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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/dev.00389

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 van Meyel, D. J.
Right arrow Articles by Agulnick, A. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Meyel, D. J.
Right arrow Articles by Agulnick, A. D.
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?

Ssdp proteins bind to LIM-interacting co-factors and regulate the activity of LIM-homeodomain protein complexes in vivo

Donald J. van Meyel1,*, John B. Thomas1,§ and Alan D. Agulnick2,{dagger}

1 The Salk Institute for Biological Studies, PO Box 85800, San Diego, CA 92186, USA
2 Department of Biology, University of California, Riverside, CA 92521, USA
* Present address: McGill University Centre for Research in Neuroscience, 1650 Cedar Avenue, Montreal, QC H3G 1A4, Canada
{dagger} Present address: CyThera, San Diego, CA 92121, USA



View larger version (67K):

[in a new window]
  		Fig. 1. Ldb1 and Ssdp2 specifically interact in vitro. Ldb1 (from mouse), Ssdp2 (human) and the LIM-HD protein Lhx3 (mouse) were transcribed and translated in rabbit reticulocyte lysates. Proteins labeled with 35S-methionine and/or tagged with the FLAG epitope were mixed in the combinations shown above each lane and then complexes were immunoprecipitated with anti-FLAG antibody-conjugated agarose beads (see Materials and Methods). Ssdp2 protein is efficiently immunoprecipitated by Ldb1 (lane 2), but not by Lhx3 (lane 4). Lhx3 can bind Ldb1 (lane 1) and can immunoprecipitate Ssdp2 in the presence of Ldb1 (lane 3). This indicates the formation of a ternary complex in which Lhx3 and Ssdp2 are each bound to Ldb1, but they do not directly interact with one another. Control experiments show that the Ldb1-Ssdp2 complex is not immunoprecipitated in the absence of the FLAG epitope (lane 5), and only a small amount of either Ssdp2 or Ldb1 binds non-specifically to the beads (lanes 5 and 6).

 


View larger version (18K):

[in a new window]
  		Fig. 2. Qualitative two-hybrid interaction assays in yeast reveal domains required for interactions between Ssdp proteins and Ldb/Chip proteins from mice and flies. Schematic diagram of recombinant proteins fused to the DNA-binding domain (DBD) or activation domain (AD) of GAL4. Mouse Ldb1 and Drosophila Chip are depicted in white, mouse Ssdp2 and Drosophila Ssdp in gray, and mouse Lhx3 as a positive control in black. Interactions between proteins were measured by ß-galactosidase activity and were scored as either positive (+) or negative (-). (A) Sequences between amino acids 201 and 255 of Ldb1 are required for interaction with Ssdp2. (B) The N-terminal 100 amino acids of Ssdp2 are sufficient for interaction with Ldb1. (C) Upon switching the configuration of the fusion proteins, the requirements of amino acids 1-100 of Ssdp2 and 201-255 of Ldb1 are reiterated, supporting the specificity of the interaction. (D) Further refinement of Ldb1 sequences required for interaction with Ssdp2 through two deletions of 10 amino acids each. Deletion of amino acids 214-223 disrupts the interaction with Ssdp2, but has no effect on the ability of Ldb1 to bind the LIM domains of Lhx3. (E) The Drosophila melanogaster (D.m.) orthologs Ssdp and Chip give similar results to those obtained for the mouse proteins. Ssdp amino acids 1-98 are sufficient for interaction with Chip, and removal of amino acids 387-426 of Chip prevents binding to Ssdp but not to the Lhx3 LIM domains.

 


View larger version (42K):

[in a new window]
  		Fig. 3. The LUFS domain of Ssdp proteins mediate interactions with Ldb/Chip that are crucial for nuclear localization. (A) Schematics comparing the amino acid composition and overall structure of mouse (M.m.) Ssdp2 and Drosophila melanogaster (D.m.) Ssdp. There is 90% identity of amino acids over the N-terminal region shown to be sufficient for interactions with Ldb1/Chip (gray box), known as the LUFS domain. The remainders of these proteins are rich in proline, glycine and methionine residues, and share three other small domains that are also highly conserved (small gray boxes, corresponding to Ssdp2 amino acids 232-240, 250-262 and 331-337). (B) Alignment of the primary amino acid sequence of LUFS domains of Ssdp proteins from mouse and Drosophila, and Arabidopsis LEUNIG, showing the LisH domain and the motif P-X-GFL-XX-WW-X-VFWD, which is notable for its conservation in all members and with yeast Flo8. (C) Schematic of Ldb1 and Chip, indicating 94% identity over residues 201-249 of Ldb1 and 387-435 of Chip (hatched boxes). We have called this region the Ldb1/Chip conserved domain (LCCD). Within the LCCD a deletion of 10 residues (Ldb1 amino acids 214-223, corresponding to Chip amino acids 400-409) disrupts the ability of Chip to interact with Ssdp without affecting its ability to homodimerize through the dimerization domain (DD) or bind LIM domains through the LIM interaction domain (LID). The position of the putative nuclear localization sequence (NLS) is indicated. (D-F) Anti-Myc immunofluorescence staining of ventral muscles of stage 16 embryo. In a wild-type background (D), apGAL4-driven expression of Myc epitope-tagged SsdpFL reveals discrete localization to the multiple nuclei in each of the muscle cells 21-24. By contrast, Ssdp{Delta}2-92 fails to localize to the nucleus and instead is found throughout the cytoplasm (E). SsdpFL fails to localize to the nucleus in a Chipe5.5 null mutant background (F).

 


View larger version (113K):

[in a new window]
  		Fig. 4. In situ hybridization of ssdp probes to whole-mount embryos and dissected larvae. (A) Embryos at germband extension (stage 11 embryo, left) have widespread expression that increasingly becomes enriched in the developing central nervous system as germband retraction proceeds (stage 12 embryo, right). (B) At stage 13-14 of embryonic development, ssdp expression is largely restricted to the brain and ventral nerve cord. (C) Higher power ventral view of the ventral nerve cord showing pan-neuronal expression within the CNS. (D) In third instar larvae, ssdp transcripts are not detectable in the ventral nerve cord (arrowhead), but moderate ssdp expression was observed in the optic lobes of the brain hemispheres (arrows in D,E). (E,F) High levels of ssdp expression are observed in imaginal discs, including the anterior region of the antennal-eye disc (E, arrowhead) and uniform levels in the wing disc (F, left) and haltere disc (F, right).

 


View larger version (48K):

[in a new window]
  		Fig. 5. The Drosophila ssdp gene and mutant alleles. (A) Schematic drawing of 11 kb of genomic DNA surrounding the ssdp locus. The ssdp gene consists of two exons, the second of which contains the entire protein coding sequence. ssdp is flanked by two predicted genes of unknown function, CG7985 and CG14313. The insertion sites of three P-elements EP(3)3097, l(3)neo48 and EP(3)3004 are shown, as are the boundaries of two deficiency alleles, ssdpL7 and ssdpL5, that were generated by imprecise excision of EP(3)3097. (B) Stage of lethality for various allelic combinations of ssdp. Viability of individuals of each genotype was assessed at three stages of development: larval first instar, larval third instar and adult eclosion. (C,D) Newly eclosed adults of the genotype ssdpEP(3)3097/ssdpl(3)neo48. Note the blistered wings (arrows in C). Other phenotypes for this allelic combination include a cleft along the midline of the notum (arrowhead in C,D), and/or misshapen, misoriented, deleted or extra macrochaetae on the notum and scutellum (arrow in D). The phenotype shown in D is frequent and relatively mild, compared with rarer individuals in which the cleft was much more severe (not shown).

 


View larger version (159K):

[in a new window]
  		Fig. 6. Ssdp modifies the activity of Chip/Ap tetrameric complexes. Newly eclosed apGAL4/+ flies carrying one copy of each UAS transgene as shown. (A) apGAL4/+ flies exhibit no wing defects. (B) apGAL4/+; UAS-ChipFL/+. Overexpression of full-length Chip (ChipFL) reduces the levels of functional Chip/Ap complexes and results in wings that are small, unfused and with a poorly demarcated margin (arrow). These defects resemble those of hypomorphic ap mutants. (C) apGAL4/+;UAS-ChipFL/ssdpL7.ssdpL7 dominantly enhances the wing defects caused by Chip overexpression, leaving little or no organized wing tissue (arrow). (D) apGAL4/+;UAS-Chip{Delta}LID:Ap{Delta}LIM/+. The fusion protein Chip{Delta}LID:Ap{Delta}LIM results in formation of hyperactive complexes. Flies overexpressing Chip{Delta}LID:Ap{Delta}LIM have blistered wings in which the dorsal and ventral surfaces fail to fuse, and which are held upward and away from the thorax in a fashion resembling LMO loss-of-function mutants. (E) apGAL4/+;UAS-Chip{Delta}LID:Ap{Delta}LIM/ssdpL7. Removal of one copy of ssdp from flies expressing Chip{Delta}LID:Ap{Delta}LIM suppresses the blistered wing phenotype; the surfaces fuse properly, although the wings remained held up. (F) apGAL4/+;UAS-Chip{Delta}LCCD/+. Expression of Chip{Delta}LCCD, which lacks amino acids 387-426 and thus cannot bind Ssdp but is still capable of homo-dimerization and LIM interaction, results in more severe wing defects than expression of ChipFL, reducing the wing to a ribbon-like process with little discernible structure (arrow).

 


View larger version (114K):

[in a new window]
  		Fig. 7. Mosaic analysis of ssdpl(3)neo48 mutant clones reveals phenotypes similar to those of Chip and ap. Mounted wings of newly eclosed adults after heatshock-induced expression of FLP recombinase and subsequent recombination at FRT sites (see Materials and Methods). (A) A control clone along the native wing margin is marked by pwn. The border of the clone is outlined in red. Next to the clone the normal arrangement of the triple row of sensory bristles along the margin is shown, including a well-spaced row of dorsal chemosensory bristles (black arrow), a second and more tightly arrayed row of dorsal mechanosensory bristles (arrowhead), and a third row of ventral bristles (out of focal plane, gray arrow). Within the clone, bristles are mutant for pwn, but are otherwise normally specified, and there are no associated mutant phenotypes. (B) Low-power image of entire wing showing phenotypes associated with dorsal clones of ssdpl(3)neo48 mutant cells in an individual in which clones were induced during second larval instar. Phenotypes include ectopic margin formation accompanied by ectopic wing outgrowth (arrow), and ectopic margins in the absence of outgrowth (arrowheads). Phenotypes were never associated with ventral ssdpl(3)neo48 clones. (C) Higher power view of the outgrowth shown in B. The tip of the outgrowth (arrowhead) has ectopic sensory bristles that resemble the wing margin. (D) Close-up view of outgrowth tip shown in C. The ssdpl(3)neo48 mutant clone (marked by pwn and outlined in red) lies near the tip of the outgrowth and is next to the ectopic margin. (E,F) Two focal planes of the same ectopic margin in the absence of wing outgrowth, the most common phenotype observed for ssdpl(3)neo48 mutant clones. The extent of the ssdpl(3)neo48 mutant clone on the dorsal surface of the wing is marked by pwn and outlined in red (E). The clone is near but not within the native wing margin, and results in the induction of ectopic bristles shown in the focal plane of F. (G) Loss of dorsal-specific bristles within an ssdpl(3)neo48 mutant clone (outlined in red) that lies on the native wing margin. Outside the clone both the dorsal-specific chemosensory (black arrow) and mechanosensory (arrowhead) bristles are intact. Within the clone, however, these bristles are lost, despite the fact that the overall structure of the wing is undisturbed. The ventral specific bristles (gray arrow) lie outside the clone and remain intact. (H) A broad ssdpl(3)neo48 mutant clone (outlined in red) that straddles the margin on both the dorsal and ventral surfaces results in complete loss of wing margin and some wing tissue, resulting in a nicked wing.

 

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 2003