Fig. 2

Fig. 2 RhoA is stringently activated at the interface between apoptotic cells and their immediate neighbors (A) AHPH in neighbors of apoptotic MCF7 cells: montage of XY and Z views (from Video S2). AHPH was present at the ZA (red arrowheads) prior to induction of apoptosis (−00:20). After apoptosis was induced, a separate pool appeared at the apoptotic:neighbor interface below the ZA pool (green arrowheads). Yellow line represents position of the Z views. See also Figure S2A. (B) AHPH in neighbor cells within the 2-dpf zebrafish periderm. A cell that did not express AHPH was laser microirradiated (asterisk); AHPH at the neighbor:apoptotic interface is marked (arrowheads). Images are sum of slices projection from z stacks. See also Video S3. (C and D) Spatial distribution of RhoA activity. Junctional GFP-AHPH was quantitated in nearest neighbor cells for MCF7 monolayers (C) and zebrafish periderm (D). We measured AHPH at the junctions that immediate neighbors made with the apoptotic cell (magenta, 1st junction), other neighbor cells in the first row around the apoptotic cell (2nd junction, green), and with epithelial cells in the second row (3rd junction, cyan). The time evolution of AHPH levels was measured for MCF7 cells (C) although AHPH was measured at 50% extrusion in the periderm (D). (E) Exogenously delivered S1P, but not apoptosis, triggers internalization of S1P₁-GFP. S1P₁ is rapidly internalized upon S1P (100 nM) treatment or addition of regular S1P-containing serum (10%); however, apoptosis does not trigger internalization of the receptor. See also Figure S2G. (F and G) Effect of S1P depletion on apoptotic cell extrusion induced by laser irradiation (F) and neighbor cell RhoA activation (G). Extrusion was quantified in cells supplemented with 10% regular FBS (control) or 10% charcoal-stripped FBS (no S1P) treated with SKI-I (10 μM) or S1P (100 nM) and SKI-I (10 μM). See also Figure S2H. Scale bars represent 15 μm. All data are means ± SEM; ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; calculated from n = 3 independent experiments analyzed with one-way ANOVA Dunnett’s multiple comparisons test (C and E–G) or Student’s t test (D). Time is mm:ss. XY panels present maximum projection of all acquired z stacks unless indicated otherwise.