The SR represents the main calcium store in striated muscle. It is highly specialized to ensure the simultaneous release of intracellular calcium in the entire cytosol of the muscle cell. The first step of SR biogenesis starts by the formation of tubular endoplasmic reticulum (ER) (30-60 nm in diameter) adjacent to the myofibril 14 . Subsequently, these tubular branches of ER develop into reticular structures surrounding the myofibrils 15 . Finally, the newly formed SR engages couplings at the A-I interface with the T-tubule originating from the sarcolemma. The molecular determinants implicated in the functional and structural organization of the SR have been reviewed elsewhere 16 .

The chronology of SR biogenesis was well investigated using electron microscopy (EM) during muscle differentiation in mouse 17 . These observations were also supported by studies employing chicken embryo 18 . In mouse, the SR is detected from as early as embryonic day 14 (E14) with punctate RyR clusters that are located in the periphery of the myofiber 17 . At this stage, the content of the feet (RyR) in the junctional SR is poor, and some SR elements without any feet are observed. At E16, RyR containing elements become abundant and start to be associated with the edges of A bands (A-I junctions) of the newly formed sarcomeres. This association results in a distinct banding pattern of a discrete SR network at the I-band with thin longitudinal connecting SR elements 17 . During the next days (E17 and E18), junctional SR acquires a predominant transverse distribution taking their final position by forming triad rows at each side of the Z-line (two SR sacs connecting one T-tubule in each triad) 17 . During the maturation of SR membranes, the frequency of feet increases, in particular, between E16 and E18, when all junctions become filled by feet. The width of the junctional gap is between 9 and 12 nm. The maturation of the RyR containing elements is accomplished at birth 17 . Elegant experiments using tagged SR proteins in differentiating myotubes showed that the SR organization was paralleled by a dynamic localization of longitudinal and junctional SR proteins 19 .

T-tubules are invaginations of the plasma membrane, which are present exclusively in striated muscle. Their role is to maintain the SR calcium store under the tight control of membrane depolarization via the voltage sensor channel DHPR 2 . Morphological studies in chicken and mouse embryos have revealed that the T-tubules start their formation after the SR 17 18 . In mouse embryos, the first defined tubules can be observed at E15. At this stage they take the aspect of short cylinders invaginating from the sarcolemma within the myotubes 17 . At E16, the newly formed T-tubules extend deeper within the myofiber, maintaining a connection with the surface by short transverse segments however they stay predominantly longitudinal. During the last days of gestation (E17, 18, 19), T-tubules progressively invade the entire fiber; the majority of them are longitudinal with some transverse connecting elements 17 18 . The transverse orientation of T-tubules is achieved during the postnatal period. Final maturation of T-tubules is completed in mouse 3 weeks after birth 17 20 .

Caveolae are subcompartments of the plasma membrane which take the aspect of 50-100 nm vesicular invaginations, and have an important role in signal transduction and vesicular transport 21 . In contrast to the other plasma membrane regions which are composed mainly of phospholipids, caveolae are considered as cholesterol-sphingolipid rich raft domains 22 . The principal protein components of the caveolae are the caveolins (CAV), which are cholesterol-binding proteins 22 . The caveolin family is represented in mammals by three members; both CAV1 and CAV2 are co-expressed in non-muscle cells especially adipocytes 21 23 whereas CAV3 is found essentially in striated muscles, and its expression is induced during muscle differentiation 24 . In skeletal muscle, CAV3 localizes at the sarcolemma where it can form a complex with dystrophin and its associated glycoproteins 25 . In addition to the sarcolemma, CAV3 is localized to the developing T-tubules 26 .

Mutations within CAV3 are associated with several muscular disorders. In particular, mutations which lead to loss of the CAV3 protein, or a decrease of more than 90% of CAV3 expression, result in autosomal dominant limb-girdle muscular dystrophy (LGMD1C), manifesting by mild to moderate proximal muscle weakness 27 . In addition, CAV3 is found mutated in rippling muscle disease 28 , familial hypertrophic cardiomyopathy 29 and long QT syndrome 9 30 . Moreover, its expression is increased in tibialis anterior from the mdx mouse, suggesting that CAV3 may contribute to the pathogenesis of DMD 31 .

Mice lacking CAV3 display a mild myopathic phenotype similar to the human pathology 32 . Besides, the ectopic expression of CAV3 in mice leads to Duchenne-like muscular dystrophic phenotype 33 . According to its localization at T-tubules, CAV3 deletion leads to disorganization of T-tubule membranes which become dilated and lose their transverse orientation (Figure 3a and 3b) 32 . This provides evidence that CAV3 is crucial for muscle function and has a role in T-tubules biogenesis. Several evidences lead to the hypothesis that similar mechanisms control the formation of the T-tubule system and the caveolae. Indeed, the mature and the developing T-tubules are associated with CAV3 and contain approximately four times more cholesterol than the plasma membrane 34 35 . Moreover, treatment of epithelial cells with Amphotericin B, a cholesterol-binding drug, results in a loss of morphologically defined caveolae at the cell surface. Similarly to CAV1 in epithelial cells, DHPR α and CAV3 are dramatically redistributed after Amphotericin B treatment of C2C12 myotubes. Interestingly, other cholesterol-rich compartments such as the trans-Golgi network do not seem affected 34 .

Mutations in BIN1 are associated to the autosomal recessive form of centronuclear myopathy 36 , a disease characterized by muscle weakness, myofiber atrophy, and abnormal positioning of nuclei within muscle fibers.

Amphiphysins are membrane bending and curvature sensing proteins able to tubulate lipid membranes via their BAR (Bin/Amphiphysin/Rvs) domain. The ubiquitous amphiphysin 2, encoded by the BIN1 gene, is highly expressed in skeletal muscle and is proposed to participate in T-tubule biogenesis. Its role in this process is provided by a polybasic amino-acid stretch encoded by exon 11, which is important for its recruitment to T-tubule membranes 37 38 . This polybasic sequence can bind specifically the phosphoinositides PtdIns(4,5)P ₂ and PtdIns4P in vitro. Interestingly, the levels of PtdIns(4,5)P ₂ and BIN1 increase simultaneously during C2C12 myotubes differentiation 37 39 .

In addition, BIN1 can tubulate membranes separately or in cooperation with dynamin 2 (DNM2), another protein mutated in centronuclear myopathy 40 41 . The cooperation between BIN1 and DNM2 is mediated by the interaction of BIN1 SH3 domain with the proline rich domain of DNM2. However, this interaction may not occur prior to BIN1 association to membranes, as the polybasic sequence binds to the SH3 domain when it is not membrane bound 42 . Indeed, PtdIns(4,5)P ₂ binding is necessary to release the SH3 domain, further enabling the interaction between the SH3 domain and DNM2 42 . While the existence of this intramolecular regulation has been deciphered in cultured cells, it is not determined whether a similar mechanism regulates T-tubules curvature. Nevertheless, myotubes expressing a BIN1 mutant lacking the polybasic sequence failed to form a normal membrane tubules network 42 .

A drosophila mutant lacking the orthologue of BIN1 (amphiphysin) exhibits a skeletal muscle defect associated to alterations in T-tubule morphology and EC coupling (Figure 3c-f) 43 . Interestingly, EM analysis of muscle biopsies from patients with BIN1 mutations revealed abnormal morphology of T-tubules 44 . In cultured COS-1 cells, overexpression of a mutation related to CNM in the BAR domain failed to form membrane tubules compared to the wild-type construct, suggesting that the lack of BIN1-mediated membrane remodeling could be at the basis of the muscle disease 36 .

Surprisingly, no skeletal muscle defect was reported in Bin1 ^-/- mice. However, these mice are dying perinatally due to cardiomyopathy, precluding a detailed analysis of skeletal muscle after birth 45 .

Dysferlin (DYSF) is a 230 kDa protein belonging to a family of genes similar to Caenorhabditis elegans ferlin. It contains a C-terminal transmembrane domain and multiple C2 domains implicated in calcium binding and calcium-dependent membrane fusion and repair. Mutations within the DYSF gene are associated with allelic muscular disorders including autosomal recessive limb-girdle muscular dystrophy type 2B 46 , Miyoshi myopathy 47 , and distal anterior compartment myopathy 48 . DYSF has a sarcolemmal localization in differentiated skeletal muscle, which is related to its role in membrane repair 49 . However, studies performed in C2C12 cells have shown that during myotubes differentiation, DYSF is associated to the T-tubules network in addition to sites of cells fusion, and can translocate to the sarcolemma upon myofiber injury 50 . Interestingly, studies performed in adult rat muscles, in which regeneration was induced by subcutaneous injection of notexin, have revealed that during early stage of muscle fiber regeneration (within the first week after notexin treatment), DYSF is mainly localized to T-tubules and translocates toward the sarcolemma in later stages of regeneration 51 .

Several mouse lines have been generated to manipulate the level and the function of DYSF, and the spontaneous SJL strain was shown to encompass an in-frame deletion in the C-terminal of the Dysf gene 49 52 53 54 55 . Similarly to CAV3 mouse mutants, mice deficient for DYSF display alterations in T-tubule structure, with more dilated and longitudinally oriented tubules 51 (Figure 3m-n). These defects are considered as primary, as they are observed at early stage of the disease when abnormalities in the myofibrillar architecture and the sarcolemma are minimal. The role of DYSF in T-tubule biogenesis is still not determined, however, it has been suggested that DYSF contributes to the fusion of caveolin 3 containing vesicles with T-tubules. This suggestion is based on i) the interaction of DYSF with DHPR in mature skeletal muscle, ii) the known interaction between DYSF and CAV3, and iii) the partial co-localization between CAV3 and DYSF during early myogenesis 56 57 . This hypothesis is also supported by the accumulation of subsarcolemmal vacuoles contiguous with the T-tubule system in dysferlinopathy patients 58 .

In addition to the proteins mentioned above, which are involved in the biogenesis of T-tubules, other proteins are implicated in the maturation of SR terminal cisternae, and the junction between T-tubules and SR. This is the case of members of the Synaptophysin family such as mitsugumin 29 (MG29 or synaptophysin-like 2, SYPL2), a transmembrane proteins highly enriched in heavy SR vesicles preparation 59 . MG29 is expressed early during myogenesis, even before the apparition of triads. It first associates to newly formed SR vesicles and then to triads, which appear later during myogenesis 59 . These observations implicate MG29 in the early formation of junctional SR and its connection to T-tubules 59 60 . In MG29-KO mice, decreased muscle mass and a slight decrease in the force generation capacity were observed 61 . Dysfunction of store-operated calcium entry (SOCE) leading to defects in intracellular calcium homeostasis, and increased muscle fatigability were also reported in these mice 62 63 . EM analysis of mutant muscles revealed morphological alterations in triad structures, including a swollen SR and longitudinal T-tubules (Figure 3g-j) 61 . However, the actual association between SR and T-tubules does not appear altered. It thus remains unclear whether such disorders in the triad structure have a link with the observed defect in SOCE.

Another mitsugumin, MG53 (also called TRIM72), has been identified as a key player in intracellular membrane trafficking and membrane repair machinery in striated muscles 64 65 . In addition to it specific expression in striated muscles, MG53 was shown to bind to dysferlin and caveolin 3, two proteins directly implicated in T-tubule biogenesis 64 66 . There are no studies demonstrating a direct implication of MG53 in the biogenesis of triad membranes; however, the current evidences sustain its implication as a potential new partner in this mechanism.

JPH family members are identified as components of junctional membranes, where they may bridge the SR via their C-terminal transmembrane domain, with the T-tubule/plasma membrane via their N-terminal domain. More specifically, the N-terminal domain can bind to membrane phospholipids including sphingomyelin and phosphatidylcholine 67 . Among the four junctophilin-like proteins in mammals (JPH1-4), JPH1 is expressed mainly in skeletal muscle while JPH2 is also expressed in cardiac muscle and implicated in hypertrophic cardiomyopathies 67 68 69 . However, JPH3 and 4 are coexpressed in brain where JPH3 is found to be associated to a Huntington-like disease 70 71 .

JPH1 deficient mice die shortly after birth due to defects in jaw muscles resulting in lack of milk suckling 72 . Electron microscopy analysis of skeletal muscle from embryos and newborn mice revealed several abnormalities in triad morphology, leading to defect in muscle contraction 72 73 . These anomalies include a reduced number of triads, swollen junctional SR, partially vacuolated longitudinal SR and irregular orientation of SR network (Figure 3k-l). Moreover, functional analysis of mutant muscles from neonate mice revealed that these muscles have an increased response to extracellular calcium stimuli, indicating a defect in EC coupling 72 . A specific role of JPH1 in the biogenesis of membrane junctions is also supported by its ability to induce junctions between the endoplasmic reticulum and the plasma membrane when overexpressed in amphibian embryonic cells 67 . In addition, the expression of JPH1 is enhanced after birth as an indicator of a role in the late biogenesis and maturation of triads 72 .

Myotubularin (MTM1) is part of a family of phosphoinositides phosphatases conserved through evolution down to yeast, and with 14 members in human 74 . While myotubularin is ubiquitously expressed, mutations within MTM1 lead to a skeletal muscle disorder: the X-linked form of centronuclear myopathy, also called myotubular myopathy, associating severe muscle atrophy and weakness at birth with abnormal positioning of nuclei 75 . This indicates that MTM1 has a muscle-specific role, which cannot be compensated by homologous proteins. In vitro and overexpression studies performed in yeast and mammalian cells have attributed to MTM1 a function in endosomal and membrane trafficking pathways and have shown that MTM1 specifically dephosphorylates PtdIns3P and PtdInsp(3,5)P ₂ into phosphatidylinositol and PtdIns5P respectively 76 77 78 79 . Recently, immunohistology and EM analysis revealed that AAV mediated overexpression of MTM1 in mouse muscle results in the formation of abnormal membrane structures 80 . These structures include vacuoles that are derived from sarcolemma and/or T-tubules, as they have positive staining for caveolin-3, dystrophin and dihydropyridine receptor (DHPR), and negative staining for laminin 2, and also contain the exogenous MTM1 protein 80 . This indicates a direct or indirect role for MTM1 in the generation or maintenance of membrane structures in skeletal muscle.

Mtm1 KO mice exhibit a progressive centronuclear myopathy 81 . EM analysis of Mtm1 KO muscles revealed alterations in T-tubules structure characterized by longitudinal orientation of T-tubules and the presence of triads deprived from T-tubule components (Figure 3o-p) 82 . These alterations become more pronounced with disease progression 44 82 . Similarly, mtm1 zebrafish morphants and mtm-depleted drosophila muscles display structural defects of the triads, and such defects have been also observed in muscles from patients with myotubular myopathy 44 83 84 . Since MTM1 expression is increased in the postnatal life in mouse and Mtm1 KO muscles display less altered T-tubules at an early stage than at a late stage of the disease, it is likely that MTM1 plays a key role in the late maturation and/or the maintenance of T-tubules rather than in their early biogenesis.

The RyR-DHPR interaction is physically linking the SR to T-tubules in skeletal muscle, and thus mediates the translation of the action potential into intracellular calcium release. RyR1, the skeletal muscle ryanodine receptor, is implicated in the susceptibility to malignant hyperthermia 85 86 , and mutated in myopathies with variable histological outcomes as central core 87 88 , multi-minicore 89 , congenital fiber type disproportion 90 91 , and/or nuclei centralization 92 93 . It was thought that the direct interaction between RyR and DHPR is necessary for T-tubule and SR assembly 94 . The concomitant expression of RyR and DHPR during myogenesis is consistent with this idea 94 . However, it has been shown by immunofluorescence and EM studies performed in mouse models lacking one or both proteins that it is unlikely to be the case. More specifically, mutant mice lacking RyR (dyspedic) or DHPR (muscular dysgenesis, mdg mouse), or even both proteins form triadic junctions with a similar architecture than wild-type mice 95 96 97 98 99 . Moreover, these myotubes have a normal disposition of other SR components such as calsequestrin and triadin, although calcium release from intracellular stores is greatly affected 96 100 . This indicates that neither RyR nor DHPR are necessary for the biogenesis of the triad structure.

In addition, the targeting of DHPR and RyR to their respective membrane is independent of each other 18 99 . This suggests that the T-tubule and SR separately posses the potential for self-assembly.