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First published online 30 March 2005
doi: 10.1242/dev.01809

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Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuléen

Department of Developmental Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, Heidelberg 69120, Germany

View larger version (50K): [in a new window]   Fig. 1. Molecular analysis of vls. (A) Location of three EMS mutations in vls. All mutations lead to a premature stop codon. WD repeats are shown in grey. (B) The vls transcript and restriction map of the genomic DNA used for generating a vls rescue transgene. Exons are drawn as boxes and the putative open reading frame is indicated in black. B, BamHI; Bs, BstEII; P, PstI; R, EcoRI. (C) Expression of the P[HA-Vls] transgene in ovaries revealed by western blotting using anti-HA antibodies. A band of 50 kDa is detected in transgenic but not in wild-type ovaries. (D) Alignment of the amino acid sequences of Vls and homologous proteins: CP8824, an incomplete Anopheles gambiae homolog; human MEP50; and MGC65780 a zebrafish protein. These proteins display strong conservation of the WD repeats (grey boxes). MEP50 and the zebrafish homolog display six potential WD repeats, whereas Drosophila Vls contains only four such repeats, as predicted by the Protein Sequence Analysis server of the BioMolecular Engineering Research Center of Boston University (http://bmerc-www.bu.edu/psa/). Multiple sequence alignment of Vls and related proteins was performed using the Pileup program of the Wisconsin Package (Genetics Computer Group). Gaps in the amino acid sequence, indicated by dots, were introduced for optimal alignment. The GenBank accession numbers of these sequences are the following: CP8824_AG, EAA14269 MEP50_HS, AAL79917 MGC65780_DR, AAH56278

View larger version (34K): [in a new window]   Fig. 2. Vls interacts physically with Csul. (A) Vls interacts with Csul in vivo. Ovarian protein extracts of homozygous vls3 females producing both TAP-Csul and HA-Vls (lanes 1 and 2), or only HA-Vls (lane 3), were directly loaded on a gel (one tenth of total input, lane1) or immunoprecipitated with Sepharose IgG (lanes 2 and 3). Following separation by SDS-PAGE electrophoresis, bound-HA-Vls was detected by immunoblotting using anti-HA antibodies. (B) Reciprocally, Vls was immunoprecipitated using anti-HA antibodies and bound Csul was detected by immunoblotting using alkaline phosphatase-conjugated anti-rabbit antibodies. (C) Binding of the C terminus of Vls to Csul. Full-length GST-Vls or derivatives were purified from bacterial extracts and incubated with S•Tag-Csul. Bound S•Tag-Csul (75 kDa) was separated by SDS-PAGE electrophoresis and detected by immunoblotting using alkaline phosphatase-conjugated S proteins. Input: one-tenth of protein extract was loaded on the gel. The amount of used GST-fusion proteins was evaluated by SDS-PAGE followed by Coomassie staining (lower panel). Representation of the GST-Vls constructs used for the mapping and summary of results are indicated on the right. The WD-repeats and the putative Csul-binding domain of Vls are indicated by grey and green boxes, respectively. (D) The C-terminal region of Csul interacts with Vls. Full-size S•Tag-Csul or derivatives were synthesized in vitro and incubated with full-size GST-Vls. Following separation by SDS-PAGE electrophoresis the bound S•Tag-Csul proteins were detected by immunoblotting using alkaline phosphatase-conjugated S proteins. Left panel: input S•Tag-Csul proteins. The arrow indicates an endogenous protein synthesized in the reticulocyte system and reactive to alkaline phosphatase-conjugated S proteins. White squares indicate the position of the different S•Tag-Csul proteins. Middle panel: S•Tag-Csul proteins bound to GST-Vls are indicated by white squares. Representation of S•Tag-Csul constructs used for the mapping and summary of the results are indicated in the right panel. The putative Vls-binding domain of Csul is depicted in blue.

View larger version (58K): [in a new window]   Fig. 3. Vls is a component of nuage and pole plasm. Fixed, whole-mount egg chambers stained with Oli-Green for DNA and anti-HA antibodies in homozygous vls3 flies expressing P{HA-Vls} (A,C,E,F,H,I,K) and in wild-type flies as controls (B,D,G,J,L). HA-Vls is detected at the posterior pole of the oocyte in transgenic vls3; P{HA-Vls} stage 10 egg chambers (A,C). (C,D) Enlarged views of similar stage 10 oocytes, as shown in A and B, respectively. HA-Vls is observed at the posterior pole of early vls3; P{HA-Vls} embryos and in pole cells (E,F,H,I). (F,G,I,J) Enlarged views of similar syncytial and blastoderm embryos, as shown in E and H, respectively. HA-Vls is detected in the nuage surrounding the nurse cell nuclei (K).

View larger version (43K): [in a new window]   Fig. 4. Defective pole plasm assembly in vls. (A,B) Absence of pole cell formation in vls3. Staining of Vas with rat anti-Vas antibodies (red) and DNA with Oli-Green (green) in wild-type (A) and vls3 (B) blastoderm embryos. (C,D) vls suppresses ectopic anterior pole cell formation induced by osk-bcd 3'UTR. Staining of Vas protein (red) and DNA (green) in osk-bcd 3'UTR (C) and vls3; osk-bcd 3'UTR (D) blastoderm embryos. No pole cells form at the anterior pole of vls3; osk-bcd 3'UTR embryos. (E,F) Osk is localized at the posterior pole of the oocyte in vls3 stage 10 egg chambers, albeit in reduced amount. (E) Wild-type and (F) vls3 egg chambers. (G) The level of Osk protein is reduced in vls. Ovarian protein extracts of wild-type (WT), vls1, aubN11/aubHN2 and osk84/Df(3R)pXT103 females were separated by SDS-PAGE electrophoresis, blotted and detected using anti-Osk antibodies. The abundance of short Osk is reduced in vls mutant ovaries, whereas the long form is maintained at the wild-type level. Both Osk forms are strongly reduced in aub ovaries, with a more substantial effect on short Osk. In contrast to aub, the phosphorylation of short Osk is not affected in vls. The same blot was successively probed for ribosomal P40, as loading control (Török et al., 1999). (H,I) Vas is localized at the posterior pole of the oocyte in wild type (H) and vls3 (I) stage 10 egg chambers. There is less localized Vas in vls3 compared with wild type. Ovaries were stained with rabbit anti-Vas antibodies (red) and Oli-Green for DNA (green). (J,K) Tud is detected in nuage and at the oocyte posterior pole in wild-type stage 10 egg chambers (J) but is not detected at these locations in vls3 stage 10 egg chambers (K). Ovaries were stained with anti-Tud antibodies (red) and Oli-Green for DNA (green).

View larger version (67K): [in a new window]   Fig. 5. Displacement of nuage components during early vls oogenesis. (A,B) GFP-Vas and (C,D) Mael accumulate in nuage in stage 7 egg chambers in wild type (A,C) and vls3 (B,D). GFP-Vas (green) was detected in fixed egg chambers stained with Hoechst 33258 for DNA (red). (E,F) Tud accumulates in nuage in wild type stage 7 nurse cells (E), whereas it is homogenously dispersed in the nurse cell cytoplasm in vls3 stage 7 egg chambers (F). Ovaries were stained using anti-Tud antibodies (red) and Oli-Green for DNA (green). Tud is found at high levels in wild type and vls3 oocytes. (G,H) Staining of DNA with Oli-Green reveals that the karyosome, which appears as a single compact dot in wild-type oocytes (G), is frequently fragmented in vls3 oocytes (H). The oocyte nuclear membrane is indicated by a broken line.

View larger version (31K): [in a new window]   Fig. 6. Vls physically interacts with Tud. (A) The eight Tudor (1-8) and two Tudor-like domains (1'-2') are depicted as purple boxes. Fragments of Tud (Golumbeski et al., 1991) used in pull-down assays are indicated below the map. N-and C-terminal amino acid residues of each fragment are indicated. (B) Western blot analysis of in vitro produced S•Tag-Tud fragments detected by using alkaline phosphatase-conjugated S proteins. (C) After incubation with GST or GST-Vsl, the bound Tud fragments were detected by immunoblotting using alkaline phosphatase-conjugated S proteins: the Tud-9A1-N and Tud-9A1-C fragments display a strong binding to GST-Vls, whereas Tud-JOZ and Tud-3ZS+L bind only weakly to it. GST alone (negative control) exhibits no binding to Tud-JOZ and Tud-9A1. Lower panels: input GST-Vls and GST proteins separated on a SDS-PAGE gel and stained with Coomassie.

View larger version (21K): [in a new window]   Fig. 7. Tud-binding domain in Vls. As performed in Fig. 2C, full-length GST-Vls or derivatives were incubated with S•Tag-Tud9A1-N. Bound S•Tag-Tud9A1-N was detected as described in Fig. 6B. Input: one-tenth of the S•Tag-Tud9A1-N extract. The smallest fragment of Vls showing binding to Tud9A1-N encompasses the first WD domain shown in green. The amount of GST-Vls proteins was visualized by Coomassie staining (lower panel). Representation of the GST-Vls fragments used for mapping and summary of the results are indicated on the right panel.