Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE WEIMBSLABORATORYPROJECT SCIENTISTSSeng Hui Low, Ph.D.Jayasri Nanduri, Ph.D.POSTDOCTORAL FELLOWXin Li, M.D., Ph.D.GRADUATE STUDENTSNikunj Sharma, MSShivakumar Vasanth, MSSENIOR TECHNOLOGISTBonnie Gorzelle, MSTECHNICIANSClaire Larson, BSMark DeGennaro, BSRESEARCH STUDENTSMin HeJunko IwasakiThomas Weimbs, Ph.D.We are interested in the molecularmechanisms of the establishment andmaintenance of epithelial cell polarity.Epithelial cells, e.g., kidney tubule cells, havetwo distinct plasma membrane (PM) domains,the apical and basolateral domains. The proteinand lipid compositions of these two PM domainsare very different from each other, which isessential for the proper function of epithelialtissues. Apical and basolateral PM proteins aretargeted to their respective domains by highlyspecific vesicular transport. The final step in eachpathway is the fusion of the transport vesicleswith the PM. A ubiquitous membrane fusionmachinery, the “SNARE machinery,” has beenidentified to be involved in most, if not all,intracellular vesicle fusion events. Soluble N-ethylmaleimide-sensitive factor attachmentprotein receptors (SNAREs) are membraneproteins localized on the vesicle membrane (v-SNAREs) and target membrane (t-SNAREs).Membrane fusion requires the binding of amatching combination of v- and t-SNAREs. Wehave previously characterized in detail thesubcellular localization and function of differentSNARE isoforms in polarized Madin Darbycanine kidney (MDCK) cells, a model cell line forthe study of membrane traffic in epithelial cells.We are currently focusing on several interrelatedquestions.SNARES/Cell PolarityWe are investigating whether the mutuallyexclusive localization of the t-SNAREs syntaxin3 and 4 on the apical and basolateral plasmamembrane domains, respectively, is necessary forcell polarity. We are identifying the targetingsignals on these syntaxins, investigating howLi, X., Low, S.H., Miura, M., and T. Weimbs (2002) SNARE expression and localizationin renal epithelial cells suggest mechanism for variability of trafficking phenotypes.Am. J. Physiol. Renal Physiol. 283:F1111-F1122.Low, S.H., Marmorstein, L.Y, Miura, M., Li, X., Kudo, N., Marmorstein, A.D., and T.Weimbs (2002) Retinal pigment epithelial cells exhibit unique expression andlocalization of plasma membrane syntaxins which may contribute to their traffickingphenotype. J. Cell Sci. 115:4545-4553 (journal cover article).Kreitzer, G., Schmoranzer, J., Low, S.H., Li, X., Gan, Y., Weimbs, T., Simon, S.M.,and E. Rodriguez-Boulan (2003) Three-dimensional analysis of post-Golgi carrierexocytosis in epithelial cells. Nat. Cell Biol. 5:126-136.Sherief, M.H., Low, S.H., Miura, M., Kudo, N., Novick, A.C., and T. Weimbs (2003)Matrix metalloproteinase activity in urine of patients with renal cell carcinoma leads todegradation of extracellular matrix proteins: possible use as a screening assay. J.Urol. 169:1530-1534.Low, S.H., Li, X., Miura, M., Kudo, N., Quinones, B., and T. Weimbs (2003) Syntaxin 2and endobrevin are required for the terminal step of cytokinesis in mammalian cells.Dev. Cell 4:753-759.Weimbs, T., Low, S.H., Li, X., and G. Kreitzer (2003) SNAREs and epithelial cells.Methods 30:191-197.The Department of Cell BiologyMembrane Trafficking Trackedin Polarized Epithelial Cellsthese SNAREs themselves reach the apical andbasolateral PM domain, and testing whetherdisruption of their targeting leads to defects inepithelial cell polarity.SNARES/Cell DivisionWe are exploring the role of SNAREs incell division (cytokinesis). We have identified, forthe first time, that two SNARE proteins localizespecifically to the so-called midbody, a narrowcytoplasmic bridge that connects the twoprospective daughter cells. Only after cleavage ofthis midbody is cytokinesis complete. Inhibitionof these SNAREs leads to failure of midbodycleavage and results in binucleated cells. This isthe first demonstration of a requirement forSNARE-mediated membrane fusion during thefinal step of the cell cycle and identifies newpotential molecular targets for tumor therapy.Polycystin-1 in Kidney DiseasePolycystin-1 is the protein affected in~85% of the cases of autosomal-dominantpolycystic kidney disease (PKD). Although it iswell established that renal epithelial cells in PKDexhibit defects in polarized protein targeting, cellproliferation and apoptosis, the function ofpolycystin-1 remains unknown. We are using anovel in vitro cell culture approach in which wecan interfere with polycystin-1 function byoverexpression of dominant-negative inhibitorsto identify the downstream signaling pathwaysinvolved in the pathogenesis of PKD. This workwill ultimately allow clinicians and researchers todevise strategies for pharmacological treatment ofthis disease.Renal Cell Carcinoma MarkersThe aim of this project is to identifyurinary protein markers for the early detection ofrenal cell carcinoma (RCC), a highly malignantcancer for which such early detection is currentlynot possible. We have discovered that the urineof RCC patients contains highly elevated levels ofmatrix metalloproteinase activity that leads to thedegradation of normally excreted extracellularmatrix proteins. Urinalysis has allowed us todetect RCC, even at the earliest stages, with asensitivity close to 100%. We are currentlydeveloping these detection assays for possiblefuture commercial use, which will allow widespreadapplication.82