Abstract
The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.
Highlights
The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction
We found that fluorescent proteins tagged with the CaaX domains from either H-ras or K-ras were robustly localised to the T-tubules, as well as to the sarcolemma at 48 hpf (Fig. 1a, b)
For this reason we obtained an existing transgenic line which strongly and robustly expresses GFP-CaaX in all cells from a ubiquitous promoter to use in our analyses[13]
Summary
The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. In the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. We use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. The transverse tubule (T-tubule) system of skeletal muscle is an extensive membrane domain that allows action potentials from innervating neurons to reach the depths of a mKate2-CaaX(tH). The T-tubule system is a vital component of the skeletal muscle, frequently disrupted in muscle disease. Merge biogenesis of this enigmatic organelle is necessary for further insight into these diseases; progress has been compromised by the absence of adequate experimental models. The T-tubules are first observed as longitudinal elements at E167 which gradually become organised into transverse elements from around birth to c myristo-mKate[2]
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