Electron Tomography of Fusiform Vesicles Figure 5. Mature FVs are only present in umbrella cells. (A) In umbrella cells, some stacks of FVs contain mFVs, which are densely labelled with anti-AUM antibody, and iFVs, which are shorter and more convex. Such stacks are surrounded by rounded uroplakin-positive (short black arrow) and uroplakin-negative (striped arrow) vesicles. (B) In the intermediate cell (ic), mFVs are not present; instead iFVs are seen below the plasma membrane (arrowhead) that borders the basolateral plasma membrane of the umbrella cell (uc). (C) Three-dimensional model of iFVs in the intermediate cell. (D) iFVs of intermediate cell (ic) are less densely labelled with anti-AUM antibody than mFVs of umbrella cell (uc). Bars: 500 nm. doi:10.1371/journal.pone.0032935.g005 joined tomograms of multiple serial semithin sections. In that way we confirmed that mFVs are flattened discs (Fig. 2) as predicted earlier [6,7]. They are composed of two opposing urothelial plaques that are connected along their rims by a flexible non- thickened membrane, which makes a distinct curvature [35]. The flattened disk shape of mFVs is in agreement with the function of urothelium. Secretory tissues contain numerous secretory vesicles, which are usually spheres with substantial intravesical volume filled with secretion products that are transported to the cell surface. Urothelium, on the other hand, does not secrete soluble products. Instead, the main products of urothelial umbrella cells are urothelial plaques, which function as blood-urine permeability barrier and help to accommodate the apical surface area of umbrella cells [5,20]. Therefore, flattened disks with minimal intravesical lumen are perfectly shaped compartments to store and transport urothelial plaques to the apical plasma membrane on one hand, and to minimise internalization of toxic substances from urine on the other hand [36]. The mechanical stability of mFVs shape is probably provided by the rigidity of urothelial plaques [22], which prevents bending and ensures minimal lumen within mFVs. To provide additional information on the higher organization of mFVs in urothelial umbrella cells, we made ET of large cell volumes. The results showed that mFVs are organized differently in the central and in the subapical cytoplasm of umbrella cells (Fig. 3). The separation of the central cytoplasm from the subapical cytoplasm is provided by a dense cytoskeletal network, formed by cytokeratins [30]. This network limits the exchange of FVs between the central and subapical cytoplasm of the cell. FVs may be transported from central to subapical cytoplasmic regions only through specialized openings in the cytokeratin network, called trajectories [24,30]. This, and possibly the organization into stacks, may provide a mechanism, which limits and regulates the traffic of FVs between the central cytoplasm and the subapical cytoplasm. In the central cytoplasm, FVs are often arranged into stacks of multiple FVs (Fig. 3). This may have two important functional consequences. First, when FVs are positioned close to each other, their maturation is facilitated. It has been suggested that FVs gradually mature by accumulation and expansion of crystalline arrays of 16-nm uroplakin particles, which derive from the trans- Golgi network [5,6,16,18,20,37]. Second, the arrangement of mFVs into stacks, together with their flattened shape provides an ideal form for packing large areas of membranes into a small cell volume. The surface area of an average mFV (Fig. 3C; diameter 2 780 nm, thickness 27 nm) is 1,02 mm , which is 3,8 times more than it would be in spherical vesicle (radius 145 nm, surface area 2 3 0,266 nm ) with the same volume (0,013 mm ). The difference is even more prominent when more mFVs are packed into a stack. Calculation shows that 10 mFVs (Fig. 3B) could be packed into approximately 10 times smaller volume that the spherical vesicles with the same surface. A similar organization can be observed in the outer segments of light-sensing cells in retina, where highly stacked membrane discs carry rhodopsin proteins [38]. These shows that mFVs are highly specialized and organized compart- ments, which provide a large storage pool of urothelial plaques needed to accommodate apical plasma membrane during distension-contraction cycles of the bladder [20,39]. PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e32935