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.
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