Figure 3.
Matrix enrichment at the B-LINK and the role of hemicentin in resisting egg-laying mechanical forces. (A) A diagram showing the RNAi feeding and timing of optogenetic muscle contraction and imaging. (B) The quantification of fold enrichment of matrix component fluorescence intensity at the B-LINK compared to the gonadal BM at the young adult stage (n = 10 each). Agrin is not shown as it was undetectable at the B-LINK and in the gonadal BM (n = 10 each). ***P < 0.0001, **P < 0.001, *P < 0.01, unpaired two-tailed Student’s t test comparing B-LINK signal to two times the gonad signal (to account for double BM at the B-LINK). Error bars represent SEM. (C) Fluorescence images of enriched matrix components and a representative non-enriched component (γ-laminin::mNG) at the young adult stage at the B-LINK and in the gonadal BM. Yellow arrowheads indicate location of fluorescence intensity measurements. White arrowhead indicates α1-type IV collagen localized in the muscle endoplasmic reticulum where it is assembled. (D) Transgenic worms expressing the ultraviolet light receptor LITE-1 in body wall and egg-laying muscles (lite-1(ce314); ceIs37 [myo-3p::lite-1 + myo-3p::GFP]) were plated on control (T444T empty vector) and hemicentin (him-4) RNAi and after 60 h were exposed to 488 nm light for 7 s to induce muscle contractions that forced egg-laying. Images show worms both pre- and post-contraction. Regions of the animals in the red box are magnified in the insert to show egg-lay and rupture (control rupture: 3%, n = 3/90; hemicentin RNAi knockdown rupture: 76%, n = 68/90). Images are stills from Videos 2 (control RNAi) and 3 (hemicentin RNAi). (E) A schematic model of utse–seam B-LINK rupture upon loss of hemicentin. Scale bars in C, 10 µm. Main scale bar in D, 100 µm; insert scale bar, 50 µm.