November 1, 2000;
is a cell surface metalloprotease expressed in cephalic neural crest
cells during early Xenopus development. The cytoplasmic domain of ADAM13
contains three potential SH3 (Src homology type 3) binding sites, suggesting that this region may support interactions with intracellular proteins. In this report we describe the identification, by a new strategy, of three proteins that bind the ADAM13
cytoplasmic domain in vitro: X-Src1
, and X-PACSIN2
. We focused our study on X-PACSIN2
protein because it colocalizes with ADAM13
in migrating neural crest
cells during embryonic development. Using pull-down experiments we show that X-PACSIN2
binds to ADAM13
in vitro. Using Xenopus XTC cells, we demonstrate that ADAM13
colocalize to membrane ruffles and cytoplasmic vesicles. We also show that X-PACSIN2
overexpression can rescue developmental alterations induced by overexpression of ADAM13
, suggesting that both proteins interact in vivo. Finally, our results suggest that X-PACSIN2
overexpression reduces endogenous ADAM13
function while a truncated X-PACSIN2
(DeltaSH3) increases this activity in cephalic neural crest
cells. We propose that X-PACSIN2
may regulate ADAM13
activity by influencing either its subcellular localization or its catalytic activity. In agreement with this model, elimination of the ADAM13
cytoplasmic domain increased developmental alterations attributable to ADAM13
[+] show captions
FIG. 3. M7/X-PACSIN2 is expressed maternally and ubiqui- tously. (A) Total RNA from 10 embryos was purified and reverse transcribed in the absence (ﰋ) or presence of MMLV polymerase. For XTC cells, 10 ﰃg of total RNA was used. cDNA underwent PCR amplification using X-PACSIN2 and integrin ﰄ1-specific oligode- oxynucleotides as described under Material and Methods. X-PACSIN2-specific amplification product appears as a single band at every stage tested as well as in XTC cells. (B) Western blot using mAb 3D8 with total protein extracts. M7/X-PACSIN2 protein is expressed in two-cell (stage 2), early blastula (stage 7), early gastrula (stage 10.5), early neurula (stage 15), and early and late tail bud (stage 22 and 32) stages. The upper form (72 kDa) is expressed in XTC cells and is increased following transfection with a full-length M7-X-PACSIN2 cDNA clone. (C) Immunolocalization of M7/X- PACSIN2 on 10-ﰃm sections using mAb 3D8. (C) Sagittal section of early gastrula (stage 101). The protein is present in the three germ layers (ectoderm, mesoderm, and endoderm). General fluores- cence appears stronger within the ectoderm and the dorsal mar- ginal zone. Membrane staining is visible near the site of involution.
FIG. 5. ADAM13 and X-PACSIN2 colocalize in Xenopus XTC cells. XTC cells were seeded on glass coverslips coated with fibronectin. After 48 h they were fixed in formaldehyde, permeabil- ized with 0.5% Triton X-100 and immunostained with the poly- clonal antibody 6615F, which recognizes the ADAM13 cytoplas- mic domain (A), and the monoclonal antibody 3D8 directed against X-PACSIN2 (B). Secondary FITCnti-mouse and Texas rednti- rabbit were used. Superposition of ADAM13 and X-PACSIN2 staining is presented in C. Area where both protein are detected with similar intensity appears in D. Overlapping staining shows that ADAM13 and X-PACSIN2 colocalize at specific areas of cell membrane as well as in some cytoplasmic vesicles (D, arrow). Both proteins are absent from areas of cellell contact (arrowhead).
FIG. 6. X-PACSIN2 rescue of alterations caused by ADAM13 overexpression. In situ hybridization of tail bud stage embryos with the cement gland-specific marker X-CG. Embryos were injected in one cell at the two-cell stage, grown up to tail bud stage (stage 22), and fixed and processed for in situ hybridization. The injected protein is localized by whole-mount immunostaining. Embryos are viewed from the injected side with the anterior to the left. (A) Embryo injected with 0.5 ng of wild-type ADAM13 shows extended cement gland (arrowhead). (B) Co-injection of 0.5 ng of wild-type PACSIN2 with 0.5 ng of ADAM13 abolishes this phenotype. (C) Co-injection of 0.5 ng of the mutant PACSIN2 lacking the SH3 domain has no effect on ADAM13-induced extension of the cement gland. Neither X-PACSIN2 (D) nor the ﰅSH3 (E) mutant alters X-CG expression. (F) Histogram of the rescue data. Numbers from four independent experiments are presented. Percentages of altered cement gland obtained with ADAM13 (lane 1), ADAM13 plus X-PACSIN2 (lane 2), ADAM13 plus ﰅSH3 X-PACSIN2 (lane 3), X-PACSIN2 (lane 4), or the ﰅSH3 mutant alone (lane 5) are plotted. Error bars represent standard deviations.
Wild-type and ﰅcyto ADAM13 perturb cement gland devel- opment. In situ hybridization of tail bud stage embryo using the cement gland marker X-CG. (A) Lateral representation of a tail bud stage Xenopus embryo. The anterior is to the left and the cement gland is indicated with an arrowhead. The same orientation and arrowhead were used for all images. Embryos were injected in one cell at the two-cell stage either with wild-type ADAM13 (0.5 ng, B) or with the truncated ﰅcyto (0.5 ng, C; 0.25 ng, D; 0.125 ng, E) mRNA. (B) Overexpression of WT ADAM13 produced embryos with cement glands extending dorsally from their normal position. (C) Similarly, ﰅcyto overexpression leads to extension of the cement gland as well as extensive epidermal blistering (arrow) and anteroposterior axis short- ening. (D and E) Lower doses of mRNA injection correlate with the disappearance of axis and cement gland alterations.
FIG. 8. Overexpression of X-PACSIN2 alters neural crest cell positioning. Embryos were injected into one blastomere at the two-cell stage with 1 ng of wild-type (A) or the truncated (B) X-PACSIN2 (ﰅSH3) transcripts. Injected embryos were cultured to tail bud stages and processed for Xslug in situ hybridization. The red arrowheads point to the injected side of each embryo. In these dorsal views, the positions of the three cephalic neural crest segments are indicated by white lines (mandibular (m), hyoid (h), and branchial (b)). X-PACSIN2 overexpression does not alter the cephalic crest segregation but appears to shorten both the hyoid and the branchial neural crest segments (A, red lines). In contrast, the cephalic neural crest cells appear as a single mass on the side injected with the truncated X-PACSIN2 transcript (B, right). (C) Schematic representation of the regulation of ADAM13 by X-PACSIN2. ADAM13 is in gray, the plasma membrane in yellow, and X-PACSIN2 in brown. In this model, X-PACSIN2 binds to the ADAM13 cytoplasmic domain through its SH3 domain and to a putative repressor (green) with its coiled-coil domain. In this conformation, ADAM13 is kept inactive. Removal of the ADAM13 cytoplasmic domain results in a constitutively active form of the protein. Deletion of X-PACSIN2 SH3 domains could block the repressor, resulting in the activation of wild-type ADAM13.