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A novel linker histone-like protein is associated with cytoplasmic filaments in Caenorhabditis elegans

¹ Georg-August University of Göttingen, Third Department of Zoology — Developmental Biology, Humboldtallee 34A, 37073 Göttingen, Germany
² Georg-August University of Göttingen, Institute for X-ray Physics, Geiststraße 11, 37073 Göttingen, Germany
³ Hannover Medical School, Neuroanatomy, Carl-Neuberg-Str.1, 30625 Hannover, Germany

View larger version (99K): [in a new window]   Fig. 2. H1.X detection in the marginal cells of the C. elegans pharynx. (A) represents an antibody labeling performed with H1.X-11; (B) represents the corresponding Nomarski micrograph. (C) represents an antibody labeling performed with H1.X-101. (D) shows H1.X::GFP expression in the marginal cells in a view lateral to the flat cell bodies. Here the tonofilaments are viewed in lateral orientation. (E) is the corresponding Nomarski micrograph. (F) shows H1.X::GFP expression in the marginal cells in an optical cross-section of the flat cell bodies. Here the tonofilaments appear in axial orientation as bright green fluorescent dots. H1.X::GFP shows a cytoplasmic as well as a nuclear localization (arrows point to the nuclei) of the fusion protein. Bar, 20 µm.

View larger version (97K): [in a new window]   Fig. 4. The expression of histone H1.X::GFP in C. elegans body muscle cells (A,C) and in the vulva muscle cells (E). H1.X::GFP is prominently localized in the cytoplasm but also enriched in the nuclei. (B,D,F) represent the corresponding Nomarski micrographs. The arrows points to individual body-wall muscle cells and the nuclei therein and to the vulva opening. Bars, 20 µm.

View larger version (136K): [in a new window]   Fig. 5. The expression of histone H1.X::GFP in head neurons and marginal cells (A) as well as in an excretory channel cell (C). H1.X::GFP is a cytoplasmic protein in both cell types. The neuronal projections are visualized by H1.X::GFP fluorescence. (B,D) are the corresponding Nomarski micrographs. Bars, 20 µm.

View larger version (81K): [in a new window]   Fig. 6. Nucleolar localization of H1.X (A) shows a Nomarski micrograph of the nuclear region of a polyploid gut cell in a hermaphrodite C. elegans with the nucleolus and its substructures visualized by differential interference contrast. (B) H1.X detected with the antibody H1.X-101 localizes to the nucleolus; (C) corresponds to DAPI DNA staining; and (D) shows the indirect immunfluorescence detection of the nucleolar protein fibrillarin with the specific monoclonal antibody P2G3 (from M. Christensen). Bar, 10 µm.

View larger version (35K): [in a new window]   Fig. 3. H1.X depletion by RNA interference and colocalization of H1.X with the intermediate filament proteins in the marginal cells of the pharynx. Indirect immunofluorescence detection of histone H1.X in the marginal cells of the pharynx (H1.X-11) shows a substantial reduction of H1.X after H1.X RNA interference (A) but not in control animals (C), which present intensely stained tonofilaments. The same two animals were additionally stained with the monoclonal anti-intermediate filament antibody IFA (B,D) (Bartnik et al., 1986), which decorates the tonofilaments. This shows a comparable permeabilization and antibody accessibility in both animals as well as a colocalization of both antigenes in the tonofilaments. Bar, 20 µm.

View larger version (59K): [in a new window]   Fig. 7. Embryonic expression and subnuclear localization of H1.X. (A,B) show H1.X::GFP fluorescence detection in a few cells in the periphery of a > 100-cell stage embryos. In (A), a fixed specimen shows a shallow fluorescence of the nucleoplasm and bright fluorescence of the nucleoli, whereas in (B), a live observation shows a shallow fluorescence in the cytoplasm and a bright fluorescence of the nucleoplasm. The brightest spots in the nucleoplasm correspond to the nucleoli. Bars, 20 µm.

View larger version (80K): [in a new window]   Fig. 8. Expression of H1.X::GFP in living (A) and in formaldehyde-fixed HeLa cells (C). The nucleoli are prominently fluorescent in the fixed and extracted cells, whereas in living cells the whole nucleoplasm fluoresces. The brightest spots in the nucleoplasm of the living cells correspond to the nucleoli. (B,D) represent the corresponding Nomarski micrographs. Bars, 10 µm.

View larger version (68K): [in a new window]   Fig. 9. C. elegans phenotypes created by H1.X RNA interference. 61 animals from 1351 scored F1 animals (4.5%) are small and dumpy (A). They reach only half of the final body length of normal C. elegans. Half of these animals (34 of 61) are additionally defective in egg laying (B). Another phenotype affected 3% of the F1 population (33 animals of 1351). These animals contain an abnormally elongated pharynx (C). A control animal is shown in (D; the arrows point to the pharynx). Bar, 20 µm.

View larger version (42K): [in a new window]   Fig. 1. Western blot analysis of histone H1.X. (A,B) shows Coomassie-bluestained SDS page gels obtained from a total lysate of C. elegans, the recombinant C. elegans proteins lamin, intermediate filament proteins A1 and B1 and recombinant H1.X. (C) shows a corresponding blot stained with the H1.X-101 antibody and (D) shows a corresponding blot stained with the H1.X-11 antibody. No antibody is reactive with intermediate filament proteins. Both antibodies recognize H1.X as a band with an apparent molecular mass of 31 kDa. H1.X-11 is crossreactive with C. elegans lamin and a further not identified protein.