Fig. 4. Rho1 activity is apically restricted during spiracular chamber formation. (A) Immunofluorescence against Rho1 protein showing its apical accumulation around the lumen of the spiracular chamber (arrows); (i) dorsal view and (ii) lateral view. (ii) The PKNG58AeGFP probe overlaps with apical Rho1, reflecting the local activation of this RhoGTPase. (B) Rho1 activity during formation of the spiracular chamber. Spiracle cells co-expressing PKNG58AeGFP and mRFP-Actin. early st11 - low levels of PKNG58AeGFP are detected throughout the spiracle primodium. The asterisk indicates the A8 tracheal pit position. Anterior is to the right; late st11 - the onset of Rho1 activity is detected in the first invaginating cells (arrow) localised posteriorly to the last tracheal pit. Notice also the higher accumulation of mRFP-Actin in these cells; b.c. - single invaginating bottle-shaped cell showing apical activation of Rho1 (arrowhead), which overlaps with apical accumulation of mRFP-Actin (apical is up); stage 13 and stage 17 - transverse and lateral views, respectively, of spiracular chambers showing accumulation of Rho1-GTP and mRFP-Actin at the luminal/apical surface (arrows). (C) Ectopic Rho1 activation blocks basolateral elongation and impairs cell invagination. The left panel shows active Rho1 (PKNG58AeGFP fluorescence) around the lumen of a wild-type spiracle (lateral view, stage 14); the dashed line outlines a single invaginated cell. The two middle panels represent spiracle cells co-expressing RhoV14 and PKNG58AeGFP (i) or GFP-Actin (ii). The right panel (iii) shows the Filzkorper defects (arrows) caused by the expression of RhoV14 (cuticle). In i, ectopic Rho1 activation is detectable on the cell membranes, as opposed to the apically restricted pattern in the wild type (same confocal settings as the WT control). ii shows a cluster of three spiracle cells with a mini-bottle shape, due to inhibition of basolateral elongation (compare with wild-type control, Fig. 3G). Scale bars: 10 µm, except B b.c, 3 µm.