Journal: PLoS ONE

Article Title: Increased Apoptosis of Myoblasts in Drosophila Model for the Walker-Warburg Syndrome

doi: 10.1371/journal.pone.0011557

Figure Lengend Snippet: (A–D and I–L) wild type ( 1151 > GFPnls ). (E–H and M–P) tw mutant ( tw , 1151 > GFPnls ). (A) and (E) Images stained by anti-α-spectrin antibody. α-spectrin is a component of cytoskeleton inside of cell membrane and bind to actin. (B), (F), (J), and (N) Images stained by anti-DE-cadherin antibody. DE-cadherin is a cell adhesion molecule located in cell surface. (C), (G), (K), and (O) Nuclei of myoblasts. (D), (H), (L), and (P) Merged images of (A–C), (E–G), (I–K), and (M–O), respectively. (I) and (M) Images stained by anti-βPS-integrin antibody. βPS-integrin is a cell adhesion molecule located in cell surface and bind to extracellular matrix. TB, arrowheads, and arrows in (A), (E), (I), and (M) show tracheoblast, the region of myoblasts, and lateral region of myoblasts, respectively. The signals of α-spectrin and βPS-integrin excessively increased in the region of myoblasts compared to the lateral region of myoblasts in tw mutant although apoptosis increased in the myoblasts of tw mutant. These signals in wild type did not change between two regions. But we could not find any difference in the signal of DE-cadherin.

Article Snippet: The primary antibodies were used in the following dilutions: anti-cleaved caspase-3 (Asp175) rabbit polyclonal antibody, 1∶300 (Cell Signaling, http://www.cellsignal.com ); anti-phospho-histone H3 (Ser10) rabbit polyclonal antibody, 1∶100 (Millipore, http://www.millipore.com ); anti-α-spectrin mouse monoclonal antibody (3A9), 1∶25 (Developmental Studies Hybridoma Bank, http://dshb.biology.uiowa.edu/ ); anti-βPS-integrin mouse monoclonal antibody (CF.6G11), 1∶200 (Developmental Studies Hybridoma Bank); anti-DE-cadherin rat monoclonal antibody (DCAD2), 1∶25 (Developmental Studies Hybridoma Bank); and anti-Dg rabbit polyclonal antibody, 1∶100 .

Techniques: Mutagenesis, Staining, Membrane

Journal: eLife

Article Title: Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction

doi: 10.7554/eLife.35518

Figure Lengend Snippet: ( A–L ) Representative traces and summary bar graph for mEJPs and EJPs recorded at 0.8 mM Ca 2+ from muscle 6 of indicated genotypes. The number of samples examined is indicated in each bar. ( A–C ) Neuronal knockdown of tnc significantly reduces the mEJPs frequency. ( D–F ) Neuronal but not muscle expression of Tnc can rescue the mEJPs frequency and EJP amplitude at tnc mutant NMJs. ( G–L ) The mean mEJPs frequency is dramatically reduced when mys/βPS integrin or if/αPS2 are knocked down in the neurons. Knockdown of if/αPS2 also induces slight reduction of the mean mEJPs amplitude and occasionally muscle attachment defects. ( M–O ) The trans-heterozygotes ( mys/+;; tnc/+) show enhancement of phenotypes compared with individual heterozygotes, indicating that tnc and mys interact genetically. Bars indicate mean ±SEM. ns, not significant (p>0.05), ***p<0.001,***p<0.001 *p<0.05. Genotypes: N > tnc RNAi (BG380-Gal4/+; UAS-tnc RNAi /+); M > tnc RNAi (UAS-tnc RNAi /+; 24B-Gal4/+); tnc rescue control ( UAS-tnc/+;tnc EP /Df(3R)BSC655); N > tnc, tnc EP/Df ( UAS-tnc/+;tnc EP /elav-Gal4,Df(3R)BSC655); M > tnc, tnc EP/Df ( UAS-tnc/+; tnc EP /24B-Gal4, Df(3R)BSC655); N > mys RNAi (BG380-Gal4/+; UAS-Dcr-2/+; UAS-mys RNAi /+); M > mys RNAi (UAS-Dcr-2/+; UAS-mys RNAi /24B-Gal4); N > if RNAi (BG380-Gal4/+; UAS-if RNAi /UAS-Dcr-2); M > if RNAi (UAS-if RNAi /UAS-Dcr-2; 24B-Gal4/+) .

Article Snippet: Primary antibodies from Developmental Studies Hybridoma Bank were used at the following dilutions: mouse anti-GluRIIA (8B4D2), 1:100; mouse anti-Dlg (4F3), 1:1000; mouse anti-Brp (Nc82), 1:200; mouse anti-α-Spectrin (3A9), 1:50; mouse anti-FasII (1D4), 1:10; mouse anti-Futsch (22C10), 1:100; mouse anti-Adducin (1B1), 1:50; mouse anti-βPS integrin (CF.6G11) 1:10; mouse anti-αPS1 integrin (DK.1A4) 1:10; mouse anti-αPS2 integrin (CF.2C7) 1:10.

Techniques: Expressing, Mutagenesis

Journal: eLife

Article Title: Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction

doi: 10.7554/eLife.35518

Figure Lengend Snippet: ( A–H ) Confocal images of third instar NMJ4 boutons from control and various tnc and mys/βPS manipulations stained for α-Spectrin (green) and HRP (magenta). Neuronal knockdown of tnc significantly increases the α-Spectrin levels; muscle knockdown mildly decreases the α-Spectrin signals (quantified in D). Thus synaptic α-Spectrin generally follows the levels of synaptic Tnc (compare with ). The recruitment of α-Spectrin appears to be dependent on Tnc/integrin complexes, since muscle knockdown of mys/βPS drastically reduces α-Spectrin accumulation at synaptic terminals (quantified in H). The number of NMJs examined is indicated in each bar. Bars indicate mean ±SEM. ns (p>0.05), ***p<0.001, **p<0.01. Scale bars: 5 μm. Genotypes: N > tnc RNAi (BG380-Gal4/+; UAS-tnc RNAi /+); M > tnc RNAi (UAS-tnc RNAi /+; 24B-Gal4/+); N > mys RNAi (BG380-Gal4/+; UAS-mys RNAi /UAS-Dcr-2); M > mys RNAi (UAS-mys RNAi /UAS-Dcr-2; 24B-Gal4/+) .

Article Snippet: Primary antibodies from Developmental Studies Hybridoma Bank were used at the following dilutions: mouse anti-GluRIIA (8B4D2), 1:100; mouse anti-Dlg (4F3), 1:1000; mouse anti-Brp (Nc82), 1:200; mouse anti-α-Spectrin (3A9), 1:50; mouse anti-FasII (1D4), 1:10; mouse anti-Futsch (22C10), 1:100; mouse anti-Adducin (1B1), 1:50; mouse anti-βPS integrin (CF.6G11) 1:10; mouse anti-αPS1 integrin (DK.1A4) 1:10; mouse anti-αPS2 integrin (CF.2C7) 1:10.

Techniques: Staining

Journal: eLife

Article Title: Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction

doi: 10.7554/eLife.35518

Figure Lengend Snippet: ( A–M ) Confocal images of NMJ4 boutons of indicated genotypes stained for βPS integrin ( A–E ) or α-Spectrin ( H–L ) (green) and HRP (magenta). The animals were reared at 25°C unless marked otherwise. When expressed in neurons, high levels of Tnc could restore the accumulation of βPS at tnc mutant NMJs (quantified in F). However, in these animals the boutons remain small, resembling the tnc mutant boutons (quantified in G). For the muscle rescue, Tnc levels were controlled using two different promoters and rearing the animals at 18°C (low expression) or 25°C (moderate). Low levels of muscle Tnc produce substantial accumulation of βPS integrin at tnc NMJs, above the control levels, and fully rescued the boutons size; high level of muscle Tnc further decreased the βPS accumulation at tnc NMJs. The α-Spectrin synaptic levels are restored only when Tnc is provided at low levels in the muscle. The number of samples examined is indicated in each bar. Bars indicate mean ±SEM. ***p<0.001. Scale bars: 5 μm. Genotypes: control ( UAS-tnc/+;tnc EP /Df(3R)BSC655); N > tnc, tnc EP/Df ( UAS-tnc/+;tnc EP /elav-Gal4, Df(3R)BSC655); M1 >tnc, tnc EP/Df ( BG487-Gal4 / UAS-tnc; tnc EP /Df(3R)BSC655); M2 >tnc, tnc EP/Df ( UAS-tnc/+; tnc EP /24B-Gal4, Df(3R)BSC655). .

Article Snippet: Primary antibodies from Developmental Studies Hybridoma Bank were used at the following dilutions: mouse anti-GluRIIA (8B4D2), 1:100; mouse anti-Dlg (4F3), 1:1000; mouse anti-Brp (Nc82), 1:200; mouse anti-α-Spectrin (3A9), 1:50; mouse anti-FasII (1D4), 1:10; mouse anti-Futsch (22C10), 1:100; mouse anti-Adducin (1B1), 1:50; mouse anti-βPS integrin (CF.6G11) 1:10; mouse anti-αPS1 integrin (DK.1A4) 1:10; mouse anti-αPS2 integrin (CF.2C7) 1:10.

Techniques: Staining, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: Local weakening of cell-ECM adhesion triggers basal tissue folding via changes in cell shape, actomyosin organization and E-cad levels

doi: 10.1101/2024.08.27.609853

Figure Lengend Snippet: views of wing imaginal discs throughout third instar larvae (A-C’) and at 2h APF (D, D’) , stained with anti-GFP (green) , anti- βPS (blue ) and the F-actin marker Rhodamine Phalloidin (red) . (A’-D’) Confocal cross-sections along the white dotted lines shown in (A-D) . Brackets indicate cell height in the wing margin (dotted line) and in ventral and dorsal domains (straight line). (E) Quantification of cell height of wing margin and adjacent cells, at different larval developmental stages. (F) Quantification of apicolateral (AL) and basolateral (BL) wing margin width at different developmental stages. (G–H’’’) Confocal views of wing discs expressing the membrane marker resille-GFP at 80h AED (G) and 96h AED (H) stained with anti-GFP (green), the F-actin marker Rhodamine Phalloidin (red), the nuclear marker Hoechst (DNA, blue ) and anti-perlecan (white) . (G’-G’’’ and H’-H’’’) confocal cross-sections along the white dotted lines shown in G and H, respectively. White arrows in H’ and H’’ point to cell detachment from the BM in the wing margin region. The statistical significance of differences was assessed with a Mann-Whitney U test, ***, ** and * p values < 0.001, <0.01, and <0.05, respectively. Scale bar in all panels, 30μm. At least 16 wing discs were assessed over three independent experiments.

Article Snippet: The following primary antibodies were used: goat anti-GFP FICT (Abcam, 1:500), mouse anti-βPS, rat anti-DE-Cad (DCAD2) and anti-wingless (DSHB, University of Iowa, USA, 1:50), rabbit anti-pMyosin light chain 2, p-Sqh (Cell signalling 1:20).

Techniques: Staining, Marker, Expressing, Membrane, MANN-WHITNEY

Journal: bioRxiv

Article Title: Local weakening of cell-ECM adhesion triggers basal tissue folding via changes in cell shape, actomyosin organization and E-cad levels

doi: 10.1101/2024.08.27.609853

Figure Lengend Snippet: (A-C’’’) Confocal views of wing imaginal discs from early to late third-instar larvae stained with anti-βPS (green in A–C’ , white in A’-C’’) , the F-actin marker Rhodamine Phalloidin (red in A–C’ , white in A’’-C’’’) and the nuclear marker Hoechst (DNA, blue in A-C’) . ( A-C ) Maximal projections of control wing disc of 80h AED (A) , 96h AED (B) and 120h AED (C) . (A’-C’’’) Confocal cross-sections along the white dotted lines shown in (A-C) . White arrows in (A’’-C’’’) point to the wing margin region. (D-F) . Quantification of βPS and F-actin levels in control wing discs of the designated developmental time points in the regions framed in A’, B’ and C’, yellow and orange denote wing margin region and adjacent cells, respectively . The statistical significance of differences was assessed with a Mann-Whitney U test, ***, ** and * p values < 0.001, <0.01 and <0.05, respectively. Scale bar in all panels, 30μm. At least 15 wing discs were assessed over three independent experiments.

Article Snippet: The following primary antibodies were used: goat anti-GFP FICT (Abcam, 1:500), mouse anti-βPS, rat anti-DE-Cad (DCAD2) and anti-wingless (DSHB, University of Iowa, USA, 1:50), rabbit anti-pMyosin light chain 2, p-Sqh (Cell signalling 1:20).

Techniques: Staining, Marker, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Local weakening of cell-ECM adhesion triggers basal tissue folding via changes in cell shape, actomyosin organization and E-cad levels

doi: 10.1101/2024.08.27.609853

Figure Lengend Snippet: ( A ) Initial simulation at 0h, showing a cross-section of the wing disc columnar epithelium perpendicular to the DV axis. On the right, a close-up of the cross-section showing the apical actin layer (green), three cell body layers (cyan) and one integrin adhesion layer (greenish-brown). (B) Interpretive scheme of the integrin adhesion layer framed in orange in (A) . Layers’ thickness in the scheme are not to scale. (C, D) Snapshot of simulation when basolateral contractility and reduction of integrin adhesion strength were applied simultaneously (C) or when the strength of the integrin adhesion was decreased prior to application of basolateral contractility (D) . Magnifications of the region framed in the snapshots are also shown. (E-F’’) Confocal cross-sections of control wing disc at 80h AED (E) and 88h AED (F) stained with anti-βPS (green in E, F and white in E’, F’) , Rhodamine Phalloidin to detect F-actin (red in E, F and white in E’’, F’’) and the nuclear marker Hoechst (DNA, blue in E, F). (G, H) Quantification of βPS and F-actin levels in control wing discs of the designated developmental time points in the regions framed in E and F (orange and yellow boxes). The statistical significance of differences was assessed with a Mann-Whitney U test, ***, ** and * p values < 0.001, <0.01, and <0.05, respectively. Scale bar in all panels, 30μm. At least 15 wing discs were assessed over three independent experiments.

Article Snippet: The following primary antibodies were used: goat anti-GFP FICT (Abcam, 1:500), mouse anti-βPS, rat anti-DE-Cad (DCAD2) and anti-wingless (DSHB, University of Iowa, USA, 1:50), rabbit anti-pMyosin light chain 2, p-Sqh (Cell signalling 1:20).

Techniques: Control, Staining, Marker, MANN-WHITNEY

Journal: bioRxiv

Article Title: Local weakening of cell-ECM adhesion triggers basal tissue folding via changes in cell shape, actomyosin organization and E-cad levels

doi: 10.1101/2024.08.27.609853

Figure Lengend Snippet: (A-B’’) Confocal views of third-instar wing imaginal discs stained with anti-GFP (white), anti-βPS (green ) , Rhodamine Phalloidin to detect F-actin (red in A, A’, B, B’ , white in A’’, A’’’, B’’, B’’’) and the nuclear marker Hoechst (DNA, blue in A, A’, B, B’) . (A) Control wing disc. (B) Wing disc co-expressing RNAis against mys and hid under the control of the ptcGal4 ( ptc>mys RNAi ;hid RNAi ). (A’, A’’, B’, B’’) Confocal cross- sections taken along the white dotted lines shown in (A, B) . (A’’’, B’’’) Super-resolution images taken in the region between the yellow and red dotted lines in (A, B). (C, D) Quantification of βPS and F-actin levels in the regions framed in A’ and B’ (orange and yellow boxes) in control (C) and ptc>mys RNAi ;hid RNAi (D) wing discs. (E) Quantification of the height of wing margin and adjacent cells in control and ptc>mys RNAi ;hid RNAi wing discs. (F-H’’’) Confocal views of 96h AED third-instar wing discs of the designated genotypes, stained with anti-DE Cad (red in F, F’, G, G’, H, H’ and white in F’’’, G’’’ and H’’’ ), the nuclear marker Hoechst (DNA, blue in F, F’, G, G’, H, H’ and white in F’’’, G’’’ and H’’’) and anti-GFP (green in G, G’, H, H’) . ( F’-F’’’, G’-G’’’, H’-H’’’) Confocal cross-sections along the white dotted lines shown in F , G and H , respectively. White arrows in ( F’’, F’’’, G’’, G’’’, H’’, H’’’ ) point to the wing margin region. (I, J, K) Quantification of anti-DE-Cad levels in controls and experimental wing discs in the regions framed in F’, G’ and H’ (orange and yellow boxes) . At least 15 wing discs were assessed over three independent experiments. The statistical significance of differences was assessed with a Mann-Whitney U test, ***, ** and * p values < 0.001, <0.01, and <0.05, respectively. Scale bar in all panels, 30μm.

Article Snippet: The following primary antibodies were used: goat anti-GFP FICT (Abcam, 1:500), mouse anti-βPS, rat anti-DE-Cad (DCAD2) and anti-wingless (DSHB, University of Iowa, USA, 1:50), rabbit anti-pMyosin light chain 2, p-Sqh (Cell signalling 1:20).

Techniques: Staining, Marker, Control, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Local weakening of cell-ECM adhesion triggers basal tissue folding via changes in cell shape, actomyosin organization and E-cad levels

doi: 10.1101/2024.08.27.609853

Figure Lengend Snippet: (A) Snapshot of a simulation where the integrin adhesion strength was reduced without inducing basolateral contractility. (B-B’’, D-D’’, F-F’’) C onfocal views of wing imaginal discs of the indicated genotypes stained with anti-βPS (green), Rhodamine Phalloidin to detect F-actin (red) and the nuclear marker Hoechst (DNA, blue). (B, D, F) Maximal projections of control (B) and wing discs expressing an abi RNAi (D) or a scar RNAi (F) under the control of wgGal4 . (B’, D’, F’) Confocal cross-s ections along the white dotted lines shown in B , D and F . (C, E, G) Quantification of βPS and F-actin levels in control (C) , wg>abi RNAi (E) and wg>scar RNAi (G) wing discs in the regions framed in B’ , D’ and F’ . (H) Quantification of the height of wing margin and adjacent cells in control, wg>abi RNA i and wg>scar RNAi wing discs. The statistical significance of differences was assessed with a Mann-Whitney U test, ***, ** and * p values < 0.001, <0.01, and <0.05, respectively. Scale bar in all panels, 30μm. At least 15 wing discs were assessed over three independent experiments.

Article Snippet: The following primary antibodies were used: goat anti-GFP FICT (Abcam, 1:500), mouse anti-βPS, rat anti-DE-Cad (DCAD2) and anti-wingless (DSHB, University of Iowa, USA, 1:50), rabbit anti-pMyosin light chain 2, p-Sqh (Cell signalling 1:20).

Techniques: Staining, Marker, Control, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Local weakening of cell-ECM adhesion triggers basal tissue folding via changes in cell shape, actomyosin organization and E-cad levels

doi: 10.1101/2024.08.27.609853

Figure Lengend Snippet: (A-B’’’) Confocal views of third-instar wing imaginal discs stained with anti-βPS (green), the F-actin marker Rhodamine Phalloidin (red in A, A’, B, B’ , white in A’’, A’’’, B’’, B’’’) and the nuclear marker Hoechst (DNA, blue). (A) Control wing disc. (B) Wing disc co-expressing an active form of the αPS2 subunit ( αPS2ΔCyt ) and the βPS subunit under the control of wgGal4 ( wg>αPS2ΔCyt; βPS ). (A’, A’’, B’, B’’) Confocal cross-sections taken along the white dotted lines shown in (A, B). (A’’’, B’’’) Super-resolution images taken in the region between the red dotted line in (A, B). (C, D) Quantification of βPS and F-actin levels in control (C) and wg>αPS2ΔCyt; βPS (D) wing discs. (E) Quantification of the height of wing margin and adjacent cells in control and wg> αPS2ΔCyt; βPS wing discs. The statistical significance of differences was assessed with a Mann-Whitney U test, ***, ** and * p values < 0.001, <0.01, and <0.05, respectively. Scale bar in all panels, 30μm. At least 15 wing discs were assessed over three independent experiments. (F) Snapshot of a simulation where no integrin adhesion weakening or basolateral contractility was applied. (G) Snapshot of a simulation where only basolateral contractility was applied, without changing integrin adhesion strength. Magnifications of regions framed in the snapshots in (F) and (G) are also shown.

Article Snippet: The following primary antibodies were used: goat anti-GFP FICT (Abcam, 1:500), mouse anti-βPS, rat anti-DE-Cad (DCAD2) and anti-wingless (DSHB, University of Iowa, USA, 1:50), rabbit anti-pMyosin light chain 2, p-Sqh (Cell signalling 1:20).

Techniques: Staining, Marker, Control, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Local weakening of cell-ECM adhesion triggers basal tissue folding via changes in cell shape, actomyosin organization and E-cad levels

doi: 10.1101/2024.08.27.609853

Figure Lengend Snippet: (A, C, E, G, I) Confocal views of 2h APF wing imaginal discs of the indicated genotypes, stained with anti-βPS (green), Rhodamine Phalloidin to detect F-actin (red) and the nuclear marker Hoechst (DNA, blue). (A, C, E) Maximal projections of control (A) and wing discs expressing an abi RNAi ( C , wg>abi RNAi ) or co-expressing an active form of the αPS2 subunit and the βPS subunit ( E , wg>αPS2ΔCyt; βPS ) under the control of wgGal4. (A’, C’, E’) Confocal cross-s ections along the white dotted lines shown in (A, C, E) . (B, D, F) Images of control (B) , wg>abi RNAi (D) and wg>αPS2ΔCyt; βPS (F) adult wings. (G, I) Maximal projections of control (G) and wing discs co-expressing RNAis against mys and hid under the control of the ptcGal4, ptc>mys RNAi ;hid RNAi (I) . (G’, I’) Confocal cross-s ection s along the white dotted line shown in (G, I) . (H, J) Images of control (H) and ptc>mys RNAi ;hid RNAi (J) adult wings. Scale bar in all panels, 30μm.

Article Snippet: The following primary antibodies were used: goat anti-GFP FICT (Abcam, 1:500), mouse anti-βPS, rat anti-DE-Cad (DCAD2) and anti-wingless (DSHB, University of Iowa, USA, 1:50), rabbit anti-pMyosin light chain 2, p-Sqh (Cell signalling 1:20).

Techniques: Staining, Marker, Control, Expressing