In the subapical cytoplasm, the orientation of mFVs depends on the distension/contraction state of urinary bladders (Fig. 4). The long axes of mFVs tend to be perpendicular to the luminal membrane in contracted bladders and parallel to it in dilated bladders. Bladders can accommodate to stretching by incorporat- ing vesicles from a cytoplasmic pool into the apical plasma membrane and it has been assumed that hinge regions facilitate membrane fusions [4,32,39]. Our observation of contacts between mFVs and the apical plasma membrane supports this hypothesis. Therefore, reorientations of mFVs might facilitate the insertion of mFVs into the apical plasma membrane. However, the methods used in this study do not allow the conclusions whether all FVs in the subapical cytoplasm are destined to be inserted into the apical plasma membrane. It is possible that some FVs are derived from the apical surface. Our results of Rab27b immunolabelling support the idea of two classes of FVs, i.e. exocytotic and endocytotic [40]. However, the bladder state has no influence on the size and shape of the plaques. Reorganization of mFVs from stacks in the central cytoplasm to individual entities in the subapical cytoplasm might reflect distinct roles of mFVs; stacked FVs enable maturation and storage of plaques, while individual mFVs facilitate the transport and fusion of plaques with the apical plasma membrane. Similar reorgani- zations of cellular compartments during protein transport was observed also in some other urothelium non-related cell systems reorganization [41]. Comparison with umbrella cells shows that intermediate cells contain vesicles that are smaller and less flattened as mFVs (Fig. 5). They are weakly labelled with antibodies against uroplakins; they contain smaller plaques and therefore they represent iFVs [18]. Interestingly, we could not detect fusions of iFVs with the plasma membrane of intermediate cells. A possible explanation for this is, that iFVs of intermediate cells lack a machinery for docking and/ or fusion like specific Rab and SNARE complexes [42]. It can be stated that the maturation of FVs coincide with the terminal differentiation of urothelial cells. Smaller, more convex iFVs are therefore characteristic of partially differentiated intermediate cells, which contain less uroplakins and also lack cell surface- associated plaques [2,10,22,43]. Large, flattened mFVs are typical compartments only of highly differentiated umbrella cells. In conclusion, we have shown here 3D ultrastructure and higher organization of FVs in the urothelial cells. mFVs, present exclusively in terminally differentiated umbrella cells, are flattened discs that are organised into stacks in the central cytoplasm. From there, individual mFVs can be transported to the subapical cytoplasm, where their orientation greatly depends on the distension-contraction cycle of the urinary bladders. Due to their shape and higher organization, mFVs act as ideal compartments, which can store and transport large amount of membranes while occupying minimal volume of umbrella cells. Materials and Methods Animals All animal experiments were approved by the Veterinary Administration of the Slovenian Ministry for Agriculture and Forestry (permission no. 34401-5/2009/4) and were in accor- dance with European guidelines and Slovenian legislation. Adult, male mice were used. They were bred under constant housing conditions, fed standard laboratory chow and water was available ad libitum. A regular circadian cycle was maintained for 12 hours; the experiments were conducted between 8 and 12 o’clock, which was the beginning of mice’s night cycle. Contracted bladders were obtained immediately after micturition. In order to obtain Electron Tomography of Fusiform Vesicles distended bladders, the ligature was made on the penis after the micturition and animals were left in the cages for 3 hours before fixation. Chemical fixation Mice were anaesthetized with ketamine/xylazine/atropine mixture (150 mg ketamine, 10 mg xylazine, and 0.1 mg atropine per kg body weight) and perfused with fixative through the left ventricle according to the method of Sprando [44]. For morphological study, 4% paraformaldehyde plus 2.5% glutaral- dehyde in cacodylate buffer and for immunocytochemistry, 2% paraformaldehyde plus 0.05% glutaraldehyde in PBS were used. After fixation, abdominal cavities were opened, bladders removed and tissues were dissected in the same fixative as used for perfusion. For morphological study, samples were put in the fixative for additional 2.5 hours, post fixed in 1% OsO4, dehydrated in graded ethanol and embedded in Epon. Ultrathin sections were counterstained with lead citrate and uranyl acetate. For immunocytochemistry, samples were fixed for additional 1 hour, dehydrated and embedded in Lowicryl HM20 resin (PolysciencesInc, Warrington, USA) in Leica AFS apparatus (Leica Microsystems, Wetzlar, Germany) by the following schedule: dehydration in 30% ethanol for 30 min at 0uC, 55% ethanol for 30 min at 215uC, 70% ethanol for 30 min at 230uC, 100% ethanol for 60 min at 250uC, infiltration at 250uC in 75% ethanol/25% HM20 for 60 min, 50% ethanol/50% HM20 for 60 min, 25% ethanol/75% HM20 for 60 min, 100% HM20 for 60 min and 100% HM20 overnight. Blocks were polymerised under UV light for 48 hours at 250uC, then 3.5 hours at a 20uC/ hour rising temperature and 24 hours at +20uC. High pressure freezing Animals were killed with CO2, abdominal cavities were opened and exposed bladders were flushed from serosa side with cold PBS containing 4% paraformaldehyde for 10 seconds, to prevent muscular contraction during sectioning. Bladders were removed, rinsed quickly with PBS and transferred to 6-hexadecene. Tissue was cut into pieces with 2 mm diameter to fit freezing disks, and immediately frozen with liquid nitrogen at 2100 bar in a Balzers HPM 010 apparatus (Boeckeler Instruments, Tuscon, USA). For morphological study and for ET, samples were freeze- substituted and embedded in Leica AFS (Leica Microsystems) apparatus according to Monaghan et al [45]: warming of samples to 290uC, freeze substitution in acetone containing 2% OsO4 at 290uC for 8 hours, at 260uC for 8 hours, and at 230uC for 8 hours. Substitution solution was changed with 100% acetone, warmed to 20uC and embedded in Epon. For immunocytochemistry, frozen samples were warmed to 290uC and freeze-substituted in pure acetone at 290uC for 24, at 280uC for 120 and at 250uC for 12 hours. Acetone was replaced with 100% ethanol for 1 hour and samples were embedded in Lowicryl HM20 as described above for chemical fixation. Immunolabelling for light microscopy Bladders were fixed with 4% paraformaldehyde in PBS for 1 hour, rinsed in PBS, embedded into 10% gelatine blocks, cryoprotected by incubation in cold 2.3 M saharose for 24 hours, and frozen in liquid nitrogen. Semithin cryo-sections (300 nm thick) were cut with Leica FCS cryo-ultramicrotom at 2120uC and collected on coverslips. Nonspecific labelling was blocked as for TEM immunolabelling. Sections were incubated in cocktail containing anti-AUM antibody and mouse monoclonal anti- cytokeratin 20 antibody (Dako, Glostrup, Denmark) overnight at PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e32935