βH-Spec and α-Spec reduce junctional myosin levels in wing discs

(A-D) Apical sections of wing imaginal discs expressing en-Gal4 UAS-RFP UAS-dcr2 sqh:GFP crossed to control (Oregon-R, OR) (A), UAS-kstRNAi (B), UAS-kst-CRISPRa (C), or UAS-α-specRNAi (D) showing the effect on Sqh:GFP (green) levels and localization in the posterior compartment (marked by RFP, blue). Scale bar = 20 μm; all images are at the same magnification. (E-H) Heat maps of relative junctional Sqh:GFP intensity of wing discs. Levels of Sqh:GFP relative to E-cad levels are shown for the different genotypes analyzed in A-D. Heat map scale is indicated on the top. Number of wing discs used for analysis: Control (OR), n=6; UAS-kstRNAi, n=5; UAS-kst-CRISPRa, n=5; UAS-α-specRNAi, n=6. (I) Average recoil velocities after laser cutting of cell junctions in anterior (A) or posterior (P) compartments of wing discs expressing UAS-kstRNAi or UAS-a-specRNAi in posterior cells (n=20). (J-K) Quantification of Sqh:GFP normalized to E-cadherin in posterior cells (P) compared to anterior cells (A) in wing disc expressing the indicated constructs, displayed as individual values, normalized by E-cad (J) or normalized by the mean intensity of Sqh:GFP (K). Data are shown as mean ± 95% CI. Statistical significance in (I) was determined by Student’s t-test between A and P. For (J) and (K), statistical significance was determined by a one-way ANOVA with Dunnett’s multiple comparison test relative to the control (Oregon-R): ns: not significant, *P<0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.

βH-Spec and α-Spec reduce junctional Jub levels in wing discs

(A-D) Apical sections of wing imaginal discs expressing en-Gal4 UAS-RFP UAS-dcr2 jub:GFP crossed to control (Oregon-R, OR) (A), and UAS-kstRNAi (B), UAS-kst-CRISPRa (C) and UAS-α-specRNAi (D) showing the effect on Jub:GFP (green) levels and localization in the posterior compartment (marked by RFP, blue). Scale bar = 20 μm. (E-H) Heat maps of relative junctional Jub:GFP intensity of wing discs. Levels of Jub:GFP relative to E-cad levels are shown for the different genotypes analyzed. Heat map scale is indicated on the top. Number of wing discs used for analysis: Control (OR), n=9; UAS-kstRNAi, n=8; UAS-kst-CRISPRa, n=6; UAS-α-specRNAi, n=8. (I-J) Quantification of Jub:GFP normalized to E-cadherin (I) or to Jub:GFP mean intensity (J) in posterior cells compared to anterior cells (P/A) in wing discs expressing the indicated constructs, displayed as individual values. Data are shown as mean ± 95% CI, error bars show 95% CI. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test relative to the control (Oregon-R): ns: not significant, *P<0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001

Jub is required for βH-Spectrin and α-Spec regulation of wing size and ex-lacZ

(A-J) Adult wings from flies cultured at 29ºC and expressing UAS transgenes altering spectrin and/or jub expression under control of a nub-Gal4 driver. Representative adult wings are shown. (K) Quantification of wing area (mean ± 95% CI, n > 20). Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. Statistical comparisons are shown relative to nub-Gal4 UAS-dcr2/+ in green or relative to nub-Gal4 UAS-dcr2 UAS-jubRNAi/+ in purple. Additional selected statistical comparisons are shown in blue. (L-Q) Third-instar wing imaginal discs expressing ex-lacZ en-Gal4 UAS-dcr2 UAS-GFP (green) crossed to OR (L), UAS-kstRNAi (M), UAS-jubRNAi (N), UAS-jubRNAi UAS-kstRNAi (O), UAS-α-specRNAi (P), UAS-jubRNAi UAS-α-specRNAi (Q) stained for expression of ex-lacZ (red/white).

βH-Spec and α-Spec localize independently in wing imaginal discs.

(A-C) Wing imaginal discs expressing Kst:YFP immunostained with α-Spec and E-cad antibodies showing the localization of βH-Spec (Kst:YFP) and α-Spec at the AJ (A), below the AJ (B) and in cross sections (C). Upper yellow arrow in cross section indicates AJ layer, lower yellow arrow indicates “Below AJ” layer. (D-F) Apical sections of wing imaginal discs expressing en-Gal4 UAS-dcr2 kstYFP along with UAS-α-specRNAi (D), UAS-β-specRNAi (E) and UAS-kstRNAi (F) immunostained with α-Spec (D and F) or β-Spec antibodies (E), validating the different RNAi lines used and showing that the apical localization of βH-Spec (Kst) is independent of the presence of α-Spec or β-Spec. Scale bar = 20 μm. The AJ layers were obtained by ImSAnE, using as a reference the E-cad channel and 7 layers were projected (2.1 μm).

βH-Spec colocalizes with myosin and is regulated by myosin activity

(A-B) Wing imaginal disc co-expressing Kst:GFP and the myosin II reporter sqh-sqh::mCherry in apical views (A) and lateral views (B), co-stained with phalloidin for F-actin (blue/white). (C-E) Apical sections of wing discs expressing en-Gal4 UAS-RFP UAS-dcr2 kst:YFP crossed to (Oregon R, OR) (C), UAS-rokRNAi (D) or UAS-sqhEE (E) in the posterior compartment (marked by RFP, blue), showing the effect of altering myosin activity on the Kst:YFP localization (gray). Scale bar = 20 μm. (F) Higher magnification of the boxed regions indicated in E. Scale bar = 10 μm (G-I) Heat maps of junctional Kst:GFP intensity relative to E-cad of wing discs (n=5, for each genotype) are shown. Heat map scale is on the right side. (J) Quantification of Kst:YFP intensity normalized to E-cadherin in posterior cells (P) compared to anterior cells (A) in wing discs expressing the indicated constructs (n=5). Data are shown as mean ± 95% CI, error bars indicate CI. Statistical significance was determined by a one-way ANOVA with Dunnett’s multiple comparison test relative to the control (Oregon-R): ns: not significant, *P<0.05; **P≤0.01.

Spectrin and myosin have overlapping binding sites on F-actin.

(A-C) Co-sedimentation assays with βH-Spec CH domains (Spectrin), F-actin, and myosin-II subfragment-1-like protein (myosin). For all sedimentation assays, A, M, Sp, E, and R refer to F-actin, myosin heavy chain, spectrin, myosin essential light chain and myosin regulatory light chain, respectively. Mw indicates marker and S and P refer to supernatant and pellet, respectively. Quantification shows mean ± S.D. of the sedimentation behavior of spectrin and myosin with F-actin (n = 3). (A) Co-sedimentation assay between spectrin (0-12 μM) and F-actin (2 μM). (B) Co-sedimentation assay between myosin (0-2.59 μM) and F-actin (2 μM). (C) Co-sedimentation assay between spectrin (12 μM), myosin (0-2.59 μM) and F-actin (2 μM). Quantification shows that myosin reduces spectrin binding by ∼50% (bottom panel). (D-D’’) Model of the Drosophila CH1 domain (yellow) bound to F-actin (grey) (D). Model of the Drosophila myosin motor domain (purple) bound to F-actin (grey) (D’). Superimposition of the CH1 and myosin motor domain on F-actin. The two strands of the actin filament are shown in light and dark grey and subdomains are indicated.

Modulation of levels of total and active myosin in wing imaginal discs by βH-Spec.

(A-C) Apical sections of third instar wing imaginal discs expressing en-Gal4 UAS-dcr2 UAS-RFP (blue) crossed with control (A), UAS-kstRNAi (B) and UAS-α-SpecRNAi (C), stained with pMLC antibody (green) and E-cad (red). (D) Quantification of pMLC normalized to E-cadherin in posterior cells (P) compared to anterior cells (A) in wing discs expressing the indicated constructs, displayed as individual values. Number of wing discs used for analysis: Control (OR), n=5; UAS-kstRNAi, n=8, UAS-α-SpecRNAi, n=5. Error bars show mean with 95% CI. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. Statistical comparisons are shown relative to the control (OR). ***P≤0.001, ****P<0.0001. (E-F) Third instar wing imaginal discs expressing en-Gal4 UAS-dcr2 UAS-RFP (blue) sqh:GFP (green) in combination with UAS-kstRNAiHMS00882 (E) and UAS-kstP[EPgy2]EY01010 (F), stained for E-cad (red). (G) Apical sections of a wing imaginal disc expressing en-Gal4 tubG80ts kst:GFP crossed with UAS-kstCRISPRa, stained for E-cad (red). Expression of Kst:GFP (green) was induced by transferring the flies from 18ºC to 29ºC for 24 h. (H) Western blots of wing disc lysates with the genotypes shown in the figure: nub-Gal4 UAS-Dcr2 kst:GFP crossed with w1118 (control) and UAS-kstCRISPRa (experimental). Kst:GFP was detected using a GFP antibody. GAPDH was used as a loading control.

α-Spec knockdown affects wing imaginal disc thickness.

(A-C) Lateral views of wing imaginal discs expressing en-Gal4 UAS-RFP UAS-dcr2 crossed with control (Oregon-R, OR) (A), UAS-kstRNAi (B) or UAS-α-specRNAi (C), immunostained with phalloidin to label F-actin (green), Hoechst to label nuclei (blue) and the posterior compartment marked by RFP (red). Dashed lines indicate 30 μm from the center. Scale bar = 20 μm (D) Quantification of wing imaginal disc thickness determined 30 μm from the center in anterior (A) and posterior (P) sides for the different genotypes indicated. Statistical significance was determined by Student’s t-test. ns: not significative; **P≤0.01; ***P≤0.001

βH-spec modulates the recruitment of Jub:GFP to AJs.

(A, B) Third instar wing imaginal discs expressing en-Gal4 UAS-dcr2 UAS-RFP (blue) jub:GFP (green) in combination with UAS-kstRNAiHMS00882 (A) or UAS-kstP[EPgy2]EY01010 (B) stained for E-cad (red). (C, D) Apical sections of wing imaginal discs expressing en-Gal4 UAS-RFP UAS-dcr2 jubGFP crossed to control (Oregon-R, OR) (C), UAS-kstRNAi (D) from Figure 2. Columns to the right show higher magnification views of the anterior (a) and posterior boxes (p).

Loss of β-Spec affects α-Spec localization in wing discs.

(A) Higher magnification of the different channels in the boxed region indicated in A (from Figure 4A). (B-E) Sections of a wing imaginal disc expressing en-Gal4 UAS-RFP UAS-dcr2 crossed with UAS-α-specRNAi (B-C) or UAS-β-specRNAi (D-E) with lateral views (C, E) stained with mouse α-Spec (green) and rabbit β-Spec (red) antibody. Scale bar = 20 μm.

Additional examination of competition between spectrin and myosin for F-actin.

(A) Quantification of the binding behavior of myosin to F-actin in the presence and absence of spectrin in co-sedimentation assays. The similarity in actin binding indicates that pre-loading spectrin does not interfere with myosin binding to F-actin under these assay conditions. (B) Model of the Drosophila CH1-CH2 domain (CH1, yellow; CH2, blue) bound to F-actin (grey) (B’). Model of the Drosophila myosin motor domain (purple) bound to F-actin (grey) (B’’). Superimposition of the CH1-CH2 and myosin motor domain on F-actin. The two strands of the actin filament are shown in light and dark grey and subdomains are indicated.