The nok NES motif is required for nuclear export. Images represent reconstructions of confocal Z-stack sections imaged on late gastrula stage whole mounts. A: Endogenous nok mostly localizes to the outer cell membrane of EVL cells whereas nuclear localization is barely detectable. B: HisMyc-tagged nok^wt fusion protein detected with anti-Myc antibody, green. The wt protein localizes to the outer cell membrane in EVL cells. C: HisMyc-tagged nok^ΔNES (green) localizes to the nucleus of EVL cells and lower levels of protein are also present at the outer cell membrane. D: HisMyc-tagged nok^ΔPatj localizes to the nucleus and lower levels are present at the outer cell membrane (green). Red arrow indicates an EVL cell nucleus, which is large compared with the smaller DL cell nuclei located just underneath. Propidium iodide (PI) nuclear counterstain in red.

Redundant mechanisms involved in nok nuclear accumulation. Images are reconstructions of confocal Z-stack sections imaged on late gastrula stage whole mounts. HisMyc-tagged nok deletion proteins are detected with anti-Myc antibody (green). A: HisMyc-tagged nok^ΔPatj strongly localizes to nuclei of EVL and DL cells (recognizable by their size) and to outer cell membranes. B: HisMyc-tagged nok^{ΔPatj NLS} protein that lacks the L27N domain and the two nuclear import signals still localizes to the nucleus, which indicates that alternatives routes of nok nuclear import must exist. C: HisMyc-tagged nok nok^{ΔPar6 Patj} protein localizes to nuclei of EVL and DL cells indicating that direct association with Par6 is not necessary for nuclear accumulation of nok. D: In contrast, HisMyc-tagged nok^{ΔPar6 Patj NLS} protein fails to localize to nuclei of EVL and DL cells suggesting redundancy of the NLS motifs and association with Par6 in nok nuclear accumulation. E: Control non-injected embryos stained with the anti-Myc antibody.

Increased levels of nuclear nok protein. Increased protein levels of HisMyc-tagged nok^{ΔPatj NLS} (expected size of 74.1 kD) compared with HisMyc-tagged nok^wt (expected size of 82.2 kD) suggest that nuclear accumulation stabilizes nok protein.

aPKC kinase catalytic activity prevents nuclear accumulation. Images are reconstructions of confocal Z-stack sections imaged on late gastrula stage whole mounts. Anti aPKC (A,B,D,E) and anti-Myc (C) stainings are false-colored. A: Endogenous aPKC localization in EVL cells is mostly at the outer cell membrane whereas (B) weak levels of wt protein are also detectable throughout the cytoplasm and nuclei of DL cells. C: HisMyc-tagged aPKCi^D57A within EVL cells localizes to the cytoplasm and is complete absent from outer cell membranes or nuclei. D: aPKCi^KD strongly localizes to the nuclei of EVL and (E) DL cells. All images are apical views onto the tissue.

nok and aPKCi are not sufficient to tether each other within the nucleus. Images are reconstructions of confocal Z-stack sections imaged on late gastrula stage whole mounts. Myc and aPKC stainings are false-colored. A,B: Embryos expressing high levels of HisMyc-tagged nok^ΔNES were counterstained with anti aPKC. A,A′: Within DL cells, high levels of nuclear HisMyc-tagged nok^ΔNES are present whereas aPKC localizes to outer cell membranes and no increased levels of nuclear aPKC are detectable (A“). B,B′: Within epithelial EVL cells, HisMyc-tagged nok^ΔNES is at outer cell membranes and within the nucleus. B”: aPKC is predominantly associated with outer cell membranes. C: Embryos overexpressing HisMyc-tagged nok^wt (green) and non-tagged aPKCi^KD (red), whereas nok^wt is absent from the nucleus (C′), aPKCi^KD is nuclear. All images are apical views onto the tissue.

Loss of nok nuclear import and export signals does not affect epithelial maintenance. Phenotypes of (A) wt, (B) nok^s305 mutant, (C) nok^s305 mutant rescued with nok^ΔNLS mRNA or with (D) nok^{ΔPatj NLS} mRNA at 36 hpf. The straight body form is rescued in nok^ΔNLS mutants (C) but not in nok^{ΔPatj NLS} mutants (D). Insets depict details of RPE from each corresponding embryo. The cobble-stone like appearance of RPE tissue surrounding the neural retina is severely disrupted and displays wholes in (B) nok^s305 but this phenotype is partially complemented in (C) nok^ΔNLS or (D) nok^{ΔPatj NLS} mutants. E: Summary of rescue efficiencies of the different deletion mutants. Mutant phenotypes for nok^ΔNES are similar to nok^{ΔPatj NLS} (not shown).