Electron Tomography of Fusiform Vesicles Figure 3. Stacks of mFV in the central cytoplasm of umbrella cells. (A) Cytoplasm of umbrella cells is divided into two regions: sub-apical (SA) and central (CE). Central cytoplasm contains majority of mFVs and other organelles. The border between the sub-apical and central cytoplasm is shown by a dashed line. (B) In the central cytoplasm, mFVs are often arranged into stacks. (C) Three-dimensional model of a stack shows that mFVs grouped into a stack have the same shape of flattened disk as individually positioned mFVs. Model of four stacked mFVs is presented. (D) Immunolabelling with anti-Rab27b antibody shows positive reactions (arrows) on some mFVs and some iFVs, while there is no labelling seen on the apical plasma membrane (arrowhead). Legend: M – mitochondrion, asterisk – mFV without anti-Rab27b labelling. Bars: 250 nm. doi:10.1371/journal.pone.0032935.g003 membrane of intermediate cells, which was in contact to the basolateral plasma membrane of umbrella cells. Three-dimen- sional reconstructions confirmed that iFVs were more convex than mFVs (Fig. 5C, Table S1 and Movies S5, S6) and they were also significantly less densely labelled with anti-AUM antibody than mFVs of umbrella cells (Fig. 5D). Discussion FVs are highly specialized compartments that transport urothelial plaques in umbrella cells of the urothelium [20,34]. Here we employed ET, freeze-fracturing and immuno-electron microscopy of the mouse urothelium in order to resolve the 3D ultrastructure and organization of FVs in their ‘close to native’ state. Our results indicate that rapid cryo-fixation is the superior method for ultrastructural studies of FVs and their morpho- functional organization in umbrella cells (Fig. 1). Until now, conclusions about the nature of FVs were based on observations of ultrathin sections and freeze-fracturing whereas 3D models were not done yet. To investigate cellular structures in 3D rather than their 2D projections, ET is the method of choice [28]. However, FVs are relatively large compartments (up to 1.2 mm), and as such they cannot be included into a single semithin (200– 300 nm) section. To make a full 3D model of mFVs, we therefore Figure 4. Orientation of mFVs in the sub-apical cytoplasm changes during bladder stretching. In umbrella cells of the contracted bladders (A–F), mFVs are oriented mostly perpendicular to the apical plasma membrane, while in the distended bladders (G), mFV are oriented mostly parallel to the apical plasma membrane. Panels B–F show representative slices from the tomogram. In panel D, a contact (arrow) between mFVs and the apical plasma membrane (arrowhead) is seen. White box in D corresponds to the area shown in B, C, E and F. Bars: 250 nm. doi:10.1371/journal.pone.0032935.g004 PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e32935