Figure 7

IFC-2 and IFB-2 interact with actin and the actin bundling protein PLST-1 in an isotype-specific fashion. (a–c) The maximum intensity fluorescence images show staining of actin filaments with Alexa Fluor 488-conjugated phalloidin (wild-type N2 in a, ifb-2(kc14) in b and ifc-2(kc15) in c). Areas demarcated by yellow dotted lines were used for quantification, which is shown in (d). The expression of actin is significantly increased in ifc-2(kc15) compared to ifb-2(kc14) and wild-type N2 (N2: 207 ± 51, n = 19; ifb-2(kc14): 203 ± 44, n = 15; ifc-2(kc15): 338 ± 99, n = 23; two independent experiments). (e,e′) The corresponding images show the interference contrast recording (e) and fluorescence micrograph detecting PLST-1::GFP produced from plst-1 allele msn190. Note the periluminal PLST-1::GFP enrichment throughout the intestine (arrowheads). (f) shows an interference contrast image of a larva harboring the loss-of-function plst-1 allele tm4255. Regions of dilated intestine are marked by arrows. (g) The immunoblot detects IFB-2 isoforms a and c and actin in lysates of plst-1(tm4255) animals either with dilated intestinal lumen (lane 1) or without (lane 2) and in lysates of wild-type N2 (lane 3). The immunosignal in the mutant animals with lumen dilation is increased coincident with a shift from IFB-2c to IFB-2a. The position and size in kDa of co-electrophoresed molecular weight markers are shown at left. (h–k) shows single plane in vivo images of PLST-1::GFP fluorescence produced from the tagged endogenous plst-1 gene in wild-type (h), ifb-2(kc14) (i) and ifc-2(kc15) (j) backgrounds. The areas delineated by yellow dotted lines were used for quantification, the results of which are depicted in (k). Loss of IFB-2 induces higher expression of PLST-1 in the apical intestine, whereas loss of IFC-2 does not (N2: 694 ± 78, n = 23; ifb-2(kc14): 796 ± 155, n = 40; ifc-2(kc15): 660 ± 111; n = 34; two independent experiments). Significance: **p < 0.01, ***p < 0.001. Scale bars: 20 µm.