XB-ART-51676
PLoS One
2015 Dec 16;1012:e0145169. doi: 10.1371/journal.pone.0145169.
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A PTK7/Ror2 Co-Receptor Complex Affects Xenopus Neural Crest Migration.
Podleschny M
,
Grund A
,
Berger H
,
Rollwitz E
,
Borchers A
.
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Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.
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Species referenced: Xenopus
Genes referenced: egfr fn1 h2bc21 myc ptk7 ror2 twist1
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Fig 1. PTK7 and Ror2 co-localize in NC cells and co-precipitate independent of the kinase-homology domain of PTK7. A Co-localization of PTK7 and Ror2. NC cells co-expressing Ror2-EGFP and PTK7-RFP show distinct areas of co-localization (yellow) as well as membrane areas where only PTK7-RFP (red) or Ror2-EGFP (green) is localized. The right panel shows a higher magnification of the single cell in the left panel (indicated by a dashed square), scale bar = 10 μm. B,C Co-immunoprecipitation of PTK7 and Ror2. Full-length myc-tagged PTK7 (PTK7-MT) and a myc-tagged PTK7 deletion construct lacking the kinase homology domain (âkPTK7-MT) as well as a full-length HA-tagged Ror2 construct were expressed in MCF7 cells. B Constructs and their protein domains are depicted in the top panel. Abbreviations are as follows: IG (immunoglobulin domain), CRD (cysteine-rich domain), K (kringle domain), TM (transmembrane domain), KH (kinase homology domain), TK (tyrosine kinase), S/T (serine/threonine-rich domain), P (proline-rich domain). C Immunoprecipitation experiment; the cell transfection scheme is indicated at the top. Co-immunoprecipitation was carried out using either anti-myc (IP α-MT, upper panel) or anti-HA antibodies (IP α-HA, middle panel). The respective cell lysates are shown in the bottom panel. Antibodies used for Western blotting and molecular weights are indicated at the right. doi:10.1371/journal.pone.0145169.g001 |
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Fig 2. The intracellular domain and the CRD domain of Ror2 are not required for PTK7/Ror2 interaction. Myc-tagged PTK7 (PTK7-MT) together with FLAG- or HA-tagged Ror2 deletions were expressed in MCF7 cells. A The respective constructs and their protein domains are depicted in the top panel. B Immunoprecipitation using anti-FLAG antibodies; the cell transfection scheme is indicated at the top. Co-precipitated PTK7 was detected using anti-myc antibodies. Immunoprecipitated PTK7 is shown in the top panel, immunoprecipitated Ror2 constructs in the middle panel and cell lysates in the bottom panel. Antibodies used for Western blotting and molecular weights are indicated at the right. C Immunoprecipitation using anti-myc antibodies; the cell transfection scheme is indicated at the top. Co-precipitated full-length Ror2 and sRor2 were detected using anti-HA antibodies (top panel). Immunoprecipitated PTK7 is shown in the middle panel and cell lysates in the bottom panel. Antibodies used for Western blotting and molecular weights are indicated at the right. doi:10.1371/journal.pone.0145169.g002 |
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Fig 3. Loss of function of PTK7 affects NC cell shape and inhibits migration of explanted NC. A Time series showing explanted NC cells injected with 7.5 ng control or PTK7 MO in combination with 50 pg mGFP RNA and 250 pg H2B-mcherry. Cranial NC explants were excised at stage 16â17 and explanted on a fibronectin matrix and incubated until they had stably adhered to the matrix. NC migration was monitored for 5 hours using spinning disk microscopy (10x objective NA 0.45). Images for representative explants injected either with control or PTK7 MO are shown at the start of the experiment (0 h) or after 1, 3 or 5 hours; scale bar = 50 μm. B Cell tracking and Delaunay triangulation for explants injected with 7.5 ng co MO. The upper panel shows a single frame of the spinning disk movie and the lower panel the Delaunay triangulation at the start of the experiment (0 h) or after 5 hours. Cells were tracked over the whole five-hour time interval using the H2B staining of single nuclei. These tracks are shown for single cells as differently colored lines in the images taken after 5 hours, scale bar = 50 μm. C Cell tracking and Delaunay triangulation for explants injected with 7.5 ng PTK7 MO. D Time series showing explants injected with 7.5 ng co MO (upper panel) or 7.5 ng PTK7 MO (lower panel) at a higher magnification. Injected NC cells were explanted at stage 17, cultured for 1.5 hours and imaged with a 63x objective (NA 1.4); scale bar = 20 μm. Images are shown at the start of the experiment (0 min) and after 45 and 90 minutes. doi:10.1371/journal.pone.0145169.g003 |
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Fig 4. Ror2 rescues the PTK7 loss of function phenotype in explanted NC cells. A NC explants injected with 7.5 ng MO in combination with 50 pg mGFP RNA, 250 pg H2B-mcherry and 150 pg Ror2 RNA. Time-lapse images (upper panel) and Delaunay triangulations (lower panel) at the start of the experiment (0 h) and after 4 or 8 hours are shown for the different conditions. B Graph summarizing percentage of migration defects of 3 independent experiments (total of 39 explants). Standard error of the means are shown. Asterisks indicates a p-value in a Studentâs t-test < 0.05. Scale bars = 200 μm. doi:10.1371/journal.pone.0145169.g004 |
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Fig 5. The kinase domain of Ror2 is required to rescue the NC migration defect in PTK7 morphant embryos. Xenopus embryos were injected with different constructs in combination with 100 pg LacZ RNA as a lineage tracer and analyzed by whole-mount in situ hybridization using a twist antisense RNA probe. A Embryo injected with 10 ng control MO and 100 pg GFP RNA shows normal NC migration. B Embryo injected with 10 ng PTK7 MO and 100 pg GFP RNA shows inhibition of NC migration on the injected side, while NC migration is normal on the uninjected side. C Co-injection of 10 ng PTK7 MO together with 100 pg Ror2 RNA rescues the NC migration defect. D Embryo injected with 10 ng PTK7 MO and 100 pg of Ror2δ469 RNA. The embryo shows a NC migration defect on the injected side. E Embryo injected with PTK7 MO and a kinase dead mutant of Ror2 (Ror2-3I) showing a NC migration defect on the injected side. F Graph summarizing the percentage of NC migration defects of a minimum of 5 independent experiments for each experimental condition. Asterisks indicates a p-value in a Student’s t-test < 0.001. Scale bar = 500 μm. doi:10.1371/journal.pone.0145169.g005 |
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S1 Fig. PTK7 and Ror2 co-precipitate each other but not EGFR or TGFÃ1R. A Full-length myc-tagged PTK7 (PTK7-MT) was co-expressed with Ror2-EGFP, EGFR-EGFP [95] or TGFÃ1R-EGFP (kind gift of A. Menke, Molecular Oncology of Solid Tumors, Giessen, Germany) as indicated in MCF7 cells. Cell lysates were precipitated using anti-myc antibodies (IP α-MT, upper panel). Precipitates are shown in the upper panels, cell lysates in the lower panel. Antibodies used for Western blotting and molecular weights are indicated at the right. B Full-length HA-tagged Ror2 was co-expressed with PTK7-MT, EGFR-EGFP or TGFÃ1R-GFP as indicated in MCF7 cells and cell lysates were precipitated using anti-HA antibodies (IP α-HA, upper panel). Precipitates are shown in the three upper panels and lysates in the two lower panels. Antibodies used for Western blotting and molecular weights are indicated at the right; * marks unspecific bands, ** Ror2 signal remaining from previous anti-HA staining, which was only partially removed by blot stripping. doi:10.1371/journal.pone.0145169.s001 |
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S2 Fig. Propidium Iodine (PI) staining to determine the viability of PTK7 loss of function NC cells. NC explants injected with 7.5 ng control MO or PTK7 MO in combination with 50 pg mGFP were treated with PI (10 μg/ml) to test for the viability of the explanted NC cells. Cell membrane integrity prevents PI staining of viable cells, while it can stain nucleic acids of apoptotic cells (red). In PTK7 morphant cells few PI-positive cells appear after 3 hours compared to 6 hours in controls (one example each is marked by a red arrow). Round-shaped PTK7 morphant NC cells appear early in the experiment and keep moving/blebbing for up to a couple of hours (white arrows, numbers indicate specific cells during the course of the experiment). Thus, the roundish cell shape is not necessarily an indication of cell death. Dashed squares show higher magnifications of specific cells. Scale bar = 50 μm. doi:10.1371/journal.pone.0145169.s002 |
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