Caveolae and protein sorting in epithelia cells
Previous and Current Research
My laboratory concentrates on two inter-connected topics: (1) biogenesis and function of caveolae and (2) the protein sorting in epithelial cells. Our interest on the caveolar structure/function was initiated through the investigations on polarized sorting of proteins in epithelial cells. In fact, the biogenesis of caveolae could be regarded as a part of a wider problem: the protein sorting in the cell.The surface of many cells is covered by small invaginations or caveolae (“small caves”). These invaginations are surrounded by a characteristic filamentous coat and also display a specific lipid composition. Caveolin-1 is a cholesterol binding protein and caveolae are enriched in cholesterol and glycosphingo-lipids. Actually, caveolae could be considered as a specific form of membrane microdomains (rafts). Although the exact function of caveolae is not known, many, in part controversial, functions, have been suggested. In our investigations we invested considerable effort to dissect molecular mechanisms governing formation of the coat, intracellular transport of caveolins (or caveolae) and to identify other resident proteins of caveolae. Using the RNA-interference approach we have recently shown that caveolin-1 in C. elegans is involved in a signal transduction process and its deficiency leads to the acceleration of meiotic cell cycle.Another problem investigated in the lab is the establishment of the polarity of the epithelial cell. This polarity is manifested by the existence of two membrane domains, apical and baso-lateral, with different biochemical and functional properties. In addition, the polarity of an embryo is fundamental for the correct development of an organism. We reasoned that the mechanisms involved in the initial polarizationof embryo and the maintenance of the epithelial polarity could be governed by the same mechanisms. In C. elegans the process of asymmetric division requires six par-genes (partition-defective). We have recently shown that a mammalian homologue of one of them, a serine-treonine kinase mPAR-1 is expressed in a wide variety of epithelial tissues and is asymmetrically localized to the lateral domain. A fusion protein lacking the kinase part shows the same localization and its prolonged expression acts in a dominant-negative fashion: lateral adhesion of the transfected cells to neighbouring cells is diminished, resulting in the "squeezing out" of the former from the monolayer. Very recently we have found that, in addition to the lateral cortex, mPAR-1 associates with centrosomes, spindle and astral microtubules. Targeted down regulation of the mPAR-1 family member EMK leads to disorientated spindles which deviate up to 90° from the normal plane. However, the integrity of tight junction and polarised distribution of proteinsare maintained in these cells at confluency. Thus mPAR-1 acts as a molecular cue for proper spindle orientation in epithelial cells.
Future Projects and Goals
Our ultimate goal remains the understanding of the function of caveolins and caveolae in a living organism. Presently we are producting caveolin \knock-out\ mice. Paralelly we will continue our work on C. elegans caveolins using dominant negative approach or producing deletion mutants. Cholesterol transport is an essential process in all multicellular organisms. We plan to apply two recently developed approaches to investigate the distribution and molecular mechanisms of cholesterol transport in C. elegans. The distribution of cholesterol in living worms will be studied by imaging its fluorescent analog, dehydroergosterol. We also plan to use a photoactivatable cholesterol analog to identify cholesterol-binding proteins in C. elegans. Our present work is aimed at the identification of the interaction partners of the mPAR-1, its upstream effectors and downstream targets. This could reveal molecular mechanisms of a fundamental problem in the cell biology: the spindle positioning in epithelial cells.
Methodological and Technical Expertise
- Isolation and purification of lipids from C. elegans
- Lipid biochemical analysis (TLC, LC/MS, etc.)
- Metabolic radio- and stable isotope labeling C. elegans for lipid/sterol/carbohydrate analysis
- Genetic analysis of the dauer stage in C. elegans
- Induction and analysis of responses to desiccation and hypoxia in C. elegans
Scheel, J., Srinivasan, J. Honnert, U., Henske, A., and Kurzchalia, T. V.
Involvement of caveolin-1 in meiotic cell cycle progression in C. elegans.
Nat. Cell Biol., 1. 117–119 (1999)
Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzchalia, T. V.
Distribution and transport of cholesterol in Caenorhabditis elegans.
Mol. Biol. Cell., 12, 1725–1736 (2001)
Drab, M., Verkade, P., Elger, M., Kasper, M., Lohn, M., Lutterbach, B., Menne, J., Lindschau, K., Mende, F., Luft, F., Schedl, A., Haller, H. and Kurzchalia, T. V.
Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.
Science, 293, 2449–2452 (2001)
Kurzchalia, T. V. and Ward, S.
Why do worms need cholesterol?
Nat. Cell Biol., 5 (2003)
Matyash, V., Entchev, E., Mende, F., Wilsch-Bräuninger, M., Thiele, C., Schmidt, A., Knölker, H.-J., Ward, S., and Kurzchalia T. V.
Sterol-derived hormone(s) control entry into diapause in Caenorabditis elegans by consecutive activation of DAF-12 and DAF-16.
PloS Biology, 2, (in press) (2004)