Cellular localization of ezrin and Y477F ezrin (VSVG tagged) in colonies growing in 3D Matrigel culture: Colonies formed from AC2M2 cells expressing pCB6 control vector or Y477F ezrin (clone C13) in day 12 embedded Matrigel cultures (see Figure 2) were fixed, and stained for ezrin (red), actin (green, phalloidin), and nuclei (blue, DAPI). Images of stained colonies were acquired using a Quorum Wave FX spinning disk confocal microscope. Panels A & B) 3D projection images of pCB6 control and Y477F ezrin expressing clones are shown (three colours merged). Inserts show single XY plane of actin-rich cellular protrusions with ezrin localized to the cytoplasmic side in pCB6 colonies. No actin-rich protrusions were evident in Y477F ezrin colonies. Panel C) Images of pCB6 colonies were deconvoluted and subjected to 3D reconstruction using Metamorph software. A middle section showed strong localization of ezrin in cellular extensions, compared to weak expression in the central core of colonies. Panel D) A middle xy plane of the pCB6 colony shown in C confirmed the absence of a hollow lumen. Panel E) Deconvoluted images of Y477F ezrin expressing colonies stained for ezrin or VSVG were subjected to 3D reconstruction as above. A middle xy plane showed a partial membranous staining which was strongest in the outer surface of colonies of Y477F ezrin-expressing cells, though some diffuse cytoplasmic staining was also evident. No hollow lumens were present. Panel F) XY planes corresponding to ezrin and VSVG staining in E show actin structures associated with the plasma membrane particularly at cell junctions. Images are representative of at least 20 colonies examined in each group. Scale bar indicates 50 μm.