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An Endocytic Capture Model for Skeletal Muscle T-tubule Formation

Thomas E Hall, Nick Martel, Nicholas Ariotti, Zherui Xiong, Harriet P Lo, Charles Ferguson, James Rae, Ye-Wheen Lim, Robert G Parton

Preprint posted on March 06, 2020 https://www.biorxiv.org/content/10.1101/2020.03.05.979666v1.full

In vivo zebrafish experimental system sets a new paradigm for skeletal muscle T-tubule development

Selected by Helena Pinheiro

Transverse (T-) tubules are extensive invaginations of the membrane that penetrate muscle cells allowing for efficient transmission of the action potential throughout the whole cell, leading to coordinated contraction. Although an aberrant T-tubule network is observed in myopathies, the biogenesis of this organelle is largely unknown.

In this preprint, the authors developed a robust system for studying T-tubule biogenesis in vivo, using zebrafish, and propose that T-tubules are stabilized by associating to myofibrils. They observed that their biogenesis depends on Rab family proteins, which are essential for the endocytic pathway in non-muscle cells.

Key Findings:

To develop the in vivo model for imaging T-tubule formation, the authors used a membrane marker that colocalizes with the T-tubule protein Bin1, a CaaX domain protein labeled with GFP, to avoid bias for any specific T-tubule protein. With this system, they were able to track T-tubule development over time from immediately after muscle cells’ fusion at 16 hpf, to 48 hpf and later, when the T- tubules are developed and adopt a regular spacing.

Then, the authors moved to transmission electron microscopy (TEM) to be able to understand T–tubule formation in a quantitative manner. Very early on, they observed tranversal oriented tubular membranes associated to the Z-lines. However, away from the Z-lines, there is also association of sarcoplasmic reticulum (SR) membranes to early forming myofibrils. By performing a time-series analysis, the authors observed that the SR and the tubules develop in a manner that allows close contact of the membranes with the myofibrils.

By assembling TEM mosaics of entire zebrafish trunk musculature at 48 hpf, 5 dpf and 10 dpf, the authors obtained enough morphometric data to develop a mathematical model of T-tubule development. They generated a predictive model relating the fiber cross sectional area with the surface area of T-tubules and sarcolemma. Sarcolemma increases in a proportional manner with the increase in fiber size, while T-tubules show exponential increase. I believe this is because T- tubules must span the interior of the fibers, and T-tubule growth should be directly proportional to the volume of the fibers.

Interestingly, this model also predicts that the increase in the surface area of T-tubules and sarcolemma would require an average of 1.03 um2/min of membrane addition in the 48h-5dpf period and 2.27 um2/min in 5d-10dpf. These values argue for a very dynamic process, and are probably underrated once they exclude the membrane necessary for SR development.

The close contact of T-tubules and SR with myofibrils led the authors to explore if the development of the myofibrils could affect the formation of the T-tubule and SR network. Thus, the authors generated zebrafish expressing fluorencent Lifeact, which labels F-actin, including sarcomeric actin, and an endoplasmic reticulum marker, KDEL. This allowed them to observe the localization of myofibrils and SR, respectively. They used these reporters to check the localization of SR and myofibrils relative to T-tubules (using CaaX reporter). To understand how myofibrils might affect membrane development the authors depleted essential sarcomeric proteins using CRISPR. The depletion of slow myosin heavy chain led to the approximation of the T-tubules but maintenance of overall structure. However, depletion of titin, which serves as a scaffold to link myosin to the Z-lines, led to a complete disruption of the membrane meshwork. These data show that T-tubule network formation depends on the appropriate development of sarcomeres.

Live imaging of CaaX at early stages shows the formation of tubular structures beneath the plasma membrane. Are these structures connected to the plasma membrane since their formation, or do they fuse with the membrane later? To answer this question the authors used a membrane impermeable dye, Alexa 647 labelled UTP, and observed that, upon injection, the dye immediately infiltrated the tubules, showing that they are connected to the extracellular space. In contrast, the uptake in the intracellular vesicles took several minutes. Taking these data into account, the authors propose that the tubules are surface connected and are analogous to endocytic tubules. Because they showed previously the importance of the myofibrils for  T-tubule network, they propose that the interaction of the vesicles and T-tubules with the developing myofibrils acts as an “endocytic capture”, stabilizing the developing tubules.

By testing the composition of the tubules in early stages, the authors discovered a similar composition to early endosomes. This observation led them to systematically test the role of all the Rab family proteins in this system, given their described importance in endocytic membrane regulation in non-muscle cells. They observed that a subset of Rab proteins are present in the T-tubule/SR junction and the overexpression of other Rab proteins, mostly endosomal, perturbs T-tubule development. To further elucidate the significance of these proteins for T-tubule development, the authors developed an in vitro system in which they transfected Bin1 in non- muscle cells. They assessed the colocalization of Bin1 with the top ten hits from Rab family proteins in the in vivo system and observed that Bin1 is able to recruit 6 out of 10 Rab protein candidates, even in the absence of sarcomeres and junctional SR. I believe this could indicate that Rab proteins are important in a very early stage of T-tubule biogenesis.

This paper describes a good system with a lot of potential for studying T-tubule development. This is particularly important in a field that is not yet understood, in part due to the lack of good experimental systems. I specially liked the effort to obtain morphometric data, which I believe is difficult when studying a complex organelle whose development depends so much on spatial organization. I also find the model proposed for T-tubule biogenesis very interesting and deserving further study.

Questions for the authors:

The Rab family proteins that have a higher colocalization with T-tubules are not the ones that lead to the stronger perturbation of the T-tubules network. Any ideas on their function in T- tubules?

Which are the most likely candidates responsible for the endocytic trapping?

Does the perturbation of sarcomere assembly also perturb membrane formation, or only its spatial organization?

 

Posted on: 30th March 2020 , updated on: 4th May 2020

doi: https://doi.org/10.1242/prelights.18005

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