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. 2009 Sep;10(9):1350-61.
doi: 10.1111/j.1600-0854.2009.00950.x. Epub 2009 May 26.

Involvement of vps33a in the fusion of uroplakin-degrading multivesicular bodies with lysosomes

Xuemei Guo  1 , Liyu Tu, Iwona Gumper, Heide Plesken, Edward K Novak, Sreenivasulu Chintala, Richard T Swank, Gregory Pastores, Paola Torres, Tetsuro Izumi, Tung-Tien Sun, David D Sabatini, Gert Kreibich
Affiliations

Involvement of vps33a in the fusion of uroplakin-degrading multivesicular bodies with lysosomes

Xuemei Guo et al. Traffic. 2009 Sep.

Abstract

The apical surface of the terminally differentiated mouse bladder urothelium is largely covered by urothelial plaques, consisting of hexagonally packed 16-nm uroplakin particles. These plaques are delivered to the cell surface by fusiform vesicles (FVs) that are the most abundant cytoplasmic organelles. We have analyzed the functional involvement of several proteins in the apical delivery and endocytic degradation of uroplakin proteins. Although FVs have an acidified lumen and Rab27b, which localizes to these organelles, is known to be involved in the targeting of lysosome-related organelles (LROs), FVs are CD63 negative and are therefore not typical LROs. Vps33a is a Sec1-related protein that plays a role in vesicular transport to the lysosomal compartment. A point mutation in mouse Vps33a (Buff mouse) causes albinism and bleeding (Hermansky-Pudlak syndrome) because of abnormalities in the trafficking of melanosomes and platelets. These Buff mice showed a novel phenotype observed in urothelial umbrella cells, where the uroplakin-delivering FVs were almost completely replaced by Rab27b-negative multivesicular bodies (MVBs) involved in uroplakin degradation. MVB accumulation leads to an increase in the amounts of uroplakins, Lysosomal-associated membrane protein (LAMP)-1/2, and the activities of beta-hexosaminidase and beta-glucocerebrosidase. These results suggest that FVs can be regarded as specialized secretory granules that deliver crystalline arrays of uroplakins to the cell surface, and that the Vps33a mutation interferes with the fusion of MVBs with mature lysosomes thus blocking uroplakin degradation.

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Figures

Figure 1
Figure 1. The lumen of umbrella cell FVs are acidified
A) Acridine orange staining of mouse urothelium. Mouse bladder explants were incubated with acridine orange and the fluorescent reagent accumulating in acidified compartments was detected by its characteristic orange emission by fluorescence microscopy. B) Immunostaining of mouse bladder sections with an anti-LAMP-1 antibody. C) Immunolocalization of DAMP on frozen sections from a mouse bladder incubated in situ with DAMP by immunofluorescence. At the ultrastructural level (D and E), gold-conjugated protein A was used. L indicates the lumen of the bladder. The location of the urothelial basement membrane is indicated by a dashed line in (B) and (C). Note acridine orange (A) and DAMP (green, C) accumulated predominantly in umbrella cells, while LAMP-1 staining (B) was rather evenly distributed in all layers of the urothelium. In panels (D and E), DAMP localized mainly to FVs (FV) and to lysosomes (Lys).
Figure 2
Figure 2. Mature FVs do not contain CD63
Paraffin sections of bladders from wild-type mice were immunostained with antibodies against UPIII (red) and CD63 (green). There is very little CD63 expressed in umbrella cells where most of the mature FVs are located. Arrows indicate colocalization of UPIII and CD63 in the intermediate cell layers. The line in the Merge panel indicates the location of the basal lamina.
Figure 3
Figure 3. Mutation of Vps33a in the Buff mouse results in the accumulation of numerous MVB-like vacuoles in urothelial umbrella cells
Light micrographs of normal (A) and Buff mouse (C) urinary bladders, embedded in epon and stained with toluidine blue. While normal urothelial umbrella cells (A) have few vacuoles, those from the Buff mouse (C) are highly vacuolated. Panels (B) and (D) represent magnified areas from the overview EMs shown in Figures S1 and S2 of the Supporting Information. Asterisks in panel B indicate MVBs in umbrella cells of normal urothelium. Panels (E) and (F) are higher magnification images of the apical plasma membrane of control and Buff mouse umbrella cells, respectively, showing the typical AUM appearance. G) Apical portion of an umbrella cell from an 8-day-old Buff mouse where FVs are still the predominant organelles.
Figure 4
Figure 4. The MVB-like vacuoles in umbrella cells of Buff mice are related to FVs and belong to the degradative pathway
Gallery of electron micrographs of cytoplasmic vacuoles in Buff mouse umbrella cells. A) The MVB-like vacuoles have rather angular contours, indicating a certain rigidity of the limiting membranes. B) At higher magnification, the asymmetric unit membrane (AUM) structure, typical of FVs and of the apical plasma membrane of normal umbrella cells, can also be seen in these MVB-like vacuoles. C–E) The luminal contents of these large MVB-like vacuoles differ widely.
Figure 5
Figure 5. Changed expression pattern of UPIII, Rab27b and LAMP-1 in umbrella cells of Buff mice
Paraffin sections of bladders from wild-type (WT; A–C, G–I) or Buff mice (Buff; D–F, J–L) were immunostained with antibodies against UPIII (A,D,G,J), Rab27b (B,E) or LAMP-1 (H,K). The line in the ‘Merge’ panels indicates the location of the basal lamina.
Figure 6
Figure 6. The large MVB-like vacuoles in umbrella cells of Buff mice are UPIII-positive, Rab27b-negative and LAMP-1-positive
Frozen thin sections of the bladder of WT (WT; A,C,E) and Buff mice (Buff; B,D,F) were immunogold labeled using primary antibodies against UPIII (A,B), Rab27b (C,D) or LAMP-1 (E,F). Note that the large MVB-like vacuoles in umbrella cells of Buff mice contain LAMP-1 and UPIII, but not Rab27b. Asterisks in panel B indicate UPIII associated with intraluminal vesicles of MVBs. Asterisks in panel C indicate Rab27b localized to fusiform vesicles. Asterisks in panel D indicate that Rab27b is rarely detected in MVBs.
Figure 7
Figure 7. The urothelium of Buff mice contains higher concentrations of LAMP-1, LAMP-2, uroplakins and Rab27b
Fifty micrograms each of total urothelial proteins obtained from wild-type (WT) or Buff (Buff) mice were separated by SDS–PAGE, and immunoblotted using antibodies against LAMP-1, LAMP-2, UPII, UPIII, Rab27b or actin. Note the significant increase in the relative amounts of both uroplakins and the LAMPs in the urothelium of the Buff mouse when compared with that of WT bladder urothelium.
Figure 8
Figure 8. Schematic representation of an urothelial umbrella cell
Emphasized are likely steps (1-15) involved in the biogenesis and targeting of FVs as well as in their degradation. The green arrows indicate steps involved in the biogenesis and exocytosis of FVs, whereas the orange ones mark the endocytic pathway affecting AUMs (see also the introductory paragraph). Blue arrows connect steps that function in the formation of lysosomes. Our results suggest that Vps33a functions in step (11) that is involved in the fusion of MVBs with mature lysosomes (Lys), but not in step (12) that regulates the delivery of lysosomal enzymes to late endosomes or MVBs. Inhibition of step (11) caused by the point mutation in Vps33a is most likely responsible for the accumulation of UPs that are contained in the MVBs.
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