Journal: Journal of Cell Science

Article Title: Drosophila Transglutaminase preserves the integrity of muscle attachments with and without mechanical strain

doi: 10.1242/jcs.264299

Figure Lengend Snippet: sr -dependent secretion of Tg is required for embryonic MTJ formation. (A) Profile of Tg mRNA or Tg protein expression across developmental stages (data from Flybase). (B) WT embryos stained with anti-Tg (magenta). Tg partially overlaps with the pattern of tendon cells marked by sr -GFP (green) in st. 13 embryos (top panel) or βPS integrin (cyan) in st. 15 (middle panel) and st. 17 (bottom panel) embryos. White asterisks mark trachea. (C) qPCR confirms knockdown of Tg mRNA in whole larvae at the L3 stage. N =3 biological replicates. Mean±s.d. (D) St. 16 embryos immunostained for Tropomyosin (TM, magenta) or βPS integrin (cyan) to visualize the somatic muscles or MASs, respectively. The average length of all MASs is shorter in sr>Tg RNAi embryos compared to controls ( sr>mCh.NLS) . (E) Violin plot with quantification of the ventral MASs in control and Tg RNAi embryos. Dashed lines highlight median and quartiles. Each data point represents the average length of MASs in hemisegments A1–A4 for muscles VL1–VL4 per embryo. n ≥14 for each genotype. Average values reflected as median. *** P <0.0005; **** P <0.0001; n.s., not significant (Kruskal–Wallis test). (F) Amino acid alignment showing the conserved cysteine in Drosophila Tg isoforms A (dTg.A) and B (dTg.B), human TGM2 (h.TG2), and human factor XIIIa (h.FactorXIIIa). Created in BioRender by Geisbrecht, E., 2026. https://BioRender.com/jfriraa . This figure was sublicensed under CC-BY 4.0 terms. (G) Western blot and bar graph showing increased levels of protein in Tg.A, but similar expression levels of Tg in da>Tg WT or da>Tg C330S whole L3 larva. Total protein is shown as a loading control. n =3 biological replicates. Mean±s.d. (H) Violin plot with quantification of the ventral MASs upon overexpression of normal or mutant Tg. Dashed lines highlight median and quartiles. Each data point represents the average length of MASs in hemisegments A1–A4 for muscles VL1–VL4 per embryo. n ≥14 for each genotype. Average values reflected as median. * P <0.05; ** P <0.001; **** P <0.0001; ns, not significant (Kruskal–Wallis test). Images in B representative of n =3 experimental repeats.

Article Snippet: Primary antibodies included mouse anti-βPS integrin CF.6G11 (1:50; Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA), rat anti-tropomyosin (TM) MAC141 (1:200; Babraham Institute, Cambridge, UK), mouse anti-HA #2367 (1:50; Cell Signaling Technology, Danvers, MA), or rabbit anti-Tg #DZ41557 (1:100; BosterBio, Pleasanton, CA).

Techniques: Expressing, Staining, Knockdown, Muscles, Control, Western Blot, Over Expression, Mutagenesis

Journal: Journal of Cell Science

Article Title: Drosophila Transglutaminase preserves the integrity of muscle attachments with and without mechanical strain

doi: 10.1242/jcs.264299

Figure Lengend Snippet: Increased mechanical tension weakens IFM attachment. (A) sr -Gal4 is driving CD8–GFP (green) to mark the tendon cells in 2-day-old adult IFMs where muscles are labeled with phalloidin (gray). Anti-Tg immunostaining (magenta) shows labeling in tendon cells (t) and the cuticle (c). (B) Phenotypic classes present in Mhc[S1]/+, sr>Tg RNAi adult flies. Anterior is to the left. Compared to a ‘WT’ notum, small and V-like indentations (white arrowsheads) are seen in phenotypes classified as ‘mild.’ These are larger and wider in the ‘severe’ category. Phalloidin staining reveals the normal complement of six IFM fibers (*) in WT samples. Mild or severe phenotypes show degenerated or detached IFMs (yellow carets). (C) Grouped bar graph depicts the percentage of female or male adult flies with WT , mild or severe phenotypes shown in B. Increased tension in Mhc[S1] ; sr>Tg RNAi enhances thoracic indentation phenotypes compared to sr>Tg RNAi individuals. N =3 biological replicates with a total of n =90 for each genotype. Mean±s.d. (D) Grouped bar graph of female or male notums showing WT, mild or severe phenotypes. Increased tension in Mhc[S1] ; sr>Tg C330S enhances thoracic indentation phenotypes compared to Mhc[S1] ; sr>Tg WT or controls. N =3 biological replicates with a total of n =90 for each genotype. Mean±s.d. (E) Schematic of optogenetics approach to induce mechanical force in the IFMs. Created in BioRender by Geisbrecht, E. (2026). https://BioRender.com/ckiahly . This figure was sublicensed under CC-BY 4.0 terms. (F) Experimental outline of temperature shifts, ATR feeding and LED exposure. C created in BioRender by Geisbrecht, E., 2026. https://BioRender.com/xd4mnoc . This figure was sublicensed under CC-BY 4.0 terms. (G) Representative confocal single plane images of sr>Tg WT RNAi IFMs subjected to optical stimulation showing either attached (−ATR, upper panel) or detached (+ATR, lower panel) IFMs (F-actin, gray) with stretched βPS integrin (magenta) staining. (H) Scatter plot showing that IFM detachment is heightened in sr>Tg WT flies after muscle contractions induced by ATR and LED exposure. n =10 for each genotype. Mean±s.d. * P <0.05; *** P <0.0005; ns, not significant (Mann–Whitney test). Images in A representative of n =3 experimental repeats.

Article Snippet: Primary antibodies included mouse anti-βPS integrin CF.6G11 (1:50; Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA), rat anti-tropomyosin (TM) MAC141 (1:200; Babraham Institute, Cambridge, UK), mouse anti-HA #2367 (1:50; Cell Signaling Technology, Danvers, MA), or rabbit anti-Tg #DZ41557 (1:100; BosterBio, Pleasanton, CA).

Techniques: Muscles, Labeling, Immunostaining, Staining, Optogenetics, MANN-WHITNEY