Electron tomography of fusiform vesicles and their organization in urothelial cells

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 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.

Figure 1. Comparison of chemically and HPF fixed umbrella cells of the urothelium.

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.

Figure 2. Structure of a mFV.

(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.

Figure 3. Stacks of mFV in the central cytoplasm of umbrella cells.

(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.

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.

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.