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Fig. 7. Levels and localization of Knk in tube-expansion mutants. (A)
A western blot with embryonic extract from stage 17 wild type and knk,
grh and rtv mutants labelled with anti-Knk reveals similar Knk
levels in embryos of all genotypes (top row). Anti-Tubulin was used for
loading control (bottom row). (B-E) Anti-Knk labelling of stage 15
wild-type (B), kkv (C), Fas2 (D) and sinu (E)
mutant DTs shows that Knk localizes to the apical surface in wild type and
kkv mutants, but occupies the entire cell surface in Fas2
and sinu mutants. (F-H) Anti-Knk also labels the apical
surface in wild-type stage 16 DT (F), but in Fas2 (G) and
mega (H) mutants Knk is detected along the lateral and basal cell
surface (arrowhead). (I-L) UAS-knkTM-expression, driven
with the tracheal Btl-GAL4 line, rescues the knk (L), but not the
Fas2 (K) mutant tracheal phenotypes, as analysed by co-labelling with
anti-Knk (red) and CBP (green). In Fas2 mutants (J) luminal chitin
appears amorphous and expanded compared with wild type (I), and
UAS-knkTM-expression in Fas2 mutants (K) does not
rescue chitin filament organization nor tube-size defects. (M) A model
illustrating the requirement of different tube expansion genes for uniform
lumen diameter expansion. Chitin chains (green) in the expanding lumen
assemble into a filamentous cable in a process that requires the apical
surface proteins Knk and Rtv, and perhaps additional components (left figure).
SJ components are needed for the correct localization of Knk and possibly
other proteins involved in chitin filament assembly. In knk and
rtv mutants, chitin fails to assemble into a filamentous cable (right
figure) and loses its function in tracheal tube-size regulation. Loss of SJ
components also leads to defects in chitin matrix assembly, but not to the
severe fusion branch constrictions seen in knk and rtv
mutants. Scale bars: 4 µm in B-E,I-L; 5 µm in F-H.
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