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doi: 10.1242/10.1242/jcs.00079
Research Article |
1 University of Konstanz, Department of Biology, PO Box 5560, 78457 Konstanz,
Germany
2 Centre de Génétique Moléculaire, CNRS, Avenue de la
Terrasse, 91198 Gif-sur-Yvette Cedex, France
Author for correspondence (e-mail:
roland.kissmehl{at}uni-konstanz.de)
Accepted 1 August 2002
N-ethylmaleimide (NEM)-sensitive factor (NSF), a regulator of soluble NSF
attachment protein receptors (SNAREs), is required for vesicular transport in
many eukaryotic cells. In the ciliated protozoon Paramecium, complex
but well-defined transport routes exist, constitutive and regulated
exocytosis, endocytosis, phagocytosis and a fluid excretory pathway through
contractile vacuoles, that can all be studied independently at the whole cell
level. To unravel the role of NSF and of the SNARE machinery in this complex
traffic, we looked for NSF genes in Paramecium, starting from a
partial sequence found in a pilot random sequencing project. We found two very
similar genes, PtNSF1 and PtNSF2, which both seem to be
expressed. Peptide-specific antibodies (Abs) recognize PtNSF as a 84 kDa band.
PtNSF gene silencing results in decreasing phagocytotic activity,
while stimulated exocytosis of dense core-vesicles (trichocysts), once firmly
attached at the cell membrane, persists. Ultrastructural analysis of silenced
cells shows deformation or disappearance of structures involved in membrane
traffic. Aggregates of numerous small, smooth vesicles intermingled with
branches of ER occur in the cytoplasm and are most intensely labeled with
anti-NSF Ab-gold. Furthermore, elongated vesicles of 
30 nm diameter can
be seen attached at cortical calcium storage compartments, the alveolar sacs,
whose unknown biogenesis may thus be revealed. Involvement of PtNSF in some
low frequency fusion events was visualized in non-silenced cells by
immuno-fluorescence, after cautious permeabilization in the presence of
ATP-
-S and NEM. Our data document that PtNSF is involved in distinct
pathways of vesicle traffic in Paramecium and that actual sensitivity
to silencing is widely different, apparently dependent on the turnover of
membrane-to-membrane attachment formation.
Key words: Ciliates, Paramecium, Secretion, Phagocytosis, Golgi, Endoplasmic reticulum
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