Electron Tomography of Fusiform Vesicles and Their Organization in Urothelial Cells

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 µm. 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

Urothelial Plaque Formation in Post-Golgi Compartments

Urothelial plaques are specialized membrane domains in urothelial superficial (umbrella) cells, composed of highly ordered uroplakin particles. We investigated membrane compartments involved in the formation of urothelial plaques in mouse umbrella cells. The Golgi apparatus did not contain uroplakins organized into plaques. In the post-Golgi region, three distinct membrane compartments containing uroplakins were characterized: i) Small rounded vesicles, located close to the Golgi apparatus, were labelled weakly with anti-uroplakin antibodies and they possessed no plaques; we termed them “uroplakin-positive transporting vesicles” (UPTVs). ii) Spherical-to-flattened vesicles, termed “immature fusiform vesicles” (iFVs), were uroplakin-positive in their central regions and contained small urothelial plaques. iii) Flattened “mature fusiform vesicles” (mFVs) contained large plaques, which were densely labelled with anti-uroplakin antibodies. Endoytotic marker horseradish peroxidase was not found in these post-Golgi compartments. We propose a detailed model of de novo urothelial plaque formation in post-Golgi compartments: UPTVs carrying individual 16-nm particles detach from the Golgi apparatus and subsequently fuse into iFV. Concentration of 16-nm particles into plaques and removal of uroplakin-negative membranes takes place in iFVs. With additional fusions and buddings, iFVs mature into mFVs, each carrying two urothelial plaques toward the apical surface of the umbrella cell

Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development

Postnatal rat urothelium was studied from day 0 to day 14, when intense cell loss as part of tissue remodeling was expected. The morphological and biochemical characteristics of urothelial cells in the tissue and released cells were investigated by light and electron microscopy, by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay, by annexin V/propidium iodide assay, and by immunofluorescent detection of active caspases and tight-junction protein occludin. Intense apoptosis and massive desquamation were detected between postnatal days 7 and 10. During this period, active caspases and TUNEL-positive cells were found in the urothelium. Disassembled cell–cell junctions were detected between cells. The majority of desquamated cells expressed no apoptotic cell morphology, but were active caspase positive and TUNEL positive. Ann+/PI− apoptotic bodies and desquamated Ann+/PI+ cells were detected in the lumen. These results indicate that apoptosis and desquamation participate in urothelial cell loss in the rat early postnatal period, indispensable for fast urothelial remodeling during development. (J Histochem Cytochem 57:721–730, 2009

Comparison of chemically and HPF fixed umbrella cells of the urothelium.

<p>mFVs are the most prevailing compartments in chemically (A, C) and in HPF fixed umbrella cells (B, D). Regarding the ultrastructure, mFVs are more dilated in chemically fixed (A, C) than in HPF fixed samples (B, D). Regarding immunolabelling with anti-AUM antibody, pattern and density of labelling is comparable on chemically (C) and HPF fixed samples (D). The organization of mFVs into stacks is better preserved in HPF fixed samples (red over-colour in B and D). Bars: 500 nm.</p

Structure of a mFV.

<p>(A) mFVs are flattened vesicles with two opposing plaques of thickened asymmetric unit membrane (AUM, blue) and slightly dilated, omega shaped hinge region of un-thickened membrane (yellow). (B) AUM and hinge regions can be seen also in the apical plasma membrane. (C) By freeze-fracturing, uroplakin particles are seen concentrated in the centre of the mFV (blue), while thin hinge region contains particle-free membranes (yellow). (D–G) Three-dimensional model of mFV shows that it has the shape of a flattened disk. In D, a slice from a tomogram is shown. In E–G, a 3D model of a mFV in different projections is shown (green). Bars: 100 nm in A, C; 50 nm in B.</p

Mature FVs are only present in umbrella cells.

<p>(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.</p

Stacks of mFV in the central cytoplasm of umbrella cells.

<p>(A) Cytoplasm of umbrella cells is divided into two regions: sub-apical (S_A) and central (C_E). 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.</p

MFUM-BrTNBC-1, a Newly Established Patient-Derived Triple-Negative Breast Cancer Cell Line: Molecular Characterisation, Genetic Stability, and Comprehensive Comparison with Commercial Breast Cancer Cell Lines

Triple-negative breast cancer (TNBC) is a breast cancer (BC) subtype that accounts for approximately 15–20% of all BC cases. Cancer cell lines (CLs) provide an efficient way to model the disease. We have recently isolated a patient-derived triple-negative BC CL MFUM-BrTNBC-1 and performed a detailed morphological and molecular characterisation and a comprehensive comparison with three commercial BC CLs (MCF-7, MDA-MB-231, MDA-MB-453). Light and fluorescence microscopy were used for morphological studies; immunocytochemical staining for hormone receptor, p53 and Ki67 status; RNA sequencing, qRT-PCR and STR analysis for molecular characterisation; and biomedical image analysis for comparative phenotypical analysis. The patient tissue-derived MFUM-BrTNBC-1 maintained the primary triple-negative receptor status. STR analysis showed a stable and unique STR profile up to the 6th passage. MFUM-BrTNBC-1 expressed EMT transition markers and displayed changes in several cancer-related pathways (MAPK, Wnt and PI3K signalling; nucleotide excision repair; and SWI/SNF chromatin remodelling). Morphologically, MFUM-BrTNBC-1 differed from the commercial TNBC CL MDA-MB-231. The advantages of MFUM-BrTNBC-1 are its isolation from a primary tumour, rather than a metastatic site; good growth characteristics; phenotype identical to primary tissue; complete records of origin; a unique identifier; complete, unique STR profile; quantifiable morphological properties; and genetic stability up to (at least) the 6th passage