Xenbase Image ID: 146127

Fig. 4. Dystroglycan mutants lacking the cytoplasmic domain (Dg-δcyto) and the tyrosine residue of the PPPY motif affect vacuolation. (A) Whole embryos are shown for wild-type (WT) and Dg-δcyto mutants at stage 32. (a,b) Stage 15 and (c,d) stage 32. Immunodetection of laminin or the Tor70 antibody in controls (a,c), in Dg-δcyto mutants (b) and in replacement of Dg with Dg-δcyto mutants (d). In mutants, cells are round instead of exhibiting a fusiform phenotype (b, arrowheads). Laminin is detected. Tor70 staining reveals a discontinuous sheath (d, arrowhead). (B) Schematic representation of Dg cytoplasmic mutants. Dg-δcyto encodes a protein that does not possess the C-terminus tail. Four amino acids after the transmembrane domain are conserved to ensure that the Dg-δcyto integrates into the membrane. Dg-FL PPPY/LALA encodes a protein in which the PPPY motif is mutated to LALA. Dg-FL Glu and Dg-FL Phe encode proteins in which the tyrosine residue is mutated to glutamate and phenylalanine, respectively. The Dg-FL KKR/ENG encodes a protein in which the KKR motif is mutated to ENG. The Dg-FL PPEY/AAAA mutant encodes a protein in which the PPEY motif is mutated to AAAA. Underlining indicates consensus binding sequences. (C) Dg-MO was co-injected with mRNAs encoding the Dg mutants. At stage 32, protein lysates were subjected to western blotting using antibodies against β-Dg and α-tubulin. Equivalent amounts of propeptides, endogenous Dg (WT), full-length Dg (Dg-FL) and the mutated β-Dg proteins are observed. R, ratios of Dg-propeptide and β-Dg to α-tubulin normalized to WT. (D) Whole embryos are shown for point mutants at stage 32 in upper panels. Overexpression of Dg-FL PPPY/LALA (a), Dg-FL Glu (b) and Dg-FL Phe (c) proteins on sections at stage 15. Replacement of Dg with Dg-FL PPPY/LALA (d), Dg-FL Glu (e) and Dg-FL Phe (f) on sections at stage 32. Immunodetection of laminin (a-c) and the Tor70 antibody (d-f). The arrowhead in e points to a vacuole. Scale bars: 500 μm in A upper panels, D upper panels; 25 μm in Aa-d,Da-f. Image published in: Buisson N et al. (2014) Copyright © 2014. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Gene Synonyms Species Stage(s) Tissue lama1.S LOC108718546 X. laevis Throughout NF stage 15 extracellular matrix Image source: Published Larger Image Printer Friendly View

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