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doi: 10.1242/10.1242/jcs.00079

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NSF regulates membrane traffic along multiple pathways in Paramecium

Roland Kissmehl^1,*,, Marine Froissard^2,*, Helmut Plattner¹, Massoud Momayezi¹ and Jean Cohen²

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

View larger version (48K): [in a new window]   Fig. 1. Paramecium NSF genes. (A) Simplified restriction map of PtNSF1 and PtNSF2 genes and position of the probes and oligonucleotides used in this study. (B) Southern blot analysis of wild-type genomic DNA probed with the long probe (primers 3-4). A star marks the bands also revealed by the short probe (primers 1-2). Bc, BclI; Bg: BglII; E, EcoRV; S, SwaI; X, XbaI. (C) Northern blot analysis of total RNA from the wild-type (WT) and from the nd9-1 mutant (1) altered in the terminal step of exocytosis. Three probes were used, the long PtNSF probe, and, as controls, probes specific for the ND9 and ND7 genes (Froissard et al., 2001). By using the long PtNSF probe, a 2.3 kb band is revealed in both strains, with similar intensity.

View larger version (118K): [in a new window]   Fig. 2. Multiple alignment of NSF genes from different species. NSF sequences from Paramecium tetraurelia (PtNSF1 and PtNSF2), Rattus norvegicus (P18708), Arabdidopsis thaliana (AAD17345), Dictyostelium discoideum (AAC48226) and Saccharomyces cerevisiae (AAA35031) were aligned using the CLUSTALW program and displayed using BOXSHADE. The conserved AAA domains of NSF ATPases are underlined. The Walker A and B domains (Patel and Latterich, 1998) are boxed in red. The limits between the N and D1, and D1 and D2 domains are indicated by vertical bars. An SRH domain is boxed in green and a divergent SRH domain is also boxed in green with dotted lines.

View larger version (51K): [in a new window]   Fig. 3. Western-blot analysis of the subcellular distribution of PtNSF using affinity-purified Abs against yeast and Paramecium NSF. Aliquots of 50 µg of whole cell homogenates (lanes 1,5), 100,000 g supernatant (lanes 2,6), 100,000 g pellet (lanes 3,7), isolated cortices (lanes 4,8), and of 100 ng of recombinant Sec18p (lane 9) were separated on a 10% SDS polyacrylamide gel and electroblotted onto nitrocellulose membranes. Immunoreactions were carried out with either affinity-purified and crossreactive polyclonal Abs against Sec18p (lanes 1-4) (Mayer et al., 1996) or with affinity-purified peptide-specific Abs against PtNSF1 (lanes 5-9), as described in the Materials and Methods. Note that each of the two Abs immunoreact with a protein band at 84 kDa (arrow) not only in whole cell homogenates but also in particulate fractions rich of ER. In addition, the polyclonal Ab against SEC18p (anti-yeast NSF) also recognizes some bands at 30 kDa (lanes 1-4), which might be the result of proteolytic activity or instability of the NSF protein itself. By using the peptide-specific Ab against Paramecium PtNSF, some higher molecular weight bands are also reactive, which may represent heteromeric complexes of NSF, which still have to be analysed in more detail.

View larger version (193K): [in a new window]   Fig. 4. Ultrastructural changes caused by PtNSF gene silencing (A-D). Panel A shows a dilated cortical cisterna, located below a ciliary basal body (bb) and flanked by alveolar sacs (as), with locally attached 0.05 µm large vesicles (asterisk). Some attached ribosomes indicate its possible origin from endoplasmic reticulum, which is frequently swollen (er, lower left). The `terminal cisterna' (early endosome) normally expected below a basal body is absent. (B) A structure similar to that seen in A has rounded-up and closed to a wheel-like structure with vesicles firmly attached at the cytosolic site trapped inside (asterisk). (C) Close to the emergence of a cilium (c), here in oblique section, at the site where alveolar sacs are interrupted, vesicles occur, instead of the usual single `parasomal sac' (clathrin-coated pit/vesicle). The arrow points to a very slim vesicle approaching the inner side of an alveolar sac (a similar vesicle can be detected in A and more are seen in Fig. 5B). The `infraciliary lattice' (il) made up of filamentous structures is unchanged. (D) Note accumulation of clumped vesicular ER elements (va; corresponding to densely immunolabeled cytoplasmic zones in Fig. 6A,B), largely devoid of ribosomes, the occurrence of numerous free ribosomes outside such vesicle aggregates and of an autophagic vacuole (av). m, mitochondria; t, trichocyst. Bars, 0.1 µm.

View larger version (145K): [in a new window]   Fig. 6. EM immuno-gold localization of PtNSF in gene-silenced (A,B,D) and control cells (C) using peptide-specific Abs against PtNSF. Note the irregular distribution of label in the cytoplasmic zones enriched in endoplasmic reticulum (er) (A,B,D), in contrast to the control (C) with rather homogenously distributed gold grains in the cytoplasm. In all cases (A-D), mitochondria (m), trichocysts (t), alveolar sacs (as) and ciliary basal bodies (bb) are essentially devoid of label. In A and B, silencing has produced the accumulation of densely labeled vesicle aggregates (va), whose morphology is more clearly identified, probably as clusters of branching ER, in panel D, which shows a higher magnification of the area marked in the lower right corner of B. Some label also occurs in bona fide lysosomes (l). Bars, 0.1 µm.

View larger version (137K): [in a new window]   Fig. 5. After PtNSF gene silencing, docking of slender vesicles of similar appearance is seen on a food vacuole membrane in A and at alveolar sacs in B. The type of vesicles highlighted by arrows, only rarely seen on food vacuoles (fv), has never been observed previously on the inner membrane of alveolar sacs (as) to whose biogenesis it may contribute. er, endoplasmic reticulum; v, vesicle. Bars, 0.1 µm.

View larger version (103K): [in a new window]   Fig. 7. Anti-NSF Ab-fluorescence labeling of normal cells. Live cells were carefully permeabilized with saponin in the presence (A-D) or absence (E,F; controls) of ATP--S and NEM, fixed in formaldehyde, with Triton X-100 added for complete permeabilization, followed by anti-PtNSF Ab labeling, as outlined in Materials and Methods. In panels A-D several structures known to undergo multiple or individual vesicle fusions are labeled (for details, see text), for example, oral apparatus (oa), cytoproct (cp), outlets (cvo) of contractile vacuoles (cv) and onsets (arrowheads) of radial canals (rc) on these vacuoles, shown with variable labeling in different stages. In controls (E,F, without additives), the outlines of some of these structures can be vaguely seen with some residual fluorescence. Bars, 10 µm.