Nataša Resnik

Desmosome Assembly and Cell-Cell Adhesion Are Membrane Raft-dependent Processes

The aim of our study was to investigate the association of desmosomal proteins with cholesterol-enriched membrane domains, commonly called membrane rafts, and the influence of cholesterol on desmosome assembly in epithelial Madin-Darby canine kidney cells (clone MDc-2). Biochemical analysis proved an association of desmosomal cadherin desmocollin 2 (Dsc2) in cholesterol-enriched fractions that contain membrane raft markers caveolin-1 and flotillin-1 and the novel raft marker ostreolysin. Cold detergent extraction of biotinylated plasma membranes revealed that ∼60% of Dsc2 associates with membrane rafts while the remainder is present in nonraft and cholesterol-poor membranes. The results of immunofluorescence microscopy confirmed colocalization of Dsc2 and ostreolysin. Partial depletion of cholesterol with methyl-β-cyclodextrin disturbs desmosome assembly, as revealed by sequential recordings of live cells. Moreover, cholesterol depletion significantly reduces the strength of cell-cell junctions and partially releases Dsc2 from membrane rafts. Our data indicate that a pool of Dsc2 is associated with membrane rafts, particularly with the ostreolysin type of membrane raft, and that intact membrane rafts are necessary for desmosome assembly. Taken together, these data suggest cholesterol as a potential regulator that promotes desmosome assembly.

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells

Background information. The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal‐ or fusiform‐shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved.

The role of cholesterol-sphingomyelin membrane nanodomains in the stability of intercellular membrane nanotubes

Intercellular membrane nanotubes (ICNs) are highly curved tubular structures that connect neighboring cells. The stability of these structures depends on the inner cytoskeleton and the cell membrane composition. Yet, due to the difficulty in the extraction of ICNs, the cell membrane composition remains elusive. In the present study, a raft marker, ostreolysin, revealed the enrichment of cholesterol-sphingomyelin membrane nanodomains along ICNs in a T24 (malignant) urothelial cancer cell line. Cholesterol depletion, due to the addition of methyl-β-cyclodextrin, caused the dispersion of cholesterol-sphingomyelin membrane nanodomains and the retraction of ICNs. The depletion of cholesterol also led to cytoskeleton reorganization and to formation of actin stress fibers. Live cell imaging data revealed the possible functional coupling between the change from polygonal to spherical shape, cell separation, and the disconnection of ICNs. The ICN was modeled as an axisymmetric tubular structure, enabling us to investigate the effects of cholesterol content on the ICN curvature. The removal of cholesterol was predicted to reduce the positive spontaneous curvature of the remaining membrane components, increasing their curvature mismatch with the tube curvature. The mechanisms by which the increased curvature mismatch could contribute to the disconnection of ICNs are discussed.

Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1–positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

Highly Selective Anti-Cancer Activity of Cholesterol-Interacting Agents Methyl-β-Cyclodextrin and Ostreolysin A/Pleurotolysin B Protein Complex on Urothelial Cancer Cells

Cholesterol content can vary distinctly between normal and cancer cells, with elevated levels in cancer cells. Here, we investigated cholesterol sequestration with methyl-β-cyclodextrin (MCD), and pore-formation with the ostreolysin A/pleurotolysin B (OlyA/PlyB) protein complex that binds to cholesterol/sphingomyelin-rich membrane domains. We evaluated the effects on viability of T24 invasive and RT4 noninvasive human urothelial cancer cells and normal porcine urothelial (NPU) cells. Cholesterol content strongly correlated with cancerous transformation, as highest in the T24 high-grade invasive urothelial cancer cells, and lowest in NPU cells. MCD treatment induced prominent cell death of T24 cells, whereas OlyA/PlyB treatment resulted in greatly decreased viability of the RT4 low-grade noninvasive carcinoma cells. Biochemical and transmission electron microscopy analyses revealed that MCD and OlyA/PlyB induce necrotic cell death in these cancer cells, while viability of NPU cells was not significantly affected by either treatment. We conclude that MCD is more toxic for T24 high-grade invasive urothelial cancer cells, and OlyA/PlyB for RT4 low-grade noninvasive urothelial cancer cells, and neither is toxic for NPU cells. The cholesterol and cholesterol/sphingomyelin-rich membrane domains in urothelial cancer cells thus constitute a selective therapeutic target for elimination of urothelial cancer cells.

Colocalization of Serum Amyloid A with Microtubules in Human Coronary Artery Endothelial Cells

Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1μm, and larger vesicles of around 10-μm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Autophagic activity in the mouse urinary bladder urothelium as a response to starvation.

The urinary bladder urothelium is subjected to mechanical forces during cycles of distension and contraction, and its superficial cells are constantly flushed by toxic urine. Yet, the urothelium shows a very slow turnover of cells and superficial cells are extremely long lived. Autophagy has a well-known role in tissue homeostasis and serves as a protective mechanism against cellular stress. Therefore, the presence of autophagy as one of possible processes of survival in an unpleasant environment and during long lifetime of superficial cells was examined in mouse urothelium. We detected and evaluated autophagic activity of superficial urothelial cells under normal and stress conditions, caused by short-term starvation of newborn and 24-h-starved adult mice. Immunolabeling and Western blotting of essential effectors of autophagy, LC3 and Beclin 1, showed a weak signal in superficial urothelial cells. On the other hand, ultrastructural analysis, which proved to be the most reliable method in our study, revealed the presence of autophagic vacuoles, some of them containing specific urothelial structures, fusiform vesicles. Quantitative analysis showed increased autophagy in newborn and starved mice in comparison to a low basic level of autophagy in the urothelium of normal mice. Interestingly, some superficial cells of adults and neonates exhibit intense immunoreactions against LC3 and Beclin 1 and the typical ultrastructural characteristics of autophagy-dependent cell death. We conclude that autophagy, despite low basic activity under physiological conditions, plays an important role in urothelial homeostasis and stability under stress.

Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models

Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking from the endoplasmic reticulum to the GA in UCs. Moreover, UPIb, UPIIIa or UPIb/UPIIIa expressing UCs revealed fragmentation and peripheral redistribution of Golgi-units. Notably, expression of UPIb or UPIb/UPIIIa triggered similar GA fragmentation in MDCK and HeLa cells that do not express UPs endogenously. The colocalization analysis of UPIb/UPIIIa-EGFP and COPI, COPII or clathrin suggested that UPs follow constitutively the post-Golgi route to the apical PM. Depolymerisation of microtubules leads to complete blockade of the UPIb/UPIIIa-EGFP post-Golgi transport, while disassembly of actin filaments shows significantly reduced delivery of UPIb/UPIIIa-EGFP to the PM. Our findings show the significant effect of the UPs expression on the GA fragmentation, which enables secretory Golgi-outpost to be distributed as close as possible to the sites of cargo delivery at the PM.

Comparative lipidomic study of urothelial cancer models: association with urothelial cancer cell invasiveness

Comparative lipidomic studies were performed across the RT4 versus T24 urothelial cancer cell lines, as models for noninvasive urothelial papilloma cells (with a relatively high level of differentiation) and invasive urothelial carcinoma cells (with low level of differentiation), respectively. The aim was to investigate the differences in lipid profile associated with different levels of urothelial cancer cell invasiveness. The cellular lipidomes were characterized using our previously developed joint methodology of liquid chromatography-mass spectrometry and high-resolution nuclear magnetic resonance, which included analysis of the phospholipids and ceramide-based glycosphingolipids. This study shows that the invasive T24 cells have 3-fold lower levels of 1-alkyl (ether)-2-acyl phosphocholine species, which are accompanied by greater length and higher unsaturation of acyl chains of several lipid classes. Moreover, d18:1-based glycosphingolipids show different profiles; in particular, α-hydroxylated glucosylceramides have lower levels in the T24 cells, along with increased lactosyl ceramides. These differences between RT4 and T24 cells suggest significantly different organization of the cellular membranes, which can affect the membrane fluidity and membrane-dependent functions, and contribute to the lower stiffness of plasma membrane and reduced cell-cell adhesion required for movement and invasiveness of these T24 urothelial carcinoma cells with a high metastatic potential.