Phone: +49-7531-88-2228
Fax: +49-7531-88-2245
Room: X 2113

Secretariate: N.N.
Phone: +49-7531-88-2231
Room: X 2112

We mainly work on secretion with Paramecium cells (ciliated protozoa), for the following reasons. These cells can be mass-cultivated sterilely, exocytosis of almost all of the numerous secretory organelles can be triggered synchronously (0.1 sec) and mutations are available on different levels of the secretory pathway (including vesicle docking at the cell membrane or membrane fusion). Paramecia can easily be microinjected and we also have established cell fractionation methods. Furthermore, we can synchronize the different steps of the secretory cycle preceding exocytosis by orders of magnitude. Synchronization is important to avoid overlaps of partly opposite effects (e.g. reversible protein phosphorylation and de-phosphorylation) and to pinpoint the role of individual parameters which sometimes have multiple effects (e.g. calcium). This can be best achieved in combination with quenched-flow analysis.

So far our most important findings with this system are the following. Intracellular transport of secretory vesicles occours along a transiently formed microtubule (MT) population in a plus-to-minus direction (as found later in polarized epithelia). The docking site is specified independently from the MT organizing center. Specific structural rearrangements of integral and peripheral proteins (including calmodulin) are required to establish membrane fusion capacity. Membrane fusion during exocytosis is of the focal type (10 nm pore size, 1 millisec), accompanied by a dispersal of protein subunits and dephosphorylation of a peripheral phosphoprotein (PP63). We found evidence for dephosphorylation by a calcium-calmodulin-activated phosphatase of the calcineurin-type. (The recent availability of protein phosphatase inhibitors has yielded similar data with other cell types). Calcium mobilized from extensive subplasmalemmal pools (which we have isolated and characterized) is sufficient to drive membrane fusion. (This is in line with the recently claimed importance of structurally non-identified subplasmalemmal stores in mammalian cells). However, this has to be superimposed by a Calcium-influx, to drive exo- and endocytosis with maximal rate. Exocytotic membrane fusion is facilitated by hydrolyzable guanosintriphosphate (GTP), whereas adenosintriphosphate exerts a priming effect (later found in other cell types). Among the second messengers analyzed only cyclic GMP rises, though only after membrane fusion has occured. A novel calcium-inhibited casein kinase or a GTP-activated protein kinase can rephosphorylate PP63 in vitro. The spectrum of phosphatases and kinases is currently analyzed as well as the identity of other proteins of the docking/fusion sites for subsequent immuno-localization. Their self-assembly is a prerequisite for exocytosis competence. Since 1995 our group has established molecular genetics and, thus, isolated two genes for PP63 isoforms.

In occasional collaborations we test some of these aspects with mammalian secretory cells. Our "focal fusion concept" (including regulatory proteins) has been generally accepted (maily on the basis of recent patch-clamp experiments by other groups) for different biomembrane fusion events and many groups now work on identifying the respective proteins involved in fusion regulation.

Selected publications:

Plattner, H., C. J. Lumpert, G. Knoll, R. Kissmehl, B. Höhne, M. Momayezi & R. Glas-Albrecht, 1991: Stimulus-secretion coupling in Paramecium cells. Eur. J. Cell Biol. 55, 3-16.

Glas-Albrecht, R., V. Schlosser & H. Plattner, 1992: Isolation of the membranes from secretory organelles (trichocysts) of Paramecium tetraurelia. Biochim. Biophys. Acta 1103, 1-7.

Plattner, H., G. Knoll & C. Erxleben, 1992: The mechanics of biological membrane fusion. Merger of aspects from electron microscopy and patch-clamp analysis. J. Cell Sci. 103, 613-618.

Knoll, G., A. Grässle, C. Braun, W. Probst, B. Houml;hne & H. Plattner, 1993: A calcium influx is neither strictly associated with nor necessary for exocytotic membrane fusion in Paramecium cells. Cell Calcium 14, 173-183.

Plattner, H. (Ed.), 1993: Membrane Traffic in Protozoa. JAI Press (Greenwich, CT, USA; London, GB). Erxleben, C. & H. Plattner, 1994: Ca2+ release from subplasmalemmal stores as a primary event during exocytosis in Paramecium cells. J. Cell Biol. 127, 935-945.

Länge, S., N. Klauke & H. Plattner, 1995: Subplasmalemmal Ca2+ stores of probable relevance for exocytosis in Paramecium. Alveolar sacs share some but not all characteristics with sarcoplasmic reticulum. Cell Calcium 17, 335-344.

Momayezi, M., D. Wloga, R. Kissmehl, H. Plattner, G. Jung, S. Klumpp & J. E. Schultz, 1996: Immuno-localization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides. J. Histochem. Cytochem. (in press).

Kissmehlr, R., T. Treptau, W. Hofer & H. Plattner, 1996: Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia. Biochem. Journal. (in press).