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. 2022 Apr 19;13:2056. doi: 10.1038/s41467-022-29741-3

Fig. 7. Optogenetic manipulation of Ezrin function in multiciliated cells of the Xenopus epidermis.

Fig. 7

a To test whether the Arabidopsis thaliana phytochrome system mediates protein interactions in MCCs in response to red light (660 nm), the phytochrome interaction factor (PIF) fused to GFP (GFP-PIF) was co-injected with phytochrome B (PHYB) fused to mCherry, localized to the plasma membrane by the C-terminal CAAX motif of Kras (PHYB-mCherry-CAAX). In response to red light, PHYB-mCherry-CAAX recruited GFP-PIF to the plasma membrane, while GFP-PIF remained in the cytoplasm after exposure to far-red light (740 nm) (scale bars, 10 µm). b The N-terminal FERM domain of Ezrin (SMART, http://smart.embl-heidelberg.de/) was fused to PIF, while the C-terminal part, containing the actin-binding domain, was fused to PHYB. While red light (660 nm) maintains the Ezrin fusion, far-red right light (740 nm) creates two Ezrin fragments, generating a dominant-negative effect. mRNAs were injected at the 4-cell stage and phycocyanobilin (PCB) (15 µM) was added at stage 6. To maintain the Ezrin in an assembled state, Xenopus embryos were exposed to red light until stage 18. At stage 20, embryos were split into two groups. One group was exposed to far-red light for 3 h to create Ezrin fragments and then returned to dark light, maintaining the dissociation. The other group was first kept in the dark, then returned to red light, keeping the Ezrin fragments assembled. c Far-red light (740 nm) disrupted rootlet tilting, revealed by shortening of the apparent rootlet length, and increased the rootlet circular standard deviation (CSD), demonstrating more randomly aligned rootlets. Depicted are the results of two independent experiments, using 5 embryos per condition. The symbols display the results for individual multiciliated cells (660 nm: 16 cells; 740 nm: 13 cells). Three cells, all derived from one embryo and depicted as black circles, were identified as outliers (ROUT, Q = 1%). The significance level was p = 0.0754 including the three outliers, and p < 0.0001 excluding the outliers (Mann–Whitney U test). Source data are provided as a Source Data file.