Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation
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eLife assessment
Lemerle et al utilize advanced correlative light and electron microscopy and molecular biology approaches to convincingly demonstrate the presence of the membrane-bending protein Bin1 and caveolae containing rings capable of membrane tubulation in developing muscle. The data is highly significant as it potentially advances our fundamental understanding of how transverse tubules are formed, a significant gap in our understanding of excitation-contraction coupling and muscle biology more broadly.
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Abstract
Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, Bin1 and caveolae composed of caveolin-3 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Bin1 expression lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations results in defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 dysfunction.
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Author Response
Reviewer #1 (Public Review):
Lemerle et al utilize elegant imaging and molecular biology approaches to convincingly demonstrate the presence of Bin1 and caveolae containing rings capable of tubulation in developing muscle. The data is of fundamental potential significance as it advances our understanding of t-tubule biogenesis, which represents a major knowledge gap in muscle biology. The paper will be of broad interest to skeletal and cardiac muscle biologists and physiologists. The paper is well written, with a comprehensive yet concise introduction, clearly presented results, and an appropriate discussion. The imaging is spectacular, and the use of CLEM provides compelling validation of the protein constituents of ring structures identified via EM. When combined with time-lapse imaging, the combination of …
Author Response
Reviewer #1 (Public Review):
Lemerle et al utilize elegant imaging and molecular biology approaches to convincingly demonstrate the presence of Bin1 and caveolae containing rings capable of tubulation in developing muscle. The data is of fundamental potential significance as it advances our understanding of t-tubule biogenesis, which represents a major knowledge gap in muscle biology. The paper will be of broad interest to skeletal and cardiac muscle biologists and physiologists. The paper is well written, with a comprehensive yet concise introduction, clearly presented results, and an appropriate discussion. The imaging is spectacular, and the use of CLEM provides compelling validation of the protein constituents of ring structures identified via EM. When combined with time-lapse imaging, the combination of approaches provides powerful nanoscale structural information alongside temporal dynamics and live-cell confirmation of tubulating ability by Bin1-Cav3 containing rings. The data indicate that Bin1 is sufficient to generate circular structures that are subsequently decorated by caveolae which facilitate tubule formation at the membrane, and they support the requirement of both Bin1 and Cav3 for efficient tubule initiation and elongation. The authors also utilize myotubes from patients with cav3 mutations to explore whether altered ring formation may contribute to muscle pathology - however, this section requires additional controls and validation to confer pathological insight. Further, additional quantification of imaging data across the study is required to increase the rigor and strength of the conclusions of this work.
We would like to thank reviewer #1 for his appreciation of our work, in particular the imaging experiments and for deeming our overall conclusions convincing. We have now performed additional experiments on patient myotubes including a rescue of Cav3, performed rigorous quantifications of rings and tubules under our different experimental conditions and re-wrote corresponding parts of the of the discussion to increase the strength of our conclusions.
Reviewer #2 (Public Review):
In this work Lemerle et al. provide long-awaited insight into how transverse tubules develop in skeletal muscle. Together with the sarcoplasmic reticulum transverse tubules form the triad, a specialized structure required for excitation-contraction coupling in skeletal muscle. Defects in transverse tubules or the triad can lead to problems such as muscular dystrophy. Whilst the involvement of specialist membrane structures (caveolae) and the membrane-bending protein Bin1 have long been recognized the precise mechanism of how caveolae and Bin1 cause transverse tubules to form and extend has remained unknown. This work provides compelling evidence, correlating antibody labelling with electron microscopy, to support the concept that caveolae rings form underneath the cell membrane which is surrounded by the endo/sarcoplasmic reticulum. These rings contain caveolin-3 and Bin1 and the authors show Bin1 enriched tubes extend from multiple points on these rings. Their data suggest that Bin1 assembles to initially form these scaffolds that then recruit the caveolae to form the ring. In addition, tubules appear continuous with the extracellular environment which is necessary for their function of facilitating calcium release during excitationcontraction coupling. In patients with mutations in caveolin-3 the caveolin ring formation as well as Bin1 tubulation were defective which may play a role in the pathology. The elegant experiments including time-lapse work clearly support the conclusions of the authors.
The ability of the authors to combine labelling studies with advanced microscopy to show the underlying structures provides very strong evidence for the proposed mechanisms. The authors suggest that the muscle-specific isoforms of BIN1 are key to tubule extension from caveolae rings but it would be interesting for them to discuss how this fits with studies suggesting that constitutive Bin1 isoforms can also form transverse tubules. It would also be interesting to understand the authors' views on whether caveolae rings are involved in the turnover of transverse tubules in adult myotubes as well as the initial formation and, additionally, if the caveolae rings are restricted to the region just under the surface membrane.
Insight into how transverse tubules are formed sets the groundwork for future therapies. This is clearly important for skeletal muscle myopathies but should also be considered in the heart. Cardiac transverse tubule loss and disorder play an important role in dysfunction in heart failure and atrial fibrillation and as such lessons learned in skeletal muscle may be successfully applied to the heart.
We would like to thank reviewer #2 for this appreciation of our work. We agree with the points raised and have updated our discussion section to highlight these points.
Reviewer #3 (Public Review):
T-tubules are an elaborate series of membrane invaginations that bring membrane voltageactivated Ca2+ channels in close apposition to the sarcoplasmic reticulum containing RyR, allowing for Ca2+-induced Ca2+ release. They serve as critical hubs of excitation-contraction coupling and play a central role in myopathies and inherited and acquired cardiomyopathies. Several membrane structures and proteins have been implicated in striated muscle t-tubule biogenesis, but the specific mechanisms of early t-tubule biogenesis are not defined. Lemerle et al here investigate the biogenesis of transverse tubules in skeletal muscle. They use skeletal myoblasts from murine and human muscle as well as sophisticated high-resolution microscopy, live cell imaging, and adenoviral targeting to forward a model of BIN1 mediated caveolae ring formation which give rise to DHPR enriched t-tubules and associate with SR. While they demonstrate that BIN1 and Cav3 enriched caveolae act together to form t-tubules, the precise pathophysiological mechanisms by which this process acts in disease remain unclear. Strengths of the study consist in the use of both murine and human skeletal muscle experiments, suggesting a conserved molecular mechanism; the innovative approach of correlative light and electron microscopy, and the use of pathological specimens. The live cell timelapse provides imaging evidence of Cav3-enriched caveolae-rings forming in centers of high BIN1 enrichment, from which t-tubules emanate. This is novel evidence in support of the biogenesis model proposed by the authors. The pathological correlation of their model is promising but limited. Specifically, while the study of Cav3 mutant specimens is used to show the Cav3 dependence of BIN 1 action (in experiments using BIN 1 overload), the authors have not tested the sufficiency of their proposed mechanism by rescuing the pathologic state. Moreover, the conditions of development likely have an important effect on the studied mechanism - such as mechanical loading, contractile state, neurohormonal environment, and so on. Furthermore, a more complete description of the precise molecular binding sites between BIN1 and Cav3 would be important. While exon11 is required for tubulation, BIN1 not expressing exon 11 appears sufficient to assemble caveolar rings, suggesting this is mediated by other specific BIN1 regions.
Overall, the study provides new details on early t-tubule biogenesis in skeletal muscle (likely shared with other striated muscle) and lays the foundations for further definition of the precise molecular mechanisms.
We would like to thank reviewer #3 for the appreciation of our work. We have now performed additional experiments on patient myotubes including rescue experiments, analysis of key excitationcontraction coupling proteins by Western blot and quantification of caveolae rings and tubules to strengthen our claims with patient myotubes.
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eLife assessment
Lemerle et al utilize advanced correlative light and electron microscopy and molecular biology approaches to convincingly demonstrate the presence of the membrane-bending protein Bin1 and caveolae containing rings capable of membrane tubulation in developing muscle. The data is highly significant as it potentially advances our fundamental understanding of how transverse tubules are formed, a significant gap in our understanding of excitation-contraction coupling and muscle biology more broadly.
-
Reviewer #1 (Public Review):
Lemerle et al utilize elegant imaging and molecular biology approaches to convincingly demonstrate the presence of Bin1 and caveolae containing rings capable of tubulation in developing muscle. The data is of fundamental potential significance as it advances our understanding of t-tubule biogenesis, which represents a major knowledge gap in muscle biology. The paper will be of broad interest to skeletal and cardiac muscle biologists and physiologists. The paper is well written, with a comprehensive yet concise introduction, clearly presented results, and an appropriate discussion. The imaging is spectacular, and the use of CLEM provides compelling validation of the protein constituents of ring structures identified via EM. When combined with time-lapse imaging, the combination of approaches provides powerful …
Reviewer #1 (Public Review):
Lemerle et al utilize elegant imaging and molecular biology approaches to convincingly demonstrate the presence of Bin1 and caveolae containing rings capable of tubulation in developing muscle. The data is of fundamental potential significance as it advances our understanding of t-tubule biogenesis, which represents a major knowledge gap in muscle biology. The paper will be of broad interest to skeletal and cardiac muscle biologists and physiologists. The paper is well written, with a comprehensive yet concise introduction, clearly presented results, and an appropriate discussion. The imaging is spectacular, and the use of CLEM provides compelling validation of the protein constituents of ring structures identified via EM. When combined with time-lapse imaging, the combination of approaches provides powerful nanoscale structural information alongside temporal dynamics and live-cell confirmation of tubulating ability by Bin1-Cav3 containing rings. The data indicate that Bin1 is sufficient to generate circular structures that are subsequently decorated by caveolae which facilitate tubule formation at the membrane, and they support the requirement of both Bin1 and Cav3 for efficient tubule initiation and elongation. The authors also utilize myotubes from patients with cav3 mutations to explore whether altered ring formation may contribute to muscle pathology - however, this section requires additional controls and validation to confer pathological insight. Further, additional quantification of imaging data across the study is required to increase the rigor and strength of the conclusions of this work.
-
Reviewer #2 (Public Review):
In this work Lemerle et al. provide long-awaited insight into how transverse tubules develop in skeletal muscle. Together with the sarcoplasmic reticulum transverse tubules form the triad, a specialized structure required for excitation-contraction coupling in skeletal muscle. Defects in transverse tubules or the triad can lead to problems such as muscular dystrophy. Whilst the involvement of specialist membrane structures (caveolae) and the membrane-bending protein Bin1 have long been recognized the precise mechanism of how caveolae and Bin1 cause transverse tubules to form and extend has remained unknown. This work provides compelling evidence, correlating antibody labelling with electron microscopy, to support the concept that caveolae rings form underneath the cell membrane which is surrounded by the …
Reviewer #2 (Public Review):
In this work Lemerle et al. provide long-awaited insight into how transverse tubules develop in skeletal muscle. Together with the sarcoplasmic reticulum transverse tubules form the triad, a specialized structure required for excitation-contraction coupling in skeletal muscle. Defects in transverse tubules or the triad can lead to problems such as muscular dystrophy. Whilst the involvement of specialist membrane structures (caveolae) and the membrane-bending protein Bin1 have long been recognized the precise mechanism of how caveolae and Bin1 cause transverse tubules to form and extend has remained unknown. This work provides compelling evidence, correlating antibody labelling with electron microscopy, to support the concept that caveolae rings form underneath the cell membrane which is surrounded by the endo/sarcoplasmic reticulum. These rings contain caveolin-3 and Bin1 and the authors show Bin1 enriched tubes extend from multiple points on these rings. Their data suggest that Bin1 assembles to initially form these scaffolds that then recruit the caveolae to form the ring. In addition, tubules appear continuous with the extracellular environment which is necessary for their function of facilitating calcium release during excitation-contraction coupling. In patients with mutations in caveolin-3 the caveolin ring formation as well as Bin1 tubulation were defective which may play a role in the pathology. The elegant experiments including time-lapse work clearly support the conclusions of the authors.
The ability of the authors to combine labelling studies with advanced microscopy to show the underlying structures provides very strong evidence for the proposed mechanisms. The authors suggest that the muscle-specific isoforms of BIN1 are key to tubule extension from caveolae rings but it would be interesting for them to discuss how this fits with studies suggesting that constitutive Bin1 isoforms can also form transverse tubules. It would also be interesting to understand the authors' views on whether caveolae rings are involved in the turnover of transverse tubules in adult myotubes as well as the initial formation and, additionally, if the caveolae rings are restricted to the region just under the surface membrane.
Insight into how transverse tubules are formed sets the groundwork for future therapies. This is clearly important for skeletal muscle myopathies but should also be considered in the heart. Cardiac transverse tubule loss and disorder play an important role in dysfunction in heart failure and atrial fibrillation and as such lessons learned in skeletal muscle may be successfully applied to the heart.
-
Reviewer #3 (Public Review):
T-tubules are an elaborate series of membrane invaginations that bring membrane voltage-activated Ca2+ channels in close apposition to the sarcoplasmic reticulum containing RyR, allowing for Ca2+-induced Ca2+ release. They serve as critical hubs of excitation-contraction coupling and play a central role in myopathies and inherited and acquired cardiomyopathies. Several membrane structures and proteins have been implicated in striated muscle t-tubule biogenesis, but the specific mechanisms of early t-tubule biogenesis are not defined.
Lemerle et al here investigate the biogenesis of transverse tubules in skeletal muscle. They use skeletal myoblasts from murine and human muscle as well as sophisticated high-resolution microscopy, live cell imaging, and adenoviral targeting to forward a model of BIN1 mediated …
Reviewer #3 (Public Review):
T-tubules are an elaborate series of membrane invaginations that bring membrane voltage-activated Ca2+ channels in close apposition to the sarcoplasmic reticulum containing RyR, allowing for Ca2+-induced Ca2+ release. They serve as critical hubs of excitation-contraction coupling and play a central role in myopathies and inherited and acquired cardiomyopathies. Several membrane structures and proteins have been implicated in striated muscle t-tubule biogenesis, but the specific mechanisms of early t-tubule biogenesis are not defined.
Lemerle et al here investigate the biogenesis of transverse tubules in skeletal muscle. They use skeletal myoblasts from murine and human muscle as well as sophisticated high-resolution microscopy, live cell imaging, and adenoviral targeting to forward a model of BIN1 mediated caveolae ring formation which give rise to DHPR enriched t-tubules and associate with SR. While they demonstrate that BIN1 and Cav3 enriched caveolae act together to form t-tubules, the precise pathophysiological mechanisms by which this process acts in disease remain unclear.
Strengths of the study consist in the use of both murine and human skeletal muscle experiments, suggesting a conserved molecular mechanism; the innovative approach of correlative light and electron microscopy, and the use of pathological specimens. The live cell timelapse provides imaging evidence of Cav3-enriched caveolae-rings forming in centers of high BIN1 enrichment, from which t-tubules emanate. This is novel evidence in support of the biogenesis model proposed by the authors.
The pathological correlation of their model is promising but limited. Specifically, while the study of Cav3 mutant specimens is used to show the Cav3 dependence of BIN 1 action (in experiments using BIN 1 overload), the authors have not tested the sufficiency of their proposed mechanism by rescuing the pathologic state. Moreover, the conditions of development likely have an important effect on the studied mechanism - such as mechanical loading, contractile state, neurohormonal environment, and so on. Furthermore, a more complete description of the precise molecular binding sites between BIN1 and Cav3 would be important. While exon11 is required for tubulation, BIN1 not expressing exon 11 appears sufficient to assemble caveolar rings, suggesting this is mediated by other specific BIN1 regions.
Overall, the study provides new details on early t-tubule biogenesis in skeletal muscle (likely shared with other striated muscle) and lays the foundations for further definition of the precise molecular mechanisms.
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