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. 2018 Sep 13;9:3737. doi: 10.1038/s41467-018-06114-3

Fig. 1.

Fig. 1

The association of SNX3 with VPS35 is essential for the endosomal sorting of Wls. a HEK293T cells were transiently transfected with GFP-tagged SNX3, SNX11 and SNX12. The lysates were subjected to a GFP-trap immuno-isolation and immuno-blotted with the indicated antibodies. b Sequence alignment of human SNX11, SNX12, SNX3 and SNX3 homologues using ESPript 3.0 (76). The residues and regions mutated in the present study are highlighted. c Merged confocal images of HeLa cells transiently transfected to express wild-type or mutated versions of GFP-SNX3 (green) and immuno-stained for endogenous VPS35 (red). The scale bar indicates 11.5 μm (see Supplementary Fig. 1 for the entire panel of images). d HEK293T cells were transiently transfected to express the indicated GFP-SNX3 construct, subjected to a GFP-nanotrap and immuno-blotted with the indicated antibodies. The fluorescent bands, from three independent biological replicates, were quantified using a Licor Odyssey Scanner to show the relative binding of the GFP-SNX3 constructs to VPS35. Error bars indicate s.e.m. e Mapping of the targeted mutations onto the NMR derived structure of full length SNX3 (33). Green residues show those mutants that lead to a loss of VPS35 association while those residues in blue have limited effects on VPS35 binding when mutated. Note that the extended amino-terminal region prior to the β1-strand is intrinsically disordered (33). f Schematic representation of the migration of the QL neuroblast descendants (QL.d) and the dependency of this migration on Wnt signalling. In wild type, the QL.d migrate to positions in the posterior part of the animal. In the absence of Wnt signalling, the QL.d migrate in the opposite, anterior direction. g Mutation of SNX-3 p.Y22A induces a defect in QL.d migration (the cells migrate towards the anterior instead of the posterior). ***p < 0.0001 (Student’s t-test from three independent biological replicates; error bars indicate s.d.). Note that complete loss of snx-3 function induces a fully penetrant defect in QL.d migration (14)