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