Volume 28, Issue 1, Pages 60-69.e8 (January 2018) Polo-like Kinase Couples Cytoplasmic Protein Gradients in the C. elegans Zygote  Bingjie Han, Katianna R. Antkowiak, Xintao Fan, Mallory Rutigliano, Sean P. Ryder, Erik E. Griffin  Current Biology  Volume 28, Issue 1, Pages 60-69.e8 (January 2018) DOI: 10.1016/j.cub.2017.11.048 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 MEX-5 RNA Binding Is Required for GFP::POS-1 and PIE-1::GFP Segregation (A) EMSA comparing the affinity of MBP-tagged MEX-5(aa 236–350) (top) and MBP-tagged MEX-5(aa 236–350; F294N; F339N) (bottom) for fluorescein-labeled TNF-ARE RNA from the TNFα 3′ UTR [10]. The target RNA is shown at the bottom with a MEX-5-binding site highlighted in gray. (B) The fraction of bound RNA is plotted as a function of MEX-5 concentration. Kd,app was determined by fitting the data to the Hill Equation [10] and is the average of at least three experiments. Error in the key is the SD. Little or no binding of MEX-5(aa 236–350; F294N; F339N) was observed at the highest concentration tested (100 nM), so we estimate the Kd,app to be greater than 100 nM. (C) Schematic of MEX-5(ZFmut) indicating the position of the TZF RNA-binding domains (ZF1 and ZF2) and the F294N and F339N substitutions. (D and E) Top: GFP::POS-1 (D) and PIE-1::GFP (E) localization in embryos of the indicated genotypes. For this and subsequent figures, images are acquired at nuclear envelope breakdown (NEBD) at the cell midplane, and embryos are oriented with the anterior to the left and the posterior to the right. Scale bar, 10 μm. Bottom: relative GFP::POS-1 and PIE-1::GFP concentration along the A/P axis in embryos of the indicated genotype (normalized to the concentration at the anterior end). (F, G, I, and J) Mean FRAP recovery curves for GFP::POS-1 (F and G) and PIE-1::GFP (I and J) in the anterior and posterior of embryos of the indicated genotype. The same wild-type (WT) data are presented in the top and bottom graphs (error bars are only shown in the top graph for visual clarity). Photobleaching was performed at time = 0. (H and K) Mean half-time of recovery (t1/2) for the indicated FRAP experiments, GFP::POS-1 (H) and PIE-1::GFP (K). A, anterior; P, posterior. In this and subsequent figures, p values are as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; n.s., not significant (>0.05). For all graphs, error bars represent SEM and the number of embryos analyzed are in parentheses. Throughout this figure, WT embryos were treated with a control RNAi (empty L4440 vector). See also Figures S1 and S2 and Table S1. Current Biology 2018 28, 60-69.e8DOI: (10.1016/j.cub.2017.11.048) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 RNA Binding Mediates POS-1 and PIE-1 Retention in the Posterior Cytoplasm (A) EMSA comparing the affinity of MBP-tagged POS-1(aa 80–180) and MBP-tagged POS-1(aa 80–180; F121N; F164N) for fluorescein-labeled SCR+ RNA from the glp-1 3′ UTR [28]. The target RNA is shown at the bottom with the POS-1-binding sites highlighted in gray. (B) The fraction of bound RNA plotted as a function of POS-1 concentration. Kd,app was determined as in Figure 1B. We did not observe saturation of POS-1(aa 80–180; F121N; F164N) binding at the highest concentration tested (2 μM). We approximate the Kd,app to be greater than 300 nM, which shows approximately half-maximal binding observed in the wild-type POS-1(aa 80–180) EMSA. (C and G) Top: schematic of POS-1 (C) and PIE-1 (G) indicating the position of the TZF RNA-binding domains (ZF1 and ZF2) and the ZFmut substitutions. Bottom: images of embryos expressing the indicated GFP::POS-1 and GFP::PIE-1 transgenes, and quantification of their relative concentrations along the A/P axis. (D, E, H, and I) FRAP recovery curves for the indicated GFP::POS-1 (D and E) and GFP::PIE-1 (H and I) transgenes. The wild-type GFP::POS-1 and GFP::PIE-1 recovery curves are also displayed in Figure 1. (F and J) Mean t1/2 for the indicated FRAP experiments, GFP::POS-1 (F) and PIE-1::GFP (J). See also Figure S3 and Table S1. Current Biology 2018 28, 60-69.e8DOI: (10.1016/j.cub.2017.11.048) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 PLK-1 Kinase Activity Is Required for POS-1 Segregation (A) Schematic of POS-1 with predicted PLK-1 phosphorylation sites within the region aa 193–244. Residues in red were mutated to either alanine (5A) or aspartic acid (5D). Gray boxes indicate the D/E/N X S/T Φ motif. (B) Relative GFP::POS-1 concentration along the A/P axis in WT (control RNAi) and plk-1(RNAi) embryos. (C) FRAP recovery curves for GFP::POS-1 in WT (control RNAi) and plk-1(RNAi) embryos. (D) Images of GFP::POS-1; plk-1(as) embryos treated with DMSO control or 1NM-PP1. (E) Posterior/anterior ratio of GFP::POS-1 concentration in DMSO control and 1NM-PP1-treated plk-1(as) embryos from 2 min before pronuclear meeting to 8 min after pronuclear meeting. (F) In vitro kinase assay with hPLK1 and the indicated substrates. Top: phosphorylation was monitored using a thiophosphate ester antibody, which recognizes alkylated ATP-γS. Bottom: Coomassie-stained gel. (G) Quantification of in vitro kinase assays (n = 4). Background was defined as the the values before kinase addition (t = 0) for each reaction. Values within each experiment were normalized to the final MBP:POS-1 time point. See also Figure S4 and Table S1. Current Biology 2018 28, 60-69.e8DOI: (10.1016/j.cub.2017.11.048) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 PLK-1 Phosphorylation Controls POS-1 Mobility (A) Top: images of embryos expressing the indicated GFP::POS-1 alleles. Bottom: relative concentrations of the indicated GFP::POS-1 alleles along the A/P axis. (B) FRAP recovery curves for GFP::POS-1 compared to GFP::POS-1(5A). (C) FRAP recovery curves for GFP::POS-1 compared to GFP::POS-1(5D). (D) Mean t1/2 for the indicated FRAP experiments. (E–G) FRAP recovery curves for the indicated GFP::POS-1 alleles in mex-5/6(RNAi) (E), plk-1(RNAi) (F), and par-1(RNAi) (G) embryos. As with all other FRAP experiments in this study, FRAP was performed simultaneously in the anterior and posterior cytoplasm. Because GFP::POS-1 mobility is uniform in mex-5/6(RNAi), plk-1(RNAi), and par-1(RNAi) embryos, the anterior and posterior recovery curves for each genotype were averaged together. (H) Mean t1/2 for the indicated FRAP experiments. See also Figure S3 and Table S1. Current Biology 2018 28, 60-69.e8DOI: (10.1016/j.cub.2017.11.048) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Characterization of mex-5(T186A);mex-6(RNAi) and PLK-1::sGFP Embryos (A) Schematic of MEX-5(T186A). (B–D) GFP::POS-1 localization (B), relative concentration along the A/P axis (C), and FRAP curves (D) in embryos of the indicated genotype. GFP::POS-1 FRAP curves are similar in control RNAi and mex-6(RNAi) embryos (see Figure S5A). (E) Mean t1/2 for the indicated FRAP experiments. (F) PLK-1::sGFP localization in WT and mex-5/6(RNAi) embryos. Top: duplicate images with different intensity normalizations (dim and bright). The centrosomal and chromosomal signals in the bright images are saturated. Bottom: the concentration of PLK-1::sGFP in the cytoplasm is not affected by mex-5/6(RNAi). (G) Relative concentration of PLK-1::sGFP along the A/P axis (normalized to the posterior end). (H) FRAP curves for PLK-1::sGFP in wild-type and mex-5/6(RNAi) embryos. (I) Working model. MEX-5 (red) and POS-1 (green) exist in fast-diffusing complexes and slow-diffusing complexes. The interaction of both MEX-5 and POS-1 with slow-diffusing complexes depends on their ability to bind RNA. PAR-1 phosphorylation of MEX-5 (depicted as “+P”) inhibits the formation of slow-diffusing MEX-5 complexes in the posterior, leading to their enrichment in the anterior. PLK-1 phosphorylation of POS-1 inhibits the formation of slow-diffusing POS-1 complexes in the anterior, where PLK-1 most likely accumulates in slow-diffusing RNA complexes due to its interaction with MEX-5, which was shown by yeast two hybrid [23]. As a consequence, slow-diffusing POS-1 complexes accumulate in the posterior, giving rise to the POS-1 concentration gradient. PLK-1 is depicted as inhibiting POS-1 association with slow-diffusing complexes for simplicity, but it could also promote POS-1 dissociation from slow-diffusing complexes. See also Figures S3 and S5 and Table S1. Current Biology 2018 28, 60-69.e8DOI: (10.1016/j.cub.2017.11.048) Copyright © 2017 Elsevier Ltd Terms and Conditions