Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates
- Harvard Medical School, United States
- University of California, Davis School of Medicine, United States
- Fred Hutchinson Cancer Research Center, United States
- Center for Reproductive Sciences, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, United States
- Institute of Molecular Embryology and Genetics, Kumamoto University, Japan
- University of California, United States
Figures
Figure 1 with 2 supplements
Phosphoproteomic screen identifies putative targets of the Wnt5a-Ror pathway.
(A) Experimental design of the six-plex mass spectrometry analysis to screen for Ror-dependent biochemical cellular changes (see the main text for details of the experimental design). (B) Volcano …
Figure 1—figure supplement 1
Conditional depletion of Ror1 and Ror2 protein in MEFs.
Immunoblots of Ror1 protein, Ror2 protein and Dvl2 protein in primary MEFs derived from the two separate E12.5 Ror1f/f; Ror2f/f; CAG-CreER mice and one E12.5 Ror1+/+; Ror2+/+; CAG-CreER mouse used …
Figure 1—figure supplement 2
Kif26b phosphorylation sites identified in the phosphoproteomic screen.
(A) A schematic illustration of mouse Kif26b showing the amino acid boundaries of the motor and coiled coil domains. The motor and coiled coil domains were identified using the following web …
Figure 2 with 1 supplement
Kif26b protein levels are regulated by the expression of Wnt5a and Ror proteins in primary mouse embryonic fibroblasts.
(A) Immunoblots of Kif26b protein, Ror1 protein, Ror2 protein and Dvl2 protein in primary MEFs derived from two E12.5 Ror1f/f; Ror2f/f; CAG-CreER mice. 4-OHT or vehicle control was added for 72 hr …
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Figure 2—source data 1
(Related to panel E) Relative mRNA expression of Kif26b and β-actin as measured by RT-qPCR in primary MEFs derived from E12.5 Wnt5a-/- mice, after 1 hr or 6 hr of Wnt5a stimulation.
- https://doi.org/10.7554/eLife.26509.007
Figure 2—figure supplement 1
Validation of anti-Kif26b antibodies.
(A) Immunoblots of Kif26b protein, EGFR protein, Ror1 protein and Dvl2 protein in primary MEFs derived from E12.5 WT mice. Indicated shRNAs were transduced via lentiviral transduction. An empty …
Figure 3 with 2 supplements
Wnt5a downregulates Kif26b levels via a ubiquitin/proteasome-dependent mechanism.
(A) Flow cytometry histograms depicting the downregulation of GFP-Kif26b fluorescence in the WRK reporter cell line after Wnt5a stimulation (0.2 μg/ml Wnt5a) for 6 hr. (B) Dose-response curve …
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Figure 3—source data 1
(Related to panel C) The kinetics of GFP-Kif26b turnover in the absence or presence of Wnt5a stimulation.
- https://doi.org/10.7554/eLife.26509.011
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Figure 3—source data 2
(Related to panel F) The effects of DMSO, epoxomicin and PYR-41 on the ability of Wnt5a to downregulate GFP-Kif26b fluorescence in the WRK reporter assay.
- https://doi.org/10.7554/eLife.26509.012
Figure 3—figure supplement 1
Expression of Wnt5a-Ror signaling components in MEFs and NIH/3T3 cells.
(A) Immunoblots of Kif26b protein, Ror1 protein, Ror2 protein and Dvl2 protein in primary MEFs derived from E12.5 WT mice. WT MEFs have a high basal level of Wnt5a-Ror signaling activity, as …
Figure 3—figure supplement 2
Characterization of the GFP-Kif26b (WRK) reporter line.
(A) Anti-Kif26b immunoblot of the GFP-Kif26b protein expressed under the control of an EF1 promoter in the WRK reporter cell line. The reporter cells were stimulated with 0.2 μg/ml Wnt5a for 0 hr, 1 …
Figure 4 with 1 supplement
Involvement of Fzd and Dvl proteins in Kif26b degradation.
(A) Flow cytometry histograms depicting the effect of ectopic Shisa2 expression on the ability of Wnt5a to induce GFP-Kif26b degradation in the WRK reporter assay. Mouse Shisa2 was expressed via …
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Figure 4—source data 1
(Related to panel B) The effects of the control virus and the Shisa2 virus on the ability of Wnt5a to downregulate GFP-Kif26b fluorescence in the WRK reporter assay.
- https://doi.org/10.7554/eLife.26509.016
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Figure 4—source data 2
(Related to panel D) The median GFP-Kif26b fluorescence in the WRK reporter cell line infected with a Fzd1 virus, a Fzd7 virus, or a Cas9 control virus.
- https://doi.org/10.7554/eLife.26509.017
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Figure 4—source data 3
(Related to panel F) Quantification of the median GFP-Kif26b fluorescence in the WRK reporter cell line infected with a Dvl1 virus or a Cas9 control virus.
- https://doi.org/10.7554/eLife.26509.018
Figure 4—figure supplement 1
Comparison of the abilities of distinct Fzd subfamily members to induce Kif26b degradation.
(A) Flow cytometry histograms depicting the effect of ectopic Fzd3 or Fzd6 expression on GFP-Kif26b levels in the WRK reporter assay. Mouse Fzd3 and Fzd6 were expressed via lentiviral transduction. …
Figure 5 with 3 supplements
Wnt5a-Kif26b signaling modulates the migratory behavior of NIH/3T3 cells.
(A) Relative wound density of two separate NIH/3T3 cell lines in which Kif26b expression is knocked out using CRISPR/Cas9 genome editing, and one control Cas9-expressing NIH/3T3 cell line in a …
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Figure 5—source data 1
(Related to panel A) Relative wound density of two separate NIH/3T3 cell lines in which Kif26b expression is knocked out using CRISPR/Cas9 genome editing, and one control Cas9-expressing NIH/3T3 cell line in a kinetic wound-healing assay.
- https://doi.org/10.7554/eLife.26509.023
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Figure 5—source data 2
(Related to panel B) Relative wound density of a GFP-Kif26b expressing NIH/3T3 cell line, treated with or without Wnt5a and a control NIH/3T3 cell line in a kinetic wound-healing assay.
- https://doi.org/10.7554/eLife.26509.024
Figure 5—figure supplement 1
Overexpression of GFP-Kif26b or knockout of Kif26b expression does not affect the proliferation rate or survival of NIH/3T3 cells.
(A) Immunoblots of Kif26b protein, Ror1 protein, Ror2 protein and Dvl2 protein in NIH/3T3 cell lines overexpressing (OE) GFP-tagged Kif26b protein using the Flp-In system or control NIH/3T3 cell …
Figure 5—figure supplement 2
Wnt5a downregulates the cellular levels of GFP-Kif26b.
Immunoblots showing levels of Kif26b in GFP-Kif26b-overexpressing NIH/3T3 cells, without or with Wnt5a stimulation (0.1 μg/ml Wnt5a for 24 hr). The level of Kif26b endogenously expressed in WT …
Figure 5—figure supplement 3
Wnt5a treatment has no effect on the wound closure efficiency of Kif26b knockout NIH/3T3 cells.
Relative wound density of two separate NIH/3T3 cell lines in which Kif26b expression is knocked out using CRISPR/Cas9 genome editing, without or with Wnt5a treatment. Traces of Kif26b knockout cell …
Figure 6
In vivo perturbation of Kif26b expression in zebrafish and mouse embryos induces phenotypes characteristic of Wnt5a-Ror signaling defects.
(A) Representative images showing the effects of Wnt5a and Kif26b mis-expression on zebrafish embryonic tissue morphogenesis. Microinjection of Wnt5a mRNA did not produce any embryos that were …
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Figure 6—source data 1
(Related to panel B) The effects of Wnt5a and Kif26b mis-expression on zebrafish embryonic tissue morphogenesis.
- https://doi.org/10.7554/eLife.26509.026
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Figure 6—source data 2
(Related to panel D) Quantification of the numbers of PGCs per gonad in E11.5 Kif26b+/+ or Kif26b-/- mouse embryos.
- https://doi.org/10.7554/eLife.26509.027
Figure 7
Model of Wnt5a-Ror-Kif26b signaling.
Schematic of canonical Wnt/β-catenin signaling (A) vs. noncanonical Wnt5a-Ror-Kif26b signaling (B). See the main text for details.
Videos
Video 1
Time-lapse fluorescent confocal microscopy reveals rapid Wnt5a-dependent downregulation of the GFP-Kif26b signal in the NIH/3T3 reporter cell line.
Bright signal reflects GFP-Kif26b fluorescence. Buffer or 0.2 μg/mL recombinant Wnt5a was added at the start of the imaging session. Cells were imaged every 10 min for 16 hr at 40x magnification.
Additional files
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Supplementary file 1
Phosphopeptides identified and quantified in the TMT/MS3 phosphoproteomic screen.
Columns include: Uniprot protein identification number, gene symbol, protein description/name, phosphosite position, phosphosite motif, localization score, spectral counts, the normalized summed signal to noise for each of the six TMT (126 to 131) channels.
- https://doi.org/10.7554/eLife.26509.029
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Supplementary file 2
Hits from the TMT/MS3 phosphoproteomic screen.
(A) Upregulated phosphopeptides that scored as ‘hits’ as defined in the text. Phosphopeptides above the bold line are ‘hits’ scored using the 2-fold cutoff filter. Phosphopeptides below the bold line are those scored between the 1.5- and 2-fold cutoffs. Columns include: gene name, protein description, fold change (4-OHT/vehicle treated samples), the p value. (B) Downregulated phosphopeptides that scored as ‘hits’ as defined in the text. Phosphopeptides above the bold line are ‘hits’ scored using the 2-fold cutoff filter. Phosphopeptides below the bold line are those scored between the 1.5- and 2-fold cutoffs. Columns include: gene name, protein description, fold change (4-OHT/vehicle treated samples), the p value.
- https://doi.org/10.7554/eLife.26509.030
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Transparent reporting form
- https://doi.org/10.7554/eLife.26509.031
