spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 19 May 2004
doi: 10.1242/dev.01154

Development 131, 2935-2945 (2004)
Published by The Company of Biologists 2004
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Belfiore, M.
Right arrow Articles by Puoti, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belfiore, M.
Right arrow Articles by Puoti, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Roles of the C. elegans cyclophilin-like protein MOG-6 in MEP-1 binding and germline fates

Marco Belfiore*, Paolo Pugnale*, Zarifja Saudan and Alessandro Puoti{dagger}

Department of Biology, Unit of Zoology, University of Fribourg, 1700 Fribourg, Switzerland



View larger version (13K):

[in a new window]
  		Fig. 1. Cloning of mog-6. (A) Genetic map. mog-6 is located on chromosome II, between unc-4 and sqt-1. (B) Deletion analysis. mnDf66, mnDf89 and mnDf90 (black) remove mog-6 while mnDf29, mnDf57, mnDf62 and mnDf87 (gray) do not. The exact breakpoints of mnDf62 and mnDf66 have not been determined (dashed lines). (C) Cosmids tested for rescue (black bars); only M106 rescued mog-6. (D) Subcloning of M106. A 3.1 kb fragment (pAP6) of M106 rescued mog-6 and contains one open reading frame. (E) The mog-6 gene. 5' and 3' flanking regions are represented as lines, exons as gray boxes, introns as lines joining the exons.

 


View larger version (40K):

[in a new window]
  		Fig. 5. Analysis of RNA splicing in mog-6 mutants. (A) RT-PCR. Genomic DNA (D) or polyA-enriched RNA (A+) from wild-type worms were used as positive controls for PCR with sets of oligonucleotides specific for either fem-3 (lanes 1 to 6), fbf-2 (lanes 7 to 12), nos-3 (lanes 13 to 18) or ceh-13 (lanes 19 to 24). Total RNA from either mog-6 or wild-type adults (wt) was used for PCR without (–) or with (+) RT. PCR on genomic DNA was used as a reference for the size of unspliced RNAs (lanes 1, 7, 13 and 19). (B) Northern analysis. Total RNA, 12.5 µg, derived from adult mog-6 or wild-type worms were run on a denaturing agarose gel and probed for mog-6, fbf, nos-3, ges-1 and actin. (C) gld-3 transcripts were analyzed on a separate blot loaded with 25 µg and 50 µg of total RNA from wild-type and mog-6 mutants, respectively. The two major gld-3 transcripts are indicated (Small and Large). Blots were also overexposed to ensure that no minor transcripts of abnormal size were present (not shown).

 


View larger version (49K):

[in a new window]
  		Fig. 3. Expression of mog-6. (A) Northern analysis of mog-6 RNA. (A, left) mog-6 RNA levels vary throughout development: E, embryos; L1 to L4, larval stages; A, adult. CeIF was the loading control. Right, RNAs from adult wild-type and glp-1 mutants were probed for mog-6 and act-1 (loading control). (B) Expression of mog-6::gfp. Top, the mog-6 gene was fused in frame with gfp. Exons are represented by boxes. The 5' and 3' flanking sequences are those of mog-6 (pAP6 in Fig. 1). Bottom, MOG-6::GFP is found in nuclei of the intestine (black arrowheads), uterus (gray arrowheads) and other somatic tissues. The black arrow points toward the vulva. Anterior is to the left. (C-I) Immunocytochemistry. (C) In wild-type L4 larvae, MOG-6 is expressed in germline nuclei throughout the mitotic and the meiotic regions. (D) To visualize nuclei, the same worm was stained with DAPI. Anti-MOG-6 antibodies stain somatic nuclei (intestine, white arrows in C,D) and mitotically or meiotically dividing germline nuclei (arrowhead in C,D). (E) Drawing of the germline shown in (C,D). Nuclei of sperm and spermatocytes are shown in white. (F) MOG-6 and MEP-1 in intestinal nuclei. The nucleolus is visible by DIC microscopy (arrow) and took up lower amounts of DAPI than the remainder of the nucleus. MOG-6 and MEP-1 are present throughout the whole nucleus, including the nucleolus. (G) MOG-6 in larvae. L3, left; L2 right; L1 before hatching, below. (H) Expression of MOG-6 in a feminized adult germline. MOG-6 is found in all nuclei, including those of mature oocytes (arrow). (I) DAPI staining of the same germline; the condensed chromosomes of accumulating oocytes are visible (white arrow). The asterisk shows the distal end of the germline. Scale bar: 20 µm.

 


View larger version (41K):

[in a new window]
  		Fig. 4. MOG-6 binds to MEP-1. (A) Interaction assays between MOG-6 and additional regulators of fem-3 in the yeast two-hybrid system. Growth on medium lacking histidine (left) or blue staining (right) represents positive interaction. MOG-6 was fused to the LexA DNA-binding domain, while the other proteins were fused to the Gal4 activation domain. Bottom lane, MOG-6 does not bind to itself. (B) Binding of MOG-6 to MEP-1 in vitro. MOG-6 bound to GST-MEP-1(FL) or GST-MEP-1(SH), but neither to GST alone nor to GST-FBF-1. (C) Functional domains in MEP-1. MEP-1(FL) is represented schematically in row 1: the Q-rich domain is shown as a blue box, and the zinc fingers are represented by vertical triangles. Row 2, MEP-1(SH) lacks the N-terminus (residues 1-407). Rows 3 to 22 represent other deletion derivatives of MEP-1. The shortest MEP-1 derivative that interacted with MOG-6 comprised 111 residues of the N-terminus (row 17, red box, residues 123 to 233) or 361 residues of the C-terminus (row 20, red box, AA 510 to 870). Internal deletions are shown by bars joining the N- and C-termini (rows 5 to 8). (D) mog-6 rescue and MEP-1 binding. The complete mog-6 gene and derivatives thereof were tested for mog-6 rescue. The wild-type mog-6 gene rescued mog-6 (row 1, 9% rescue, n=31 rolling unc-4mog-6 worms analyzed; self-fertile rolling Uncs were considered as rescued). mog-6 constructions bearing point mutations in the CBD (bars in rows 2 to 4) or lacking the entire CBD (AA275-434; row 5) rescued mog-6 at least to the same extent as the wild-type mog-6 gene. MOG-6 truncations retaining either the entire CBD (row 6), the CBD plus the C-terminus (row 7), or the C-terminus alone (row 8) did not rescue mog-6. Similarly, the entire N-terminal extension of MOG-6 did not rescue mog-6 (row 9). MOG-6 derivatives were also assayed for MEP-1 binding in the yeast two-hybrid system. Right; MOG-6 requirements for MEP-1(FL) binding. Full length MOG-6 bound to MEP-1 (row 1, see also Fig. 4A). A MOG-6 protein that lacked the CBD interacted with MEP-1 (row 5), but neither the CBD alone (row 6), nor the CBD fused to the C-terminus (row 7) were sufficient for MEP-1 binding. If taken separately, the C- and N-termini of MOG-6 did not interact with MEP-1 (rows 8 and 9).

 


View larger version (41K):

[in a new window]
  		Fig. 2. (A) The MOG-6 protein. The CBD is highlighted in gray (AA275-434). Residues that are involved in Cyclosporin A (CsA)-binding in canonical cyclophilins are shown in bold. The nuclear localization signal is overlined. The starting point of the deletion in mog-6(q465) is shown by an arrow. The sequences of MOG-6 and of its human ortholog hCyp-60 are aligned. Conserved residues are boxed. (B) Western analysis. A 64 kD protein was detected with affinity-purified anti-MOG-6 antibodies in wild-type adults and masculinized adults (fem-3(q96gf)), but was absent in mog-6(q465) mutant adults. A minor 42 kD protein (open arrow) was absent in q465 extracts but was also detected with preimmune serum (not shown). White arrowheads indicate cross-reacting bands that are oogenesis-dependent, since absent in masculinized animals.

 


View larger version (48K):

[in a new window]
  		Fig. 6. mog-6 is not required for the correct localization of GLD-3 and FBF. Germlines from adult hermaphrodites were immunostained for GLD-3 (A,C) and FBF (E,G). The corresponding DAPI stainings indicate the start of the spermatogenic region in masculinized mutants (shown to the right of white arrowheads in B,D,H). (E,F) Wild-type germlines produce oocytes in the proximal region (arrow). Scale bar: 20 µm.

 


View larger version (81K):

[in a new window]
  		Fig. 7. gld-3 mog-6 double mutants have tumorous germlines. Adult hermaphrodites were analyzed for germline phenotypes by DIC. (A) gld-3(RNAi) caused feminization of mog-6/+ heterozygotes (arrow points toward a stacking oocyte). (B) Germlines of mog-6 homozygotes were masculinized (sperm is shown between the two arrows). (C) gld-3(RNAi) mog-6 hermaphrodites are tumorous. (D) Dissected tumorous germline stained for DNA (top) or for the mitotic marker phosphohistone H3 (PH3) (bottom). Mitotic germ cells are present throughout the entire germline, including the proximal part of the germline (white arrowheads). The distal end is on the left. (E) Magnification of the proximal region shown in D (top) in a different focal plane. Asterisks indicate distal ends of germlines. Black arrowheads point toward the vulva. Scale bar: 20 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2004