Fig. 5. Tubules are stabilised by endocytic capture.

a–c Different tubule morphologies by confocal microscopy (EGFP-CaaX, inverted). a Longitudinal elements are frequently seen between forming tubules (arrows) and additional elements appear to connect to the sarcolemma (arrowheads). b Glancing optical sections across the sarcolemma at 16 hpf immediately before stabilised transverse orientated tubules become visible, show CaaX-positive tubules directly beneath. c At 48 hpf putative surface-connected elements (arrows) are frequently seen in glancing optical sections across the sarcolemma. Images are representative of 12 individual cells within different individual animals. d–f Intramuscular injection of Alexa-647 conjugated UTP into EGFP-CaaX fish results in immediate infiltration into the developing T-system. (d) 16 hpf, (e) 24 hpf, (f) 48 hpf. Images are representative of three different individual animals. g Timelapse microscopy of 10,000 MW-dextran-Alexa-647 injected EGFP-CaaX fish shows immediate infiltration into the tubules, and uptake into intracellular vesicles within 10 min (see also Supplementary Movie 5). Images are representative of three different individual animals. h Serial blockface electron microscopy and 3D reconstruction shows tubules connecting stabilised, sarcomere associated tubules to the sarcolemma. Images were derived from two individual animals and images shown are representative of both. See also Supplementary Fig. 5a. Individual Z planes are shown in h1–h6. Scale bars; (a–c) 5 µm, (d–f) 10 µm, (g, h) 5 µm.