Fig. 8

Functional consequences of tyrosine-34 glyosylation of RhoA.

(a) GlcNAcylation of RhoA by Afp18^G does not alter nucleotide binding. Fluorimetric analysis of mant-GDP (mGDP) or mant-GppNHp (mGppNHp) binding to WT RhoA or glycosylated RhoA (RhoA-GlcNAc) bound to GDP. Nucleotide exchange was monitored by the increase in fluorescence on mant-nucleotide binding to RhoA. Data are representative of two independent experiments. (b) Nucleotide exchange was measured by mant-GppNHp exchange with WT RhoA, Afp18^G-GlcNAcylated RhoA in the presence or absence of LARG. Data are represented as means±s.d. of three technical replicates. (c) Western blot analysis of RhoA, Rac1, Cdc42 pull-down experiments with zebrafish (ZF4) cells treated with Afp18^G (plus PA for delivery), Afp18^G NxN (plus PA for delivery) and C. difficile toxin B (TcdB). After Rho GTPase activation with CNF1, active RhoA was pulled down with ROCK II-coupled beads and Rac1 and Cdc42 with PAK-coupled beads. Bound GTPases were detected by western blotting using anti-RhoA, anti-Rac1, and anti-Cdc42 antibodies, respectively. Immunoblot of total RhoA is the loading control. (d) Live images at 30% epiboly (4.7 h.p.f.) of control non-injected, GFP mRNA (50 pg per embryo), RHOA mRNA (50 pg per embryo), RHOA Y34F (50 pg per embryo)+ Afp18^G mRNA (0.5 pg per embryo), Afp18^G mRNA (0.5 pg per embryo) + RHOA mRNA (50 pg per embryo) or Afp18^G (0.5 pg per embryo) + RHOA Y34F mRNA (50 pg per embryo) injected embryos. GFP mRNA (50 pg per embryo) was also included in each injection mix, and GFP fluorescence used to examine for homogenous expression: panels show transmitted light images in left columns and corresponding GFP fluorescence images in right columns. GFP, RHOA and RHOA Y34F injected embryos developed indistinguishable from non-injected control embryos. Scale bar, 500 µm. (e) Quantification of percentage embryos that develop normally or were severely affected regarding the organization of the blastoderm in experiment shown in d. Co-injection of RHOA or RHOA Y34F mRNA together with Afp18^G mRNA significantly reduces the fraction of disrupted and degraded blastoderm phenotypes, and increases the fraction of embryos developing normally at 30% epiboly. Values are average mean±s.e.m. of four biological replicates. Significance (P values <0.001) of changes in phenotype distribution was valuated using the Kruskal–Wallis test.