Figure 3. EXC-6 Localizes to, and Regulates, Leading-Edge F-Actin in the EC(A-A00) Maximum projection of a confocal z stack from a transgenic animal immunostained to visualize (A) Venus::EXC-6 and (A') ERM-1, which binds apical F-actin (Gobel et al., 2004). Merge of Venus::EXC-6 and ERM-1 is shown in (A''). In this region of the EC, which includes the cell body (the EC nucleus is marked),Venus::EXC-6 accumulates in basolateral microfilaments and does not colocalize with apical ERM-1.(B-B0000) Maximum projection of spinning disk confocal z sections of a live wild-type hermaphrodite showing (B) cytoplasmic CFP, (B0) LifeAct::TagRFP, and (B00) Venus::EXC-6. Area boxed in (B-B00) is magnified in (B000 and B0000). Arrowhead marks the extent of apical lumen, as seen with cytoplasmic CFP (B). Note leading edge F-actin structure past this point of lumen extension in (B0), magnified in (B000). Venus::EXC-6 (B00) colocalizes with F-actin only at this leading-edge structure (B0000). Post., posterior.(C-C0000) Maximum projection of spinning disk confocal z sections of a live
exc-6(0) hermaphrodite showing (C) cytoplasmic CFP, (C0) LifeAct::TagRFP, and (C00) Venus::EXC-6(I213A). Mutation of the conserved actin-binding Ile213 abolishes rescue of
exc-6(0) (Figure 1F) and colocalization with the aberrant leading-edge Factin structure (C0-C0000), without altering Venus::EXC-6 accumulation on basolateral microfilaments (arrows in C00). Ant., anterior. (D-E0) In
exc-5(0), apical F-actin is greatly decreased (compare to Figures 1B00, 2A0, 2B0, 3B0, and 3C0), most evident in cystic (''c'') regions (compare to cysts in Figures 2C0 and 2D0), and the leading-edge F-actin structure is aberrant and no longer localizes to the EC canal tip. (F and G) In (F) and (F0), the
exc-6(0);
exc-5(0) double mutant displays disorganized or absent F-actin in the EC and greatly reduced EC canal extension, which is quantified in (G), demonstrating a significant decrease in outgrowth in the double mutant as compared to
exc-5(0) and
exc-6(0) singles. (H) Quantification of the multiple lumen phenotype in
exc-5(0),
exc-6(0) singles, and an
exc-6(0);
exc-5(0) double. Note significant suppression of the
exc-6(0)phenotype in the double. For (G) and (H), n = 30 animals of each genotype were scored. Significance was calculated by a Fisher's exact two-tailed test.Scale bars, 5 um. Error bars indicate SD. *p % 0.05; **p % 0.01; ***p % 0.001.