Urothelial plaque formation in post-Golgi compartments

Abstract

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.

Figure 1. Uroplakins are not accumulated in the Golgi cisternae, but in the post-Golgi compartments.

A) Top-view on the umbrella cell immunolabelled with anti-GM130 (green) shows a GA network spread across the whole cytoplasm. B) Plaques of asymmetric thickened membrane are not seen in the Golgi cisternae. C) Inset shows higher magnification of GA part, which has membranes 8–9 nm thick. D and E) Optical sections created from electron tomogram reconstruction, with superimposed models of the GA. D) GA forms flattened, occasionally fenestrated cisternae of various lengths (violet). E) From the rims of trans-Golgi cisterne and from the trans-Golgi network emanate tubulo-vesicular extensions (gold; GA cisternae are violet). F) Double immunolabelling with anti-gianti antibody (green, arrows) and anti-AUM antibody against mature uroplakins (red), shows no colocalization in the umbrella cell. GA (arrows) lies lateral to the nucleus, while uroplakins are seen throughout the cytoplasm (*) and on the apical surface (arrow-head). G) Labelling of cryo-ultrathin sections with anti-AUM antibody is negative in the GA and positive in post-Golgi compartments (arrows). Legend: M – mitochondria; blue – nucleus (DAPI). Bars 10 µm in A, F, 200 nm in B, G. Tomogram for D, E was taken at 6500×.

Figure 2. Mature uroplakins are first detected in UPTVs.

A) UPTVs are post-Golgi compartments (black arrows), which are positioned next to the GA. Rims of some GA cisternae have coated membranes (white arrow). B) Projection of 3-D model shows that UPTVs are individual rounded membrane compartments (green; trans-Golgi network is gold). C) Sparse mature uroplakins are detected in UPTVs (arrows) and in the small iFVs (bigger arrows) next to the GA. Some iFVs are densely anti-AUM labelled. D) Freeze-fracture of umbrella cells shows UPTVs (arrows), which are positioned close to each other suggesting fusions of vesicles (white arrow). Larger iFVs carry numerous protein particles gathered into urothelial plaques (*). Arrow in circle indicates direction of Pt shadowing. Bars 200 nm.

Figure 3. Spatial and size correlations between UPTVs, iFVs and mFVs suggest gradual formation of urothelial plaques in the biosynthetic pathway.

A) UPTVs (arrows) and small iFVs (1) accumulate next to the polarised stack of larger iFVs (2) and mFVs (3). B) Freeze-fracture replica showing polarized stack of iFVs. Arrow in circle indicates direction of Pt shadowing. C) Uroplakin particles accumulate progressively from UPTVs, over iFVs to mFVs. UPTVs and smaller iFVs (black arrows) are detected next to larger iFVs. The central region of these vesicles shows heavy anti-AUM labelling (big arrow), while the rims remain uroplakin negative (white arrows). Note anti-AUM negative rounded vesicles (striped arrows) that might be involved in plaque formation by removing the excess of non-thickened membranes from maturing FVs. D) iFVs have vesicular dilatations (arrow-heads) and vesicles are seen in their vicinity (arrows), suggesting intense vesicular traffic . E) Various sizes of plaques of asymmetrically thickened membrane (black arrows) in iFVs are seen on ultra-thin section. Asymmetrically thickened membrane (12 nm thick) lines the central regions of iFVs, while non-thickened membranes (arrow-heads) line the rims of iFVs and separates plaques. Domains of non-thickened membrane appear dilated. F) At higher magnification, 12 nm thick plaque (arrow) and 9 nm thick hinge regions (arrow-head) are seen. G) Smaller iFVs (arrow-heads) and three larger iFVs, which contain a remarkably varied number of protein particles (black arrows) in their plaques. iFVs that contain a high number of particles (*) in their plaques, contain only a small amount of particle-free membranes (φ). White arrow shows close proximity of a smaller and bigger iFV, a position suggesting their fusion, thus increasing the number of uroplakin particles. H and I) Optical sections created from electron tomogram reconstruction, with superimposed models of iFVs. H) UPTVs are positioned close to each other (green), and next to small iFV (blue). I) Small iFVs (blue) are spherical or ellipsoid, while bigger iFVs (yellow) are flattened. Bars 200 nm, 500 nm in B, G.

Figure 4. mFVs present the final stage of urothelial plaque formation.

A) On the micrograph are shown mFVs organized into a stack. mFVs have a narrow intravesicular lumen and un-dilated rims. B) Projection of 3-D model shows difference in size and shape between bigger iFV (yellow) and mFVs (red). C) mFVs have urothelial plaques (*) positioned centrally. Note flattened, coin-like shape of the mFV. Arrow in circle indicates direction of Pt shadowing. D) After two hours of endocytosis, the HRP reaction product is present in a multivesicular body (arrow), while the majority of multivesicular bodies (arrow-heads) and all of the FVs remain un-labelled. Bars 500 nm.

Figure 5. Model of post-Golgi formation of urothelial plaques.

1) UPTVs with sparse uroplakin particles (red) leave trans-Golgi network (GA). 2) iFVs are formed by homotypic fusion of UPTVs (arrows). 3) In iFVs, the number of particles increases and small plaques are formed, which leads to a flattening of the iFVs. Small iFVs further fuse with each other (arrows) or with UPTVs to form large iFVs that eventually constitute a polarized stack of iFVs. 4) In iFVs membrane sorting and particle concentration take place. At the vesicle's rims, fusions with UPTVs and iFVs incorporate additional uroplakin particles (arrow in), while budding removes excess of non-thickened membrane (arrow out and 5). In the central part of the vesicle, uroplakin particles are concentrated into growing plaque (red line). 6) When the size of the plaque reaches dynamic equilibrium , mFVs are formed. 7) mFVs are detached from the stack and can be transported to the apical plasma membrane. Legend: green, blue and red lines represent initial-, central- and final stage of plaque formation, characterized by UPTVs, iFVs and mFVs, respectively.