FMNL2 is required for actin turnover at the cell–cell interface. (A) Western blot analysis of MCF10A cells expressing dox-inducible control or FMNL2 shRNAs. (B) Live MCF10A cells expressing LifeAct-GFP with the indicated shRNAs (monitored through RFP) seeded in Matrigel. Arrows highlight the altered distribution of actin in FMNL2 shRNA cells. (C and D) Quantification of aberrant junctional actin as visualized by Lifeact-GFP in shRNA-expressing (C) or siRNA-treated (D) MCF10A cell pairs. (E) Western blot showing that FMNL2ΔDAD-GFP is resistant to siRNA against FMNL2 UTR. Active FMNL2 rescues junctional actin in FMNL2-depleted MCF10A cells in 3D. (F) MCF10A cell pair expressing GFP-actin. In 3D FRAP experiments, junctional GFP-actin was photobleached (white circle). Magnifications illustrate GFP-actin recovery. (G) FRAP curves comparing the effects of DMSO, Jasplakinolide, or Latrunculin B on junctional actin. (H) FRAP curves of GFP-actin MCF10A cells expressing indicated shRNAs. (I) Corresponding experiments to Fig. 2 F showing FMNL2-independent recovery of cytosolic and plasma membrane GFP-actin. (J) MCF10A cells expressing the indicated shRNAs grown for 14 d before staining for F-actin. The top right panels show an enlargement of individual cells. The bottom right panels show shRNAs as RFP. (K) Quantification of J. (L) Caco-2 cells expressing either control or FMNL2 shRNA grown in 3D for 7 d. After staining for F-actin, lumen formation was quantified (M). Error bars indicate SEM. *, P ≤ 0.05.