In vivo cell biological screening identifies an endocytic capture mechanism for T-tubule formation

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.

Muscle-specific Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle

“…To date, detailed analyses of T-tubule development have been difficult in mammalian systems due to a slow and asynchronous process of T-tubule development. We have recently characterized this process extensively in the developing zebrafish embryo using light microscopy, three-dimensional (3D) electron microscopy and semi-automated quantitative assays of T-tubule development (Hall et al, 2020); this system overcomes many of the challenges of the mammalian system and is amenable to precise mechanistic dissection. We therefore generated knockout zebrafish models for both cavin4a and cavin4b , and crossed these lines to obtain a zebrafish line completely lacking Cavin4 ( cavin4a;cavin4b double mutant, hereafter referred to as cavin4 -/- ; see Figures S3-S4 and Methods section for a detailed description of the generation and characterization of individual cavin4a -/- and cavin4b -/- lines).…”

“…However, while we were able to generate adult cavin4 -/- zebrafish, they were poor breeders, hampering the generation of homozygous clutches; experiments were therefore performed using homozygous x heterozygous crosses, with pre-or post-genotyping to identify cavin4 -/- embryos. To visualize the muscle fibers, we crossed the cavin4 -/- line to a stable transgenic line expressing ubiquitous GFP-CaaX, which effectively delineates the sarcolemma and T-tubules (Hall et al, 2020; Williams et al, 2011). Imaging of transverse sections from 5 dpf WT embryos revealed GFP localization at the sarcolemma and at the T-tubules in a characteristic radial “spoke-like” pattern (Figure 3A).…”

“…In view of the effect of the loss of Cavin4 on the T-tubule system in mouse skeletal muscle, we next examined the distribution of Cav3. In zebrafish embryos, T-tubules are observed to penetrate from the sarcolemma to the fiber midline by 48 hpf (Hall et al, 2020). Imaging of a stable transgenic line expressing Cav3-GFP revealed that Cav3 was strongly associated with early T-tubules during zebrafish development (31 hpf, Figure 3B).…”

“…In view of these findings and the observed upregulation of BIN1 expression in the absence of CAVIN4 (Figures S2D and S2H), we hypothesized that a functional interaction exists between Cavin4 and Bin1 during T-tubule development. We first utilized a model cellular system as done previously (Hall et al, 2020; Lee et al, 2002) to examine the possible association of Cavin4 with Bin1-induced tubules in non-muscle cells. Co-expression of Bin1b and Cavin4b produced numerous membrane tubular structures in BHK cells that were positive for both Bin1b and Cavin4b (Figure 5D).…”

“…We first utilized a model cellular system as done previously (Hall et al, 2020;Lee et al, 2002) to examine the possible association of Cavin4 with Bin1-induced tubules in non-muscle cells. Co-expression of Bin1b and Cavin4b produced numerous membrane tubular structures in BHK cells that were positive for both Bin1b and Cavin4b ( Figure 5D).…”