Figure 6. Sec5 phosphorylation contributes to cellular organization and organism development.

(a) Perturbation of the Sec5 phosphorylation cycle leads to defects in epithelial cells. MDCK cells stably expressing the indicated HA-Sec5 proteins were fixed in formalin and stained with ZO-1(green), HA (red), and DAPI (blue). Images were at the same magnification; bar = 10μm. (b) Quantification of multi-nucleated cells expressing the indicated form of Sec5 from 3 different experiments (n=3) performed as in (a). A minimum of 600 cells in total were counted. Errors represent SEM. Left, WB to confirm replacement of endogenous Sec5 with cells lysates prepared from the same experiments. (c) Perturbation of the Sec5 phosphorylation cycle in zebrafish embryos causes severe deformation and lethality. Zebrafish embryos were injected with 0.5 mM MO oligos alone or in combination with indicated mRNAs encoding different versions of mouse Sec5 around 4-8 cell stages. Embryos were monitored up to 96 hpf. (d) Quantification embryos with normal morphology or only minimal degeneration as in (c). A minimum of ~150 embryos in total were phenotyped in a double-blind manner from 3-4 independent experiments (n=4 for MO alone and WT, n=3 for mutants). The error bars represent SD.