The primary passage was then split into two 10 cm plates at six million cells per plate (day 0)

The primary passage was then split into two 10 cm plates at six million cells per plate (day 0). virus on the ability of Wnt5a to downregulate GFP-Kif26b fluorescence in the WRK reporter assay. elife-26509-fig4-data1.xlsx (13K) DOI:?10.7554/eLife.26509.016 Figure 4source data 2: (Related to panel D) The median GFP-Kif26b fluorescence in the WRK reporter cell line infected Benznidazole with a Fzd1 virus, a Fzd7 virus, or a Cas9 control virus. elife-26509-fig4-data2.xlsx (15K) DOI:?10.7554/eLife.26509.017 Figure 4source 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. elife-26509-fig4-data3.xlsx (13K) DOI:?10.7554/eLife.26509.018 Figure 5source 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. elife-26509-fig5-data1.xlsx (19K) DOI:?10.7554/eLife.26509.023 Figure 5source data 2: (Related to panel B) Relative wound density of MLNR 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. elife-26509-fig5-data2.xlsx (22K) DOI:?10.7554/eLife.26509.024 Figure 6source data 1: (Related to panel B) The effects of Wnt5a and Kif26b mis-expression on zebrafish embryonic tissue morphogenesis. elife-26509-fig6-data1.xlsx (12K) DOI:?10.7554/eLife.26509.026 Figure 6source data 2: (Related to panel D) Quantification of the numbers of PGCs per gonad in Benznidazole E11.5 or mouse embryos. elife-26509-fig6-data2.xlsx (13K) DOI:?10.7554/eLife.26509.027 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. elife-26509-supp1.xlsx (1.8M) DOI:?10.7554/eLife.26509.029 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 can cause Robinow syndrome, a congenital disorder characterized by short-limbed dwarfism and morphological defects in craniofacial and genital structures, demonstrating that the Wnt5a-Ror-Dvl pathway regulates morphogenesis during human development (Afzal et al., 2000; van Bokhoven et al., 2000; Person et al., 2010; Bunn et al., 2015; White et al., 2015, 2016). However, since the function of Dvl phosphorylation is not clear, and Dvl is a common component of several signaling pathways including the canonical Wnt signaling pathway and the planar cell polarity (PCP) pathway, how the Wnt5a-Ror pathway signals to carry out its biological functions remains incompletely understood. In this study, we conducted a whole phosphoproteome-scale mass spectrometric screen comparing wild-type cells with cells lacking the Ror family of proteins in an effort to identify additional effectors of Wnt5a-Ror signaling. The screen identified a number of candidate proteins whose levels or phosphorylation status was influenced by Wnt5a-Ror signaling, including factors involved in cytoskeletal regulation and cell adhesion, processes crucial for the morphogenesis of tissues. We then focused the remainder of the study on characterizing Kif26b, a Benznidazole member of the kinesin microtubule motor Benznidazole superfamily, which stood out as a particularly promising target of Wnt5a-Ror Benznidazole signaling for the following reasons. Mutations in the orthologs of and produce similar neuronal migration and axon guidance phenotypes, suggesting that these molecules might function in a common molecular pathway (Wightman et al., 1996; Forrester et al., 1998). Moreover, recent studies demonstrated that Kif26b plays crucial roles in regulating cytoskeleton-driven processes, including cell migration, polarization and adhesion, raising the possibility that Kif26b could function specifically as a cytoskeletal effector of the Wnt5a-Ror pathway (Uchiyama et al., 2010; Guillabert-Gourgues et al., 2016). Through a series of biochemical studies, we demonstrate that Wnt5a-Ror signaling regulates the steady-state abundance of Kif26b in cells via a mechanism involving the ubiquitin-proteasome system that is.