Electron Tomography of Fusiform Vesicles and Their Organization in Urothelial Cells Samo Hudoklin1, Kristijan Jezernik1, Josef Neumu¨ ller2, Margit Pavelka2, Rok Romih1* 1 Faculty of Medicine, Institute of Cell Biology, University of Ljubljana, Ljubljana, Slovenia, 2 Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria Abstract The formation of fusiform vesicles (FVs) is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella) cells during the distension-contraction cycle. We have analysed the three-dimensional (3D) structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 mm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4–15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm. Citation: Hudoklin S, Jezernik K, Neumu¨ ller J, Pavelka M, Romih R (2012) Electron Tomography of Fusiform Vesicles and Their Organization in Urothelial Cells. PLoS ONE 7(3): e32935. doi:10.1371/journal.pone.0032935 Editor: Wanjin Hong, Institute of Molecular and Cell Biology, Singapore Received October 4, 2011; Accepted February 1, 2012; Published March 12, 2012 Copyright: 2012 Hudoklin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Ministry of Education and Sport, Government of Republic of Slovenia, Slovenia (grant no. 3311-04-831450), and by the Slovenian Research Agency (project: BI-AT/07-08-019). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rok.romih@mf.uni-lj.si Introduction Superficial urothelial cells (umbrella cells) of the urinary bladder contain numerous fusiform vesicles (FVs), called also fusiform vacuoles or discoidal vesicles [1,2,3]. FVs have been described, depending on mammalian species, as being either fusiform or discoidal in cross-section [4]. According to Staehelin et al., they have a form of biconvex discs with a diameter 0.5–1 mm [5,6]. Minsky and Chlapowsky proposed that FVs are pancake-like flattened spheres, but this has never been confirmed by ultrastructural 3D analyses [7]. FVs are lined by an asymmetric unit membrane (AUM), which contains four major integral proteins, uroplakins (UPs) Ia, Ib, II and IIIa [8,9,10,11,12,13]. Uroplakins form 16-nm intramembra- nous uroplakin particles, which are hexagonally arranged in urothelial plaques. Plaques measure between 0.3 and 1 mm in diameter [5], and they are connected by a non-thickened membrane, called hinge region [1,14,15]. UPs are synthesized in the endoplasmic reticulum, where UPIa and UPIb form heterodimers with UPII and UPIIIa, respectively. Conformational changes in the Golgi apparatus enable the formation of 16-nm intramembranous particles [1,6,15,16,17], which are hexagonally arranged into 2D crystalline plaques in the post-Golgi compart- ments [18]. While the structure of the 16-nm particles is largely known [19], the information on the 3D structure of mature FVs is missing. The plaque composition of mature FVs is identical to that of the apical plasma membrane of umbrella cells, therefore it has been proposed that FVs are transported from the Golgi apparatus towards the apical cell surface where they fuse with the plasma membrane [1,2,15,20,21,22]. According to one hypothesis, FVs are inserted into the apical plasma membrane during bladder distension (filling with urine) and retrieved during bladder contraction (micturition). This membrane recycling therefore provides a mechanism to adjust surface area of umbrella cells during distension-contraction cycles of the urinary bladder [1,7,15,23,24]. Alternative hypothesis says that FVs are not retrieved during contraction of the bladder; instead the apical surface area is accommodated only by the apical plasma membrane infolding [2]. The analyses of morpho-functional organization of FVs are therefore essential for understanding their role in the intracellular membrane traffic and in the turn-over of the apical plasma membrane. Electron tomography (ET), which allows 3D reconstructions of objects with the resolution below 10 nm, has greatly contributed to the understanding of subcellular structures and compartments PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32935