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First published online 23 April 2008
doi: 10.1242/dev.017202

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Myopic acts in the endocytic pathway to enhance signaling by the Drosophila EGF receptor

Grant I. Miura^*, Jean-Yves Roignant, Michel Wassef and Jessica E. Treisman

Kimmel Center for Biology and Medicine of the Skirball Institute, NYU School of Medicine, Department of Cell Biology, 540 First Avenue, New York, NY 10016, USA.

View larger version (97K): [in this window] [in a new window]   Fig. 1. mop is required for EGFR signaling. (A-F') Third instar Drosophila eye discs. (A,A') mopT612 mutant clones marked by the absence of GFP (green in A'). Photoreceptors are stained with anti-Elav (A, magenta in A'). (B-B'',C-C'') Eye discs with large mopT612 mutant clones generated in a Minute background and marked by the absence of GFP (B',C', green in B'',C''). R8 photoreceptors are stained with anti-Ato (B, magenta in B'') or anti-Sens (C, magenta in C''). mop has little effect on R8 differentiation. (D-F') mopT612 mutant clones marked by the absence of GFP (green in D',E',F'). Activated Caspase 3 staining (D, magenta in D') marks apoptotic cells and Cyclin B staining (E, magenta in E') marks cells in G2 or M phase. Posterior mop mutant clones contain reduced numbers of photoreceptors and show increased cell death and cell cycle re-entry. Phospho-MAPK staining (F, magenta in F') is reduced in mop mutant clones in the morphogenetic furrow (long arrow) and posteriorly (short arrow). (G,H) Embryos stained with anti-β-galactosidase reflecting aos-lacZ expression. (G) Wild type; (H) maternal/zygotic mop mutant. aos expression is strongly reduced in the absence of mop. (I) An adult wing containing mopT612 mutant clones shows loss of wing vein material (arrow). (J) A third instar wing disc with mopT612 clones made in a Minute background and marked by the absence of GFP (green), stained with anti-β-galactosidase reflecting aos-lacZ expression (magenta).

View larger version (106K): [in this window] [in a new window]   Fig. 2. mop acts on internalized EGFR. All panels show third instar Drosophila eye discs. Photoreceptors are stained with anti-Elav (A,C,E,G,I,K,M,O; magenta in B,D,F,H,J,L,N,P). (A,B) mopT482 mutant clones are positively marked by GFP expression (green in B, outlined in A). (C,D) Clones expressing EGFRtop are positively marked by GFP expression (green in D, outlined in C). (E,F) mopT612 mutant clones expressing EGFRtop are positively marked by GFP expression (green in F, outlined in E). (G,H) mopT612 mutant clones expressing Rasv12 are positively marked by GFP expression (green in H, outlined in G). (I-P) Large clones generated in a Minute background are marked by the absence of GFP (green in J,L,N,P, outlined in I,K,M,O). (I,J) mopT612; (K,L) mopT612 aos7; (M,N) mopT612 CblF165; (O,P) mopT612 sty5. Although EGFRtop induces photoreceptor differentiation in a wild-type background, it does not rescue mop mutant clones; removing aos also fails to rescue. Photoreceptor differentiation can be restored to mop mutant clones by expressing Rasv12 or removing Cbl or sty.

View larger version (84K): [in this window] [in a new window]   Fig. 3. Structure and expression of the Drosophila Mop protein. (A) Mop contains regions of homology to Bro1 and to tyrosine phosphatases. The positions of stop codons introduced by three mop mutant alleles are indicated. (B) Comparison of the Mop tyrosine phosphatase domain with the consensus sequences for the ten functional motifs defined for tyrosine phosphatases (boxed). Amino acids that differ from the consensus sequence are in red; the arrow indicates the predicted active site cysteine. (C-F) Third instar eye imaginal discs with clones positively marked by GFP expression (green in D,F) stained with anti-Elav (C,E, magenta in D,F). (C,D) mopT482 clones expressing a wild-type UAS-mop transgene; (E,F) mopT482 clones expressing a phosphatase-dead transgene (UAS-mopCS). Both transgenes fully rescue photoreceptor differentiation. (G-J) Third instar wing imaginal discs expressing aos-lacZ and ap-GAL4 and stained overnight with X-Gal. ap-GAL4 drives expression in the dorsal compartment (outlined in G). (G) Wild type; aos is weakly expressed in the wing vein primordia (bracket). (H) UAS-FlagMop; (I) UAS-EGFRtop; (J) UAS-FlagMop and UAS-EGFRtop. Mop overexpression gave very weak ectopic aos expression (arrow, H), but strongly enhanced the effect of activated EGFR.

View larger version (102K): [in this window] [in a new window]   Fig. 4. Mop is an endosomal protein. (A) Drosophila S2R+ cells stained for endogenous Mop. (B,C) GFP-Rab7 fluorescence in live S2R+ cells treated with dsRNA targeting lacZ (B) or mop (C). Mop depletion causes enlargement of Rab7-containing endosomes. (D-I) Wing discs expressing UAS-FlagMop with ap-GAL4 and stained for Flag (magenta in D,E; green in G,I), coexpressed Rab5-GFP (green in D), Rab11 (green in E,F), or Dor (magenta in G,H). (J-L) S2R+ cells expressing UAS-FlagMop and UAS-GFPRab5 (J), UAS-GFPRab7 (K) or UAS-GFPRab11 (L) with Actin-GAL4. Flag staining is shown in magenta and GFP in green. Mop is present on vesicles that are adjacent to Rab5-containing vesicles (arrowheads in D,J), shows partial colocalization with Dor and Rab7 (arrowheads in G,K), and does not colocalize with Rab11. (M,N) Wing discs with mopT612 clones marked by the absence of GFP (green in N), stained with anti-Hrs (M, magenta in N). Hrs shows reduced levels and punctate localization (arrows, M) in mop mutant clones. (O) An eye disc with a clone of cells expressing UAS-FlagMop and UAS-EGFRtop, stained with anti-Flag (green) and anti-EGFR (magenta). Most of the activated EGFR is present in vesicles that do not contain Mop. Scale bars: 10 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Mop is required for MAPK phosphorylation and EGFR cleavage. (A-C) Drosophila eye disc with mopT612 mutant clones marked by the absence of GFP (A, green in C) and stained with anti-EGFR (B, magenta in C). EGFR levels are slightly increased in mop mutant clones. (D) Western blot with Mop antibody of extracts from S2 cells transfected with Actin-GAL4 and UAS-mop, untreated (wt) or treated with mop dsRNA. mop is expressed in S2 cells and its levels can be significantly reduced by RNAi. (E) D2F cells were treated with lacZ or mop dsRNA and incubated with sSpi-conditioned media for 0 or 30 minutes. Protein lysates were blotted with antibodies to diphospho-MAPK and total MAPK. mop dsRNA treatment resulted in a decrease in MAPK phosphorylation. (F) Western blot with anti-EGFR of lysates from D2F cells treated with lacZ or mop dsRNA and incubated with sSpi-conditioned media for the indicated times. Cells treated with mop dsRNA failed to accumulate a faster-migrating band recognized by the EGFR antibody (asterisk). The position of full-length EGFR is indicated by an arrow. (G) S2R+ cells were treated with lacZ, mop, Cbl and/or sty dsRNA as indicated, and transfected with Actin-GAL4, UAS-GFP and UAS-EGFRtop, and UAS-CblL in the indicated lanes. Protein lysates were blotted with antibodies to EGFR and GFP (transfection control). A smaller band recognized by the EGFR antibody that is the same size as the smaller band in F, is indicated by an asterisk; the arrow indicates full-length EGFRtop. The proportion of the smaller band is increased by Cbl cotransfection or sty RNAi and decreased by mop RNAi. The lower three panels show RT-PCR quantification of mop, Cbl and sty mRNA, demonstrating the efficiency of the RNAi treatment.

View larger version (67K): [in this window] [in a new window]   Fig. 6. Progression through endocytosis promotes EGFR signaling. (A,B) An HrsD28/Df(2L)Exel6277 Drosophila eye disc stained with anti-Elav (A, green in B) and anti-Sens (magenta in B). The arrow in B indicates a group of ommatidia containing only R8 photoreceptors. (C,D) aos-lacZ expression in wild-type (C) and HrsD28/Df(2L)Exel6277 (D) wing discs stained in parallel with X-Gal. R1-7 differentiation and aos expression are reduced in Hrs mutants. (E) Semi-quantitative RT-PCR for Actin, mop, Tsg101 and Vps28 mRNA in D2F cells treated with mop, Tsg101 or Vps28 dsRNA, demonstrating efficient knockdown in each case. (F) Western blot with antibodies to Tubulin and diphospho-MAPK of lysates from D2F cells treated with lacZ, mop, Tsg101 or Vps28 dsRNA and incubated with Spi for 0 or 30 minutes. Depletion of either Mop or the ESCRT-I complex components reduced MAPK phosphorylation.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Model for the effect of Mop on EGFR endocytosis in Drosophila. Upon activation of EGFR, ubiquitylation by Cbl induces EGFR internalization through clathrin-coated vesicles. These vesicles fuse with early endosomes and the EGFR is passed from the Hrs complex to the ESCRT-I, ESCRT-II and ESCRT-III complexes as the endosomes are transformed into multivesicular bodies (MVBs). ESCRT-III promotes EGFR deubiquitylation and entry into the internal vesicles of MVBs; fusion of MVBs with lysosomes results in EGFR degradation. Sprouty prevents the EGFR from progressing into late endosomes. We propose that Mop is required for EGFR progression through the endocytic pathway, perhaps through its effect on Hrs. This progression may allow EGFR to encounter crucial downstream components located on late endosomes (X), or to be recycled to the plasma membrane to prolong signaling. Cleavage of the receptor must occur at a stage after the requirement for mop.

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