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Fig. 5. Knk is an apical GPI-anchored protein expressed in the developing
trachea and epidermis. (A) The wild-type Knk protein (top) is
predicted to contain an N-terminal signal peptide (green), two tandem DM13
domains (blue), one DOMON (red) domain and a C-terminal GPI-anchor (black).
Seven knk alleles, which all cause the same phenotypes, harbour
premature stop codons to produce truncated Knk proteins of various lengths
(illustrated below the wild Knk protein). The closest relatives to Knk in
plant and worms are represented by Arabidopsis thaliana (Accession
number BAB08762), with four transmembrane domains (TM, black) and a cytochrome
BS61 domain (yellow), and Caenorhabditis elegans (Accession number
AAG49388). (B) Western blot analysis of stage 17 embryonic extracts
show that Knk protein is present in both the membrane (pel) and the soluble
fraction (sup), compared with the transmembrane Syntaxin1A protein, which only
precipitates with the membrane fraction. (C) Incubation of the membrane
fraction in (B; pel) with Phospholipase C releases some Knk into the
membrane-free supernatant (sup), indicating that Knk is a GPI-anchored
protein. By contrast, Tout-velu (Ttv), which has a C-terminal type 1b
transmembrane domain, is resistant to Phospholipase C treatment. (D-F)
In-situ hybridization with knk anti-sense RNA probes detects
knk transcripts in the developing trachea from stage 13 (arrows in
D-F). From stage 15 the Knk transcript is also detected in the pharynx,
hindgut and epidermis (D). (G-L) Co-labelling with anti-Knk and
anti-Fas3 reveals apical Knk localization in the trachea and epidermis. In
stage 15 wild type tracheal epithelium anti-Knk (G and J; red) highlights the
apical cell surface and parts of the apico-lateral surface as seen from the
slight overlap with Fas3 (J; green). Knk-labelling is absent in knk
mutant trachea (H,K). Stage 16 wild-type epidermis (I) also displays apical
Knk labelling (green) compared with the lateral Fas3 (red), which is absent in
the knk mutant epidermis (L). (M-O) Stage 15 embryos labelled
with the 2A12 antibody shows that the tracheal phenotype of knk
mutants (M) is rescued by ectopic expression of UAS-knk (N) and
UAS-knkTM (O) driven with Btl-GAL4. Scale bars: 50 µm in D;
25 µm in E,F,M-O; 7 µm in G-L.
