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Drosophila EGFR signaling is modulated by differential compartmentalization of
Rhomboid intra-membrane proteases
Shaul Yogev, Eyal D. Schejter and Ben-Zion Shilo
Supplementary Figures
Figure S1. Rho-1 does not localize to the Golgi or endosomes
GFP tagged Rho-1 (green) was expressed in the eye disc using GMR-Gal4, and its
localization was compared to that of known compartment markers (red).
(A) No co-localization was observed with the Golgi marker dGMAP. Similar results
were obtained with an anti-Drosophila Golgi antibody (not shown).
(B), (C) and (D) Rho-1GFP shows no significant co-localization with Rab5, Rab7 or
Rab11 positive puncta, which mark the early, late and recycling endosomes,
respectively.
Figure S2. Rho-1KDEL expression induces very low levels of EGFR signaling
Rho-1KDEL and Rho-3KDEL are enzymatically active, as assayed in cell culture (Fig.
2), and by their ability to cleave LexA-tagged Star in embryos (not shown), utilizing a
previously described assay (Tsruya et al., 2007). Yet, Rho-1KDEL does not give rise to
pronounced activation of EGFR in various tissues, in contrast to native Rho-1.
(A) Wild-type eyes show a regular array of facets (ommatidia), each with a single
bristle.
(B) Expression of Rho-1 yields a very rough eye, with many fused and misrotated
ommatidia.
(C) Rho-1KDEL expression in the eye leads only to minor defects such as misrotated
ommatidia.
(D) A wild type wing shows five narrow longitudinal veins, and two cross veins at
typical positions.
(E) Expression of Rho-1 in the wing imaginal disc leads to the formation of excessive
vein tissue, resulting in small and blistered wings.
(F) Rho-1KDEL ectopic expression in the wing disc leads to very little extra vein
tissue (arrows).
(G) The phenotype of Rho-3KDEL expression in the wing is identical to that of Rho1KDEL, indicating that the enzymes do not differ in affinities for their substrates.
Note: Due to the location of MS1096-Gal4 on the X chromosome, wings of females were
assayed to assure equal intermediate levels of expression.
Figure S3. Attenuated activation of EGFR by Rho-3 is epistatic to activation by
Rho-1.
Activation of EGFR was monitored by induction of pointedP1 expression. Normally,
pntP1 is expressed in two cell rows on each side of the midline. Upon expression of
Rhomboid proteins by prd-Gal4, the pattern of ectopic pntP1 expression was monitored
within the stripes, and adjacent to them.
(A) Rho-1 induces pronounced expression of pntP1 within the stripes, and several
cell rows adjacent to them.
(B) Rho-3 induces pntP1 expression within the stripes, but a reduced level. In
addition, no expression of pntP1 is detected outside the stripes.
(C) Co-expression of Rho-1 and Rho-3 results in a pattern identical to Rho-3
alone.
Figure S4. Rho-3 induced EGFR activation is sensitive to Star levels
(A) Ventral view of a stage 9-10 embryo in which Rho-3 was expressed under the
control of prd-Gal4. Phosphorylation of MAPK (dpERK-red) is observed
throughout the prd stripe (white outlines, marked with CD8GFP -green), as well
as in the ventral midline, where Rho-1 is endogenously expressed.
(B) Halving spi gene dosage does not significantly modify the dpERK pattern
induced by prd-Gal4/Rho-3 expression.
(C) Rho-3 dependent EGFR activation (dpERK-red) is largely suppressed when one
copy of Star is removed.
Figure S5. Rho-1 induced EGFR activation is insensitive to spi or Star gene dosage
Ventral views of stage 9-10 embryos of the indicated genotypes, stained for Yan (red)
and GFP (green).
(A) Expression of Rho-1 in the prd stripe induces degradation of Yan throughout the
stripe and in adjacent cells.
(B-C). The effects of ectopic Rho-1 expression on Yan degradation persist spi (B) or
Star (C) heterozygous background.
Figure S6. rho-3 expression in the eye disc is induced by EGFR activation.
(A) rho-3 expression in wt eye discs carrying the enhancer trap element Inga, was
monitored by anti-Gal staining.
(B) Hyper activation of EGFR signaling by expression of cSpi-GFP under GMRGal4, gave rise to an expanded pattern of Inga expression.
Figure S7. Rho-2 is partially localized to the ER in-vivo, and is a mild activator of
the EGFR
(A) Ventral view of a stage 9-10 embryo, expressing Rho-2 under the control of prdGal4, and stained for Yan (red) and GFP (green). Many cells within the prd stripe
still display Yan and no activation is observed beyond the prd stripes, indicating
that only mild EGFR activation levels are generated, as is the case with Rho-3
expression.
(B) Lateral view of a stage 13 embryo, expressing GFP-tagged Rho-2 under the
control of en-Gal4. GFP immunofluorescence (green) is detected mostly around
nuclei, indicative of ER localization, as well as in some puncta.
(C) Female gonad from a late third instar larva expressing UAS-Rho-2GFP (green)
via a heat shock inducible Gal4 driver. Traffic Jam (TJ; blue) and 1B1 (red) mark
somatic intermingled cell nuclei and germ-cell fusomes, respectively. The
characteristic perinuclear ER localization of Rho-2 is observed in both the
germline and soma. Note higher expression of the UAS-induced construct in the
soma compared to the germline. The inset shows a magnification of two germ
cells (asterisks), identified by their fusomes and lack of TJ expression.
References
Tsruya, R., Wojtalla, A., Carmon, S., Yogev, S., Reich, A., Bibi, E., Merdes, G., Schejter,
E. and Shilo, B.Z. (2007) Rhomboid cleaves Star to regulate the levels of secreted
Spitz. Embo J, 26, 1211-1220.