Hts is required for recruiting α-Spec to cell cortex and loss of Hts phenocopies spectrin mutants. The z-projections at lateral domain (A–E) and AJs (F and G), respectively. (A and A’) A Triton X-100–permeabilized pupal eye disc containing GFP-positive hts^null MARCM clones was stained for phalloidin. Decreased cortical F-actin level in hts^null mutant PECs was most obvious in lateral domain (A and A’) but less obvious in apical domain (see Fig. S3, A and A’). Quantification of phalloidin membrane intensity is shown in H. (B and B’) Similar to A and A’ except that α-Spec staining is shown. Note the decreased cortical α-Spec level in hts^null mutant PECs. Quantification of α-Spec membrane intensity is shown in I. (C and C’) A third instar wing disc containing RFP-negative hts^null mutant clones was imaged for GFP–α-Spec. Note the dramatically decreased level of cortical GFP–α-Spec in hts^null mutant clones. Quantification of GFP–α-Spec membrane intensity is shown in J. (D and D’) A third instar wing disc containing DsRed-positive clones expressing α-spec RNAi was imaged for GFP-Hts. Note the similar level of cortical GFP-Hts inside and outside the mutant clones. Quantification of GFP-Hts membrane intensity is shown in K. (E and E’) A pupal eye disc containing GFP-positive MARCM clones with the hts^null mutation was stained for p-MLC. Note the increased level of cortical p-MLC in the hts^null mutant cones. Quantification of p-MLC membrane intensity is shown in L. (F and G) Pupal eye discs of the indicated genotypes were stained for DE-cad. Note the extra interommatidial cells in hts mutant eye discs (yellow asterisks in G). 20 ommatidia of each genotype were used for counting interommatidial cells, and the number on the lower right of each panel indicates the number of extra cells per cluster. (H–L) Quantification of mean membrane intensity of the indicated proteins analyzed in A–E. Data are means ± SEM (n ≥ 20 cells, representative of five animals). ****, P < 0.0001. Scale bars, 5 µm. N.S., no significance.