The ELMO1-DOCK1-Rac1 complex influences ERM phosphorylation and tight control of phospho-Ezrin is required for proper ciliogenesis. (A) Immunoblot of 24 hpf zebrafish lysates showing elevated phospho-Ezrin (pERM) levels in embryos injected with TB-MO rac1 (4ng)/rac1l (0.5ng), SB-MO elmo1 (2ng) and SB-MO dock1 (2ng) as compared with Co-MO (4.5ng). (B) Immunoblot of 24 hpf zebrafish lysates showing reduced pERM levels in zebrafish embryos injected with elmo1 mRNA (20pg) or rac1l mRNA (20pg) as compared with control embryos. Anti-γ-Tubulin immunoblots served as a loading control. Immunoblots represent results from one of three independent experiments with similar results. (C-F) Overexpression of Ezrin by transient transgenesis demonstrates the importance of tight regulation of phosphorylation at threonine 564 for the function of zebrafish Ezrin. cadherin 17 (cdh17) promoter-driven expression of either the nonphosphorylatable T564A mutation or the phosphorylation mimetic mutant T564D caused cyst formation on the side of the pronephros where the MCCs in the mid-portion of the pronephric tubule are most affected. Arrowheads indicate the tubular mid-portion, with the cells expressing mutant Ezrin on the side of the formed cyst. The number of individual embryos analysed is indicated above each bar. (G) Quantification of pronephric cyst formation of 48 hpf zebrafish embryos injected with SB-MO elmo1 (2 ng) with or without ezrin(T564A) mRNA (10 pg) (**P=0.006), SB-MO dock1 (2 ng) with or without ezrin(T564A) mRNA (10 pg) (**P=0.009) or TB-MO rac1 (4 ng)/rac1l (0.5 ng) with or without ezrin(T564A) mRNA (10 pg) (*P=0.02). Scale bars: 50 μm.
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