Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity

The Notch signaling pathway governs cell-to-cell communication in higher eukaryotes. In Drosophila, after cleavage of the transmembrane receptor Notch, the intracellular domain of Notch (ICN) binds to the transducer Suppressor of Hairless (Su(H)) and shuttles into the nucleus to activate Notch target genes. Similarly, the Notch antagonist Hairless transfers Su(H) into the nucleus to repress Notch target genes. With the aim to prevent Su(H) nuclear translocation, Hairless was fused to a transmembrane domain to anchor the protein at membranes. Indeed, endogenous Su(H) co-localized with membrane-anchored Hairless, demonstrating their binding in the cytoplasm. Moreover, adult phenotypes uncovered a loss of Notch activity, in support of membrane-anchored Hairless sequestering Su(H) in the cytosol. A combined overexpression of membrane-anchored Hairless with Su(H) lead to tissue proliferation, which is in contrast to the observed apoptosis after ectopic co-overexpression of the wild-type genes, indicating a shift to a gain of Notch activity. A mixed response, general de-repression of Notch signaling output, plus inhibition at places of highest Notch activity, perhaps reflects Su(H)’s role as activator and repressor, supported by results obtained with the Hairless-binding deficient Su(H)LLL mutant, inducing activation only. Overall, the results strengthen the idea of Su(H) and Hairless complex formation within the cytosolic compartment.


Membrane-anchored Hairless protein restrains Notch signaling activity
Maier D.
University of Hohenheim, Institute of Biology, Dept. of General Genetics, Garbenstr. The Delta signal peptide was subcloned into the pUAST attB vector (pUAST SP). The transmembrane domain was PCR amplified and cloned into the pBluescript (pBT TM) vector. Afterwards Hairless without IRES was cloned into the pBT TM vector, and then into the pUAST SP vector. Restriction sites used for cloning are shown above; red ones were introduced by PCR. SP, signal peptide; MNNL, Notch ligand domain; TM, transmembrane domain; DSL, Delta-Serrate-Lag2 domain; M1/2, regular translation start in Hairless, M3, IRES-mediated translation start in Hairless, SBD, Suppressor of Hairless binding domain with NT and CT highly conserved sequences [74,75]; GBD, Groucho binding domain; CBD, binding domain of the C-terminal binding protein; myc tag. (b,c) Western Blots; approximate size is given in kDa using a prestained marker. Protein extracts derived from Oregon R wild type embryos, and embryos derived from a cross of da-Gal4 and either UAS-H myc for control or UAS-SPTM-H myc . Blots were probed with anti-Hairless (b) and anti-myc antibodies (c), respectively. Two protein isoforms of approximately 150 kDa and 120 kDa are derived from the Hairless locus, the smaller one by use of an IRES [42,43] (arrows). The respective calculated molecular weight is 111 kDa and 96 kDa. Membrane-anchored SPTM-H myc protein, however, is detected as a single band of approximately 130 kDa (asterisk) with a calculated molecular weight of 112 kDa (b,c). Specificity was confirmed by detection with anti-Myc antibodies (c).

Figure S3: Effect of the combined activity of SPTM-H myc and Su(H) on Wingless expression
(a,a') omb-Gal4 was used to drive co-expression of SPTM-H myc (green) and Su(H) (blue) in the central domain of wing imaginal discs. Wingless (red) expression was downregulated along the dorso-ventral boundary (arrow), but derepressed within the omb-domain (asterisk). The disc appeared slightly enlarged. Although less distinct, Su(H) staining follows to a large degree the dotted staining of SPTM-H myc (a'), which appears cyan in the merge; (b) Co-induction of SPTM-H myc with Su(H) LLL results in increased disc size, however, Wingless expression was not reduced. Su(H) LLL protein expression was weaker and did not overlap with SPTM-H myc protein. Size bars: 100µm (a,b) and 50µm (a').

Figure S4: Effect of the combined activity of SPTM-H myc and Su(H) on Cut expression
(a,a') SPTM-H myc (green) and Su(H) (blue) were co-expressed in the central domain of wing imaginal discs using the omb-Gal4 driver. Cut (red) expression was repressed along the dorso-ventral boundary (arrow), and expanded elsewhere within the omb-domain. The disc appeared slightly enlarged. Vesicular co-localization of Su(H) and of SPTM-H myc (a') is apparent in the merge (cyan color), albeit Su(H) protein accumulation appears more uniform; (b,b') The combined overexpression of SPTM-H myc (green) and Su(H) LLL (blue) caused enlarged discs, without impeding Cut (red) expression along the boundary or inducing it outside (arrow). The Cut expression domain, however, comprised more than the normal 2-3 rows of nuclei (b', compare with a'). Mutant Su(H) LLL protein accumulated uniformly at a remarkably low level, and did not overlap with SPTM-H myc . Size bars: 100µm (a,b); 50µm (a',b').

Figure S5: Effects on the size of the omb-expression domain
Overexpression within the omb-domain of the given constructs. (a) Pictures were taken from Figure 5 (upper row) and Figure 7 (lower row) to demonstrate size measurements. The width and length of the omb expression domain was recorded from several specimens; the average number relative to GFPcontrol is shown above and below, respectively. For width measurements, vg BE expression was used as lead. Box blot representation of the statistical analysis on width (b) and length (c) measurements. Center lines show medians, box limits indicate 25 th and 75 th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles; outliers are represented by dots. n= 5, 4, 4, 4, 14, 6 sample points. ANOVA two-tailed Dunnet's test for multiple comparisons was applied relative to GFP-control (*** <0.001; * <0.01; ns >0.05).