Biogenesis of Transverse Tubules and Triads: Immunolocalization of the 1,4-Dihydropyridine Receptor, TS28, and the Ryanodine Receptor in Rabbit Skeletal Muscle Developing in Situ

• Published in: The Journal of cell biology 1991, Vol.112 (2), p.289-301
• Main Authors: Yuan, Shaohua, Arnold, Wayne, Jorgensen, Annelise O.
• Format: Article
• Language: English
• Subjects: Animals; Animals, Newborn; Antibodies; Calcium Channels; Caveolae; Cytosol; Fluorescent Antibody Technique; Fluorescent antibody techniques; Immunoblotting; Microscopy; Models, Biological; Muscle Development; Muscle fibers; Muscle Proteins - analysis; Muscle Proteins - metabolism; Muscles - cytology; Muscles - embryology; Muscles - metabolism; Rabbits; Receptors; Receptors, Cholinergic - analysis; Receptors, Cholinergic - metabolism; Receptors, Nicotinic - analysis; Receptors, Nicotinic - metabolism; ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic reticulum; Skeletal muscle; tubules
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.112.2.289

Our previous immunofluorescence studies support the conclusion that the temporal appearance and subcellular distribution of TS28 (a marker of transverse (T) tubules and caveolae in adult skeletal muscle, correspond very closely to those of T-tubules forming de novo in developing rabbit skeletal muscle. To extend our morphological studies of the biogenesis of T-tubules and triads, the temporal appearance and subcellular distribution of the α 1-subunit of the 1,4-dihydropyridine receptor (a marker of the T-tubules and caveolae) was compared to (a) that of TS28; and (b) that of the ryanodine receptor (a marker of the junctional sarcoplasmic reticulum) in rabbit skeletal muscle cells developing in situ (day 19 of gestation to 10 d newborn) by double immunofluorescence labeling. The results presented show that the temporal appearance and relative subcellular distribution of the α 2-subunit of the 1,4-dihydropyridine receptor (α 1-DHPR) are distinct from those of TS28 at the onset of the biogenesis of T-tubules. Thus, in a particular developing myotube the α 1-DHPR appeared before TS28 (secondary myotubes; day 19-24 of gestation). Furthermore, the α 1-DHPR was distributed in discrete foci at the outer zone of the cytosol, while TS28 was confined to foci and rod-like structures at the cell periphery. As development proceeded (primary myotubes; day 24 of gestation) ∼50% of the foci were positively labeled for both TS28 and the α 1-DHPR, while ∼20 and 30% of the foci were uniquely labeled for TS28 and the α 1-DHPR, respectively. The foci labeled for both TS28 and the α 1-DHPR and the foci uniquely labeled for TS28 were generally confined to the cell periphery, while the foci uniquely labeled for the α 1-DHPR were mostly confined to the outer zone of the cytosol. 1-2 d after birth, TS28 was distributed in a chickenwire-like network throughout the cytosol, while the α 1-DHPR was confined to cytosolic foci. In contrast, the temporal appearance and subcellular distribution of the α 1-DHPR and the ryanodine receptor were very similar, if not identical, throughout all the stages of the de novo biogenesis of T-tubules and triads examined. Assuming that the subcellular distribution of TS28 represents the distribution of forming T-tubules the results presented are consistent with the following plausible scheme for the biogenesis of T-tubules and triads. Before the onset of T-tubule formation, α 1-DHPR-containing cytosolic vesicles form a complex with a ryanodine receptor-co