JCS033167 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Localization of Nok deletion proteins during zebrafish gastrula stages. Images represent reconstructions of confocal Z-stack sections imaged on late gastrula stage whole mounts (A-H) or sections (I-N). (A-H,I’-N’) Myc-tagged Nok fusion proteins, green. (A’-H’) aPKC. (I-N) aPKC, red; Myc-tagged Nok, green. aPKC was used as a counterstain to visualize apical cell membranes (also shown in A’-H’). All Nok deletion mutants correctly localize to the apical membrane. (E,M) Nok^ΔPar6 also has increased levels of cytoplasmic protein. White arrows (G,G’) highlight the presence of Nok^ΔPatj at the membrane. Arrowheads (N) indicate correct colocalization of Nok^ΔPatj with aPKC at apical junctions. (F,G,N) Nok^ΔPatj and Nok^ΔPatjΔLin7 also display nuclear localization localization which can be attributed to the deletion of a nuclear export signal within the L27N domain (red asterisks) (Bit-Avragim et al., 2008). (H) The Nok^L27 deletion protein comprises only the two L27 domains and lacks the rest of the Nok protein; it correctly associates with the membrane. Therefore, the bipartite L27 domains are sufficient but not necessary for correct membrane association. Importantly, under these WT conditions, none of the deletion proteins tested completely lacks membrane association. Possibly, alternative combinations of protein associations or other functionally redundant mechanisms contribute to tethering Nok at the apical membrane during gastrula stages. Orientation: (A-H) apical view, (I-N) apical to the top.