panels show 3D reconstruction of ‘spheroid’ SCC invasion assays in control and RhoE depleted A431 cells. Grid spacing is 50μm. (e) Immunofluorescence for myc (green) and actin (red) showing RhoE-myc localisation in two DDR1 stable knock down clones (shDDR1#3 and shDDR1#4) or control cells (shCtr). Scale bars are 10 μm. (f) the % of cells with RhoE localised to the plasma membrane is shown. Average of three experiments is shown: 20-30 cells were counted per experiment. (g) Representative images showing DDR1 localisation in A431 cells transfected with either control or RhoE siRNA. Scale bars are 10 μm. (h) Representative images of F-actin and pS19-MLC in control and DDR1 siRNA transfected cells treated with 5μm Y27632 (ROCK inhibitor). Scale bars are 10 μm.

Figure 6

Schematic model showing the acto-myosin organisation in collective movement in normal conditions (i) or when interfering with DDR1, Par3 or RhoE (ii).