Drosophila EGFR signalling is modulated by differential compartmentalization of Rhomboid intramembrane proteases

Abstract

We explore the role of differential compartmentalization of Rhomboid (Rho) proteases that process the Drosophila EGF receptor ligands, in modulating the amount of secreted ligand and consequently the level of EGF receptor (EGFR) activation. The mSpitz ligand precursor is retained in the ER, and is trafficked by the chaperone Star to a late compartment of the secretory pathway, where Rho-1 resides. This work demonstrates that two other Rho proteins, Rho-2 and Rho-3, which are expressed in the germ line and in the developing eye, respectively, cleave the Spitz precursor and Star already in the ER, in addition to their activity in the late compartment. This property attenuates EGFR activation, primarily by compromising the amount of chaperone that can productively traffic the ligand precursor to the late compartment, where cleavage and subsequent secretion take place. These observations identify changes in intracellular compartment localization of Rho proteins as a basis for signal attenuation, in tissues where EGFR activation must be highly restricted in space and time.

Figure 1

Subcellular localization of Rho-1 and Rho-3 in the eye imaginal disc (A–E) GMR-Gal4 drives expression of the indicated UAS constructs in all cells posterior to the morphogenetic furrow. ElaV (Blue) marks the differentiated photoreceptors, FasIII (red) stains plasma membranes and BiP marks the ER. (A) mSpi–GFP (green) localizes exclusively to the perinuclear ER within photoreceptor cells, where it co-localizes with BiP (red). (B) Optical sections through the apical (top) and middle (bottom) portions of ommatidia reveal that Rho-1-HA (red) does not co-localize with mSpi but is found within more apical punctate structures. (C) Rho-3–GFP (green) is detected both around the nuclei, in a pattern reminiscent of mSpi–GFP, and in punctate structures. (D) Rho-3–GFP (green) co-localizes with Rho-1–HA (red) in apical punctate structures. (E) Rho-3–GFP (green) and mSpi–HRP (red) co-localize in the peri-nuclear ER. A full-colour version of this figure is available at the EMBO Journal Online.

Figure 2

Rhos can cleave both Spitz and Star in the ER. (A) Addition of a KDEL ER-retrieval sequence to HA-tagged Rho-1 re-localizes it in S2R⁺ cells, to the perinuclear ER. (B) Cell lysates from Schneider cells transfected with the indicated constructs and probed with an anti-GFP antibody. Even when diluted 400-fold compared with mSpi–GFP, Rho–KDEL constructs efficiently produce the faster migrating band, which corresponds to the cleaved ligand (arrowhead). The membrane was reblotted with an anti-actin antibody to ensure equal loading for all lanes. (C) Star–HA is also efficiently cleaved by Rho-1–KDEL and Rho-3–KDEL, such that most of the precursor is converted to a faster-migrating band (arrowhead). The cleaved C-terminal, extracellular domain of Star is detected by anti-HA. (D–F) To monitor specifically the capacity of Rho proteins to cleave Star in the ER in embryos, a Star protein tagged at the cytoplasmic domain with a LexA VP16 transcriptional activator and a LexA-lacZ reporter were used. Upon expression of Rho-1–KDEL or Rho-3–KDEL by the prd-Gal4 driver, prominent cleavage of Star was detected by X-Gal staining.

Figure 3

ER cleavage by Rhos induces low levels of EGFR activation. (A, A′) Lateral view of a stage-14 embryo. UAS–Rho-1–HA (green) expressed under the control of en-Gal4 localizes to discrete punctate structures, reminiscent of its localization in the eye imaginal disc. (B, B′) Similarly expressed Rho-3–GFP (green) localizes in the embryo around the nuclei (consistent with ER localization), as well as in punctate structures. (C) Ventral view of a stage 9–10 WT embryo (anterior to the left). Yan protein (red) is degraded in the 1–2 cell rows bordering the ventral midline (vm), which expresses rho-1. (D) Ectopic expression of UAS–Rho-1–KDEL driven by prd-Gal4. The EGFR pathway does not show signs of ectopic activation within the prd stripes (defined by co-expression of UAS–GFP, marked by white lines), as assayed by Yan degradation. (E) Following similar expression of Rho-1, Yan is fully degraded throughout the prd stripe, as well as in adjacent cells. (F, F′) Intermediate levels of EGFR activation are observed upon ectopic expression of Rho-3. Many nuclei within the prd stripe still display Yan, and the non-autonomous effect is hardly detected. (G) Co-expression of Rho-1 and Rho-3. The partial Yan degradation pattern in prd stripes resembles that observed upon expression of Rho-3 alone (F). (H–K) Ventral views of stage 9–10 embryos expressing the indicated Rho in the ventral midline (arrowheads), where Rho-1 is endogenously expressed. (H) Yan is degraded in 1–2 cells bordering the ventral midline in WT embryos. (I) Expression of Rho-1 in the midline by btl-Gal4 does not modify the endogenous Yan degradation pattern. (J) Rho-1–KDEL expression is epistatic to the endogenous Rho-1 and leads to a reduced range of EGFR activation, manifested by Yan presence in cells bordering the midline. (K) Expression of Rho-3 is also epistatic to that of Rho-1. A full-colour version of this figure is available at the EMBO Journal Online.

Figure 4

ER cleavage sensitizes EGFR signalling to the level of Star. UAS–Rho-3–GFP and UAS–mCD8–GFP were jointly expressed under the control of prd-Gal4. (A) Degradation of Yan (red) following Rho-3 expression in spi heterozygous embryos displays a similar pattern to the one observed upon Rho-3 expression in WT embryos. (B) Star heterozygosity largely suppresses the EGFR hyperactivation caused by Rho-3 expression. (C) Simultaneous expression of mSpi with Rho-3 does not significantly alter the Yan degradation pattern. (D) Co-expression of Star and Rho-3 significantly elevate EGFR activation levels. The degradation of Yan reaches the extent observed upon expression of Rho-1 (Figure 3E). A full-colour version of this figure is available at the EMBO Journal Online.

Figure 5

ER-active Rhos display mild EGFR activation in the wing, which is sensitive to the levels of Star. Rho-proteases were expressed in female wing imaginal discs using MS1096-Gal4 in the genetic backgrounds indicated. (A) WT wings. (B) Rho-1–KDEL expression yields very subtle phenotypes, except when it is co-expressed with Star, where EGFR activation becomes comparable to that seen with Rho-1. (C) Rho-1 expression leads to very pronounced EGFR hyperactivation phenotypes. There is no sensitivity to Star gene dosage. (D) Expression of Rho-2 leads to intermediate phenotypes, namely vein expansion. This phenotype is suppressed upon reduction in Star gene dosage and becomes more severe when Star is co-expressed with the protease. (E) Rho-3 expression produces intermediate phenotypes. Importantly, Star heterozygosity or overexpression strongly suppresses or enhances the phenotype, respectively. A full-colour version of this figure is available at the EMBO Journal Online.

Figure 6

Expression pattern of Rho-1 and Rho-3 and hyperactivation of the EGFR pathway in rho-3 mutant eyes. (A) Expression of the rho-1 enhancer trap X81 is detected by anti-β-galactosidase staining (red) in R8, R2 and R5 photoreceptor cells, in ommatidia throughout the eye disc. Photoreceptors, visualized with the neuronal nuclear marker ElaV (blue), form discrete eight-cell clusters . The morphogenetic furrow (mf) is indicated. (B) Expression of the rho-3 enhancer trap Inga (anti-β-galactosidase, red) is observed in the R8 cells of photoreceptor clusters near the mf, and expands to the other cell types in the ommatidium. ElaV (blue) and FasIII (green) mark neuronal nuclei and outlines of cells in the eye disc, respectively. Insets in (A) and (B) show magnified views of marked regions. (C) Scheme representing the expression pattern of both rho genes in the eye disc. Rho-1 and Rho-3 are co-expressed in R8, R2 and R5, whereas Rho-3 is expressed alone in all other cells. (D–E) The R4 photoreceptor cell fate, monitored in eye imaginal discs by expression of mδ0.5-LacZ. β-gal (red), is detected in only one cell per ommatidium in WT discs (D; and Cooper and Bray, 1999), but is often found in several cells of a single ommatidium in rho-3-null (ru^PLLb) mutant discs (E). FasIII (green). (F–G) The R7 cell fate is monitored by the co-expression of Prospero (red) and the neuronal marker ElaV (blue). Cells that express Prospero but not ElaV are cone cell precursors. Only one nucleus in each WT ommatidium shows co-expression of the two markers (F), whereas induction of ectopic R7 cell fates is observed in rho-3^– (arrows in G). (H) Mutant clones of rho-3 were generated (detected by absence of GFP), and expression of the cone cell marker, Cut, was followed. A decrease in Cut expression is observed within the mutant clones. Arrow shows the position posterior to the morphogenetic furrow (mf, arrowhead), at which Cut begins to be expressed in the WT cells. (I–K′) Sections through the retinas of adult flies of the indicated genotypes. In bottom schemes of the data, trapezoids indicate a full complement of photoreceptors and their spatial orientation, whereas red and green circles indicate ommatidia with missing or extra photoreceptors, respectively. (H) Expression of the apoptosis inhibitor p35 under control of GMR-Gal4 does not alter photoreceptor number and ommatidial patterns. (I) In rho-3⁻ retinas, only 17% of the ommatidia show a full wild-type complement of photoreceptors. (J) Bypassing apoptosis by expressing p35 in rho-3⁻ eyes reveals hyperactivation of EGFR. Most ommatidia (54%) now show the WT array of seven photoreceptors, and in some cases (22%) extra photoreceptors are detected. (J′) Examples of single ommatidia from GMR>p35; rho-3⁻ eyes, showing missing photoreceptors (a) or extra photoreceptors (b) and (c). (L) Quantification of photoreceptor numbers in adult ommatidia from the indicated genotypes. Note: the death of photoreceptor cells in eye imaginal discs expressing only Rho-1, which was uncovered by co-expression of p35, may stem either from insufficient phosphorylation of HID by MAPK, or from death of mis-specified photoreceptor cells. A full-colour version of this figure is available at the EMBO Journal Online.

Figure 7

ER localization of Rho-2 in the female larval gonad attenuates EGFR signalling. (A) Cleaved Spi is produced by Rho-2 in the primordial germ cells (PGC-blue), leading to activation of EGFR in the somatic intermingled cells (IC-green), promoting their survival and preventing an unknown signal that inhibits PGC proliferation (Gilboa and Lehmann, 2006). (B) In WT gonads, ICs, marked by anti-Traffic-Jam (TJ, green) are found between and around germ cells (anti-Vasa, blue). Membranes of somatic cells, as well as the fusome, a germline-specific organelle, are labelled with 1B1 antibody (red). (C) Star heterozygous gonads are characterized by increased number of Vasa-positive cells, which are tightly clustered, and fewer intermingled cells, which are no longer spatially mixed with the germ line. (D) Overexpression of UAS-Star in the germ line using nanos-Gal4-VP16 leads to an increase in the number of ICs, with a concomitant reduction in PGC numbers. (E) sl-null mutant female gonads are small, with a lower number of germ cells, and elevated numbers of ICs, indicative of EGFR hyperactivation. (F) Quantification of germ cell (Vasa-positive, blue) and IC (TJ-positive, green) mean numbers in gonads of the indicated genotypes. Eight specimens were examined per genotype. Error bars represent standard deviation. A full-colour version of this figure is available at the EMBO Journal Online.

Figure 8

ER targeting of Rhos reduces active ligand secretion. (A) The canonical Spitz processing by Rho-1. Cleavage in the late compartment occurs after the chaperone, Star, has accomplished at least one round of Spitz trafficking and results in high levels of secreted ligand. (B) ER activity of Rho-2 or Rho-3 reduces the amounts of active, secreted Spitz. Star cleavage in the ER (circled) prevents the chaperone from trafficking Spitz to the late compartment. ER-cleaved Spitz is retained by a specific, Sl-dependent mechanism. Thus, only the residual Star molecules, which ‘escaped' cleavage by the protease in the ER, are capable of trafficking Spitz precursor (and possibly also cleaved Spitz) to the late compartment, where the activity of Rho-2 or Rho-3 produces the secreted ligand, at reduced levels. A full-colour version of this figure is available at the EMBO Journal Online.