Figure 8.

Effects of EpCAM depletion on actin cytoskeleton organization, myosin phosphorylation, and protrusive activity. (A–D) Confocal images of phalloidin-stained BCR explants. (A) Typical punctate phalloidin pattern (arrowheads) in control cells (COMO) with prominent accumulation at tricellular corners (large arrows). (B) Concentration at corners in EpCAM MO cells (large arrows), and decrease of the signal along the membranes. (C) Rescue of membrane staining and disappearance of the signal at corners upon coinjection of dominant-negative PKC-δ mRNA. (D) Homogenous membrane staining of EpCAM-overexpressing cells. (E–H) Live confocal images of the surface of membrane GFP-expressing BCR cells. Arrows: large protrusion. Arrowheads: small protrusions. (I) Quantitation of protrusive activity from time-lapse movies (see selected frames in Fig. S4). EpCAM MO-injected cells showed much fewer long-lasting protrusions than controls (P = 2,5E⁻⁰⁷; Student’s t test) but many more short-lived extensions (P = 5,4E⁻⁰⁷). Most protrusions emanating from EpCAM-overexpressing cells were long lived (P = 6,9E⁻⁰⁶ compared with GFP controls). (J) Increased myosin light chain (MLC) phosphorylation in EpCAM MO BCRs, and partial rescue by coexpression of dominant-negative PKC-δ. (K and L) Rescue of tissue separation by coexpression of constitutively active RhoA. EpCAM mRNA (200 pg) was injected alone or with V14RhoA mRNA (25 pg). Numbers on top indicate total number of explants/number of experiments. *, P < 0.05 compared with EpCAM alone (Student’s t test).