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Sarcoplasmic reticulum: Structural determinants and protein dynamics
The International Journal of Biochemistry & Cell Biology 43 (2011) 1075–1078
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Organelles in focus
Sarcoplasmic reticulum: Structural determinants and protein dynamics Vincenzo Sorrentino∗ Molecular Medicine Section, Department of Neuroscience and Interuniversity Institute of Myology (IIM), University of Siena, via A. Moro, 53100 Siena, Italy
a r t i c l e
i n f o
Article history: Received 18 December 2010 Received in revised form 21 March 2011 Accepted 8 April 2011 Available online 14 April 2011 Keywords: Sarcoplasmic reticulum Excitation–contraction coupling Striated muscle Calcium signalling Muscle cell biology
a b s t r a c t The sarcoplasmic reticulum is a unique organelle found in muscle cells that is dedicated to the regulation of Ca2+ homeostasis and activation of myofilament contraction. The functional requirement for an efficient and synchronous activation of Ca2+ release from the SR, following the depolarization of the plasma membrane, accounts for the complex three-dimensional organization of internal membranes observed in muscle cells and for the localization of proteins at specific sites of the SR. Recent advancements in understanding the molecular basis of SR structure and function have greatly increased our understanding of muscle cellular physiology and biology. Parallel work has revealed that several human diseases affecting skeletal and cardiac tissues are linked to either mutations or altered post-translational modifications of SR proteins. © 2011 Elsevier Ltd. All rights reserved.
Organelle facts • The SR is a dynamic Ca2+ storage organelle found in muscle cells. • The SR maintains specialized associations with the myofibrils and T tubules. • The longitudinal SR is dedicated to Ca2+ uptake. • The junctional SR is the site specialized for Ca2+ release. • Mutations in SR proteins are associated with several human diseases.
1. Introduction Muscle cells contain a unique form of endoplasmic reticulum called sarcoplasmic reticulum (SR) that is specialized in the regulation of Ca2+ homeostasis and control of muscle contraction (Kaisto and Metsikkö, 2003; Franzini-Armstrong, 2004; Rossi et al., 2008). In order to efficiently release Ca2+ close to the contractile apparatus, the SR is organized in a complex three-dimensional network that encircles each of the thousands of myofibrils that pack muscle cells. In this elaborate arrangement, the SR is accompanied by a second system of internal membranes formed by thousands of narrow tubules, called T tubules, which are continuous with
∗ Tel.: +39 0577 234 079; fax: +39 0577 234 191. E-mail address: [email protected] 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.04.004
the sarcolemma from where they spread into the cytoplasm of muscle cells. T tubules also surround each single myofibril with a very precise pattern whereby each T tubule is positioned close to the enlargements of the SR network called terminal cisternae (Franzini-Armstrong, 2004). This complex organization is required to provide the structural basis for the efficient transduction of the depolarization-induced signal from the plasma membrane to the SR to achieve synchronous activation of Ca2+ release in cells that may be up to several cm long and hundreds of m thick (Rios et al., 1991). In this review we will mainly address the molecular aspects underlying the organization of the SR with respect to myofibrils and T tubules. SR Ca2+ regulatory proteins and Ca2+ signalling in muscle cells will be only partially discussed here, but are the focus of excellent reviews (Franzini-Armstrong and Protasi, 1997; Rossi and Dirksen, 2006; Endo M, 2009).
2. SR function The SR appears as a network of tubules and cisternae that surrounds the myofibrils. The vast majority of SR tubules form the so-called longitudinal SR (L-SR), that covers most of the length of the sarcomere and regularly merge into larger membrane structures, known as terminal cisternae. The L-SR tubules and terminal cisternae are organized in a highly stereotyped pattern that is aligned with the underlying sarcomere (Fig. 1B). Regarding the organization of the SR, but also of other structures, skeletal and cardiac muscles may present several subtle differences: in this review, unless specified, we shall refer to skeletal muscle. In skeletal muscles, two terminal cisternae positioned on the opposite sides of one T tubule form the so-called “triad” (Franzini-Armstrong,
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Fig. 1. (A) Proteins of the SR are localized in distinct regions of the skeletal muscle fibers. Longitudinal cryosections of skeletal muscle fibers were stained with antibodies against SERCA, ank1.5, RyRs, JP1, DHPR and ␣-actinin. As shown, SERCA staining is mainly co-localized with ␣-actinin, which identifies the Z-disk region of the sarcomere. On the contrary, ank1.5 yields a strong band that alternates with ␣-actinin, in a staining compatible with M-band localization. Staining for RyRs identifies a distinct region, corresponding to the border between the A–I band, which also stains for all j-SR proteins (like JP1) and for the DPHR on T tubule. It should be mentioned that, at the level of resolution of confocal microscope, each of the single bands observed when staining for jSR proteins corresponds to a single triad, i.e. corresponds to two terminal cisternae. (B) Schematic representation of SR and myofilaments in a mammalian skeletal muscle fiber: longitudinal SR (L-SR) tubules, Terminal cisternae (TC) and T tubules (TT) are reported in parallel to specific sarcomere regions to indicate their relative localization. Bar = 5 m.
2004). The membrane of the terminal cisternae facing the T tubule is called the junctional SR (jSR). The jSR is the site where the Ca2+ release channels, also called ryanodine receptors (RyRs), and other SR proteins, like triadin, junctin and calsequestrin are localized (Franzini-Armstrong, 2004). Specialized voltage-sensitive Ca2+ channels, the dihydropyridine receptors (DHPRs), are located in the region of the T tubule membrane facing the jSR. Following the depolarization of the plasmamembrane, activation of DHPRs on the T tubule triggers the opening of the RyRs on the SR. Activation of RyRs releases massive amounts of Ca2+ from the SR that start muscle con-
traction, hence the entire process is called excitation–contraction (E–C) coupling (Rios et al., 1991; Lanner et al., 2010). Maintenance of Ca2+ homeostasis requires that following activation of Ca2+ release, Ca2+ is re-uptaken again and stored into the SR until the next events of release occur. A large number of proteins participate in these events; among them, we shall only mention the most known ones. After each cycle of Ca2+ release, the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) pumps lead to muscle relaxation, by restoring low levels of resting Ca2+ in the cytosol (Rossi and Dirksen, 2006). Storage of Ca2+ is facilitated by
V. Sorrentino / The International Journal of Biochemistry & Cell Biology 43 (2011) 1075–1078
the presence in the SR lumen of a high number of Ca2+ -binding proteins, including calsequestrin, sarcalumenin, histidine-rich protein and junctate (Franzini-Armstrong and Protasi, 1997; Lanner et al., 2010). Calsequestrin is the most abundant Ca2+ -binding protein of the SR. In the presence of high concentrations of Ca2+ , Calsequestrin forms large polymers localized near the jSR membrane of the terminal cisternae, where it assembles through its interaction with triadin and junctin (Franzini-Armstrong and Protasi, 1997). Since triadin and junctin can also interact with the RyRs, it has been proposed that these two proteins provide a bridge that keeps high concentrations of Calsequestrin, and hence of Ca2+ , close to the Ca2+ release channels (Györke et al., 2004; Goonasekera et al., 2007).
3. SR physiology 3.1. Assembly of the SR around myofibrils and organization of proteins in the longitudinal SR In spite of the evident high level of complexity of the threedimensional organization of the SR, only recently, two molecules, ank1.5 and obscurin, that appear to be involved in the association of the SR with the myofibrils, have been identified. Ank1.5 is a small muscle-specific isoform of ankyrin1 (Bagnato et al., 2003; Kontrogianni et al., 2003) that is situated on the SR. It has been demonstrated that ank1.5 can bind with its cytosolic tail the Cterminal region of obscurin, a large protein on the myofilaments (Young et al., 2001). In adult skeletal muscle, obscurin is localized at the M-band of the sarcomere. Interestingly, ank1.5 is found in the region of the L-SR that surrounds the M-band and this localization depends on ank1.5 ability to bind to obscurin (Bagnato et al., 2003; Cusimano et al., 2009). Mice knockout for the obscurin gene have normal sarcomeric structure and function but present signs of mild myopathy, altered localization of ank1.5, and a reduced amount of longitudinal SR around the myofibrils (Lange et al., 2009). Ank1.5 knockout mice have a reduced contractile performance accompanied by alterations in longitudinal SR structure and in the myofibrillar apparatus (Sorrentino et al., unpublished). Although the complete puzzle is not yet solved, we can envision a scheme where SR organization results from a process of membrane assembly and restructuring that ends in the formation of a network of tubules that is held close to the myofibrils thanks to specific contacts established between ank1.5 on the L-SR and obscurin at the M-band (Flucher et al., 1993; Giacomello and Sorrentino, 2009). In skeletal muscle fibers, the L-SR covers most of the A and I bands of the sarcomere, with a differential density of membranes that is higher in correspondence of the Z-disk (Fig. 1B). Accordingly, immunostaining of L-SR proteins with specific antibodies and analysis by fluorescence or confocal microscopes consistently yields a stronger signal in the region of the SR that overlaps the Z-disk, and a much weaker signal in correspondence of the M-band (Salanova et al., 2002). This contrasts with the preferential localization of ank1.5 at the M band (compare SERCA and ank1.5 signals in Fig. 1A). The dynamic properties of L-SR proteins have been further analysed by the Fluorescence Recovery After Photobleaching (FRAP) technique. These studies have shown that SERCA pumps are highly mobile while ank1.5 displays a relatively lower mobility within the L-SR membrane (Cusimano et al., 2009). The reduced mobility of ank1.5 appears to depend on its ability to bind to obscurin, since deletion of the obscurin binding site in ank1.5 resulted in an increased mobility and in re-localization of the mutant protein to the Z-disk (Cusimano et al., 2009). These data thus suggest that proteins that are free to move in the L-SR membrane, like SERCA or mutant ank1.5, are mainly observed around the Z-disk probably because of the higher density of SR membranes in this region, while alternative localizations, such as that of ank1.5 at
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the M-band, seem to be dependent on an active retention mechanism. 3.2. Organization of the jSR and of triads In mammalian skeletal muscle fibers, terminal cisternae and T-tubules are located at the junctions between A–I bands (FranziniArmstrong, 2004). The assembly of triad junctions has been demonstrated to be mediated by Junctophilins (JPs), a family of SR transmembrane proteins that extend from the surface of the SR to the sarcolemma and that play a central role in holding together these two membrane compartments (Ito et al., 2001). Overexpression of JPs cause the SR to move close to the plasmamembrane, while abrogation of the expression of JP1 and JP2, by means of gene targeting techniques, reduces the establishment of couplings between SR and T tubules in knockout mice (Takeshima et al., 2000). Several types of evidence indicate that jSR proteins are assembled in a stable macromolecular complex of high molecular mass organized around the RyR Ca2+ -release channels (Lanner et al., 2010). Interestingly, since JP1 has been shown to interact with RyR1 (Phimister et al., 2007) and RyR1 on its own can interact with other jSR proteins like including triadin and DHPR, it appears possible that the junctional SR proteins may be recruited at triads by sequential events mediated by protein-protein interaction (Lanner et al., 2010). This is also in agreement with data from FRAP-based experiments that indicated that junctional proteins, like JP1, RyR1, triadin and DHPR, all displayed a progressive reduction in their mobility following the assembly into junctional domains (Cusimano et al., 2009). However, although we can draw a working model for assembly of SR domains and for protein localization in these areas, we still have no clue about exactly how triads are positioned at the A–I border in skeletal muscle. Experiments following embryonic development and in vitro differentiation of skeletal muscle cells, have shown that DHPRs and RyRs co-localize and become functionally coupled a few days before triads are formed and aligned with the A–I band border (Flucher et al., 1993; Takekura et al., 2001; Cusimano et al., 2009). This means that the organization of mature triads follows the organization of junctional proteins and that the final localization of triads at the A–I border may require the establishment of additional interactions with the contractile apparatus. The identity of the proteins responsible for the positioning of triads in correspondence to specific regions of the sarcomere remains an important, yet unanswered, question. 4. SR in human pathology Mutations in SR proteins involved in Ca2+ homeostasis have been detected in several human diseases affecting skeletal and cardiac muscle tissues. Mutations in the RYR1 gene have been found in patients predisposed to Malignant Hyperthermia (MH), a life-threatening crisis commonly triggered by exposure to common anaesthetics or exertional/environmental heat stroke, and in patients with central core disease (CCD), a rare myopathy with hypotonia and proximal muscle weakness. Identification of a subset of MH patients with mutations in the DHPR but not in the RYR1 gene, clearly points to MH as a dysfunction of the skeletal muscle e–c coupling machinery (Betzenhauser and Marks, 2010). Mutations in either RYR2 or Calsequestrin2 genes have been found in patients with severe stress-induced ventricular tachycardia, where an aberrant Ca2+ release from the SR during diastole triggers ventricular arrhythmia resulting in cardiac arrest and sudden death (Györke et al., 2009; Kushnir and Marks, 2010). Mutations in the SERCA1a gene, resulting in a slow-down in the reuptake of Ca2+ from the cytosol with delayed relaxation and skeletal muscle
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cramping, have been linked to Brody’s disease (Brini and Carafoli, 2009). In addition to genetic diseases, multiple evidence indicates that -adrenergic stimulation regulates the activity of several SR proteins, including SERCA and RyRs. Among these, a direct phosphorylation of RyR2 by PKA, while playing a positive effect on cardiac performance in healthy individuals, seems to contribute to further deterioration in the cardiac performance in failing heart (Wehrens et al., 2003; Shan et al., 2010). Such studies have opened the way to search for new therapeutic approaches to treating cardiac diseases (Watanabe et al., 2009; Kushnir and Marks, 2010). In conclusion, the identification of mutations in the obscurin-binding domain of titin suggests that alterations in proteins involved in the organization of the SR may also cause muscular diseases (Fukuzawa et al., 2008). 5. Future outlook The past years have seen a major advancement in our understanding of the role of SR proteins involved in regulation of Ca2+ signalling in both physiological and pathological conditions. More recently, new data have started to describe how the threedimensional network of SR membranes is organized with respect to T tubules and to the myofilaments. However, many details are still missing. Identification of additional proteins responsible for the organization of the three-dimensional arrangement of the SR and T tubules, and of proteins, either present in the contractile apparatus or the extra-sarcomeric cytoskeleton, that organize the stereotyped pattern of triads at the A–I border may provide novel information in muscle physiology. Acknowledgements I thank Daniela Rossi and Emiliana Giacomello for comments on the manuscript and for help with Fig. 1. This work was supported in part by a grant from Telethon (GGP08153). References Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V. Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles. J Cell Biol 2003;160:245–53. Betzenhauser MJ, Marks AR. Ryanodine receptor channelopathies. Pflugers Arch 2010;460:467–80. Brini M, Carafoli E. Calcium pumps in health and disease. Physiol Rev 2009;89:1341–78. Cusimano V, Pampinella F, Giacomello E, Sorrentino V. Assembly and dynamics of proteins of the longitudinal and junctional sarcoplasmic reticulum in skeletal muscle cells. Proc Natl Acad Sci U S A 2009;106:4695–700. Endo M. Calcium induced calcium release in skeletal muscle. Physiol Rev 2009;89:1153–76. Flucher BE, Takekura H, Franzini-Armstrong C. Development of the excitation–contraction coupling apparatus in skeletal muscle: association of sarcoplasmic reticulum and transverse tubules with myofibrils. Dev Biol 1993;160:135–47.
Franzini-Armstrong C. The membrane systems of muscle cells. In: Myology. Mc Graw-Hill; 2004. pp. 232–256. Franzini-Armstrong C, Protasi F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol Rev 1997;77:699–729. Fukuzawa A, Lange S, Holt M, Vihola A, Carmignac V, Ferreiro A, et al. Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band – implications for hereditary myopathies. J Cell Sci 2008;121:1841–51. Giacomello E, Sorrentino V. Localization of ank1.5 in the sarcoplasmic reticulum precedes that of SERCA and RyR: relationship with the organization of obscurin in developing sarcomeres. Histochem Cell Biol 2009;131:371–82. Goonasekera SA, Beard NA, Groom L, Kimura T, Lyfenko AD, Rosenfeld A, et al. Triadin binding to the C-terminal luminal loop of the ryanodine receptor is important for skeletal muscle excitation contraction coupling. J Gen Physiol 2007;130: 365–78. Györke I, Hester N, Jones LR, Györke S. The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium. Biophys J 2004;86:2121–8. Györke S. Molecular basis of catecholaminergic polymorphic ventricular tachycardia. Heart Rhythm 2009;6:123–39. Ito K, Komazaki S, Sasamoto K, Yoshida M, Nishi M, Kitamura K, et al. Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1. Cell Biol 2001;154:1059–67. Kaisto T, Metsikkö K. Distribution of the endoplasmic reticulum and its relationship with the sarcoplasmic reticulum in skeletal myofibers. Exp Cell Res 2003;289:47–57. Kontrogianni-Konstantopoulos A, Jones EM, Van Rossum DB, Bloch RJ. Obscurin is a ligand for small ankyrin 1 in skeletal muscle. Mol Biol Cell 2003;14:1138–48. Kushnir A, Marks AR. The ryanodine receptor in cardiac physiology and disease. Adv Pharmacol 2010;259:1–30. Lange S, Ouyang K, Meyer G, Cui L, Cheng H, Lieber RL, et al. Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum. J Cell Sci 2009;122:2640–50. Lanner JT, Georgiou DK, Joshi AD, Hamilton SL. Ryanodine receptors: structure, expression, molecular details, and function in calcium release. Cold Spring Harb Perspect Biol 2010;11:a003996, Epub 2010 Oct 20. Phimister AJ, et al. Conformation-dependent stability of junctophilin 1 (JP1) and ryanodine receptor type 1 (RyR1) channel complex is mediated by their hyperreactive thiols. J Biol Chem 2007;282:8667–77. Rios E, Ma JJ, González A. A The mechanical hypothesis of excitation–contraction (EC) coupling in skeletal muscle. J Muscle Res Cell Motil 1991;12:127–35. Rossi AE, Dirksen RT. Sarcoplasmic reticulum: the dynamic calcium governor of muscle. Muscle Nerve 2006;33:715–31. Rossi D, Barone V, Giacomello E, Cusimano V, Sorrentino V. The sarcoplasmic reticulum: an organized patchwork of specialized domains. Traffic 2008;9: 1044–9. Salanova M, Priori G, Barone V, Intravaia E, Flucher B, Ciruela F, et al. Homer proteins and InsP3 receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres. Cell Calcium 2002;32:193–200. Shan J, Betzenhauser MJ, Kushnir A, Reiken S, Meli AC, Wronska A, et al. Role of chronic ryanodine receptor phosphorylation in heart failure and -adrenergic receptor blockade in mice. J Clin Invest 2010;120:4375–87. Takekura H, Flucher BE, Franzini-Armstrong C. Sequential docking, molecular differentiation, and positioning of T-Tubule/SR junctions in developing mouse skeletal muscle. Dev Biol 2001;239:204–14. Takeshima H, Komazaki S, Nishi M, Iino M, Kangawa K. Junctophilins: a novel family of junctional membrane complex proteins. Mol Cell 2000;6:11–22. Young P, Ehler E, Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol 2001;154:123–36. Watanabe H, Chopra N, Laver D, Hwang HS, Davies SS, Roach DE, et al. Flecanide prevents catecholaminergic polymorphic ventricular tachycardia in mice and humans. Nat Med 2009;15:380–3. Wehrens XH, Lehnart SE, Huang F, Vest JA, Reiken SR, Mohler PJ, et al. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell 2003;113: 829–40.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Organelles in focus
Sarcoplasmic reticulum: Structural determinants and protein dynamics Vincenzo Sorrentino∗ Molecular Medicine Section, Department of Neuroscience and Interuniversity Institute of Myology (IIM), University of Siena, via A. Moro, 53100 Siena, Italy
a r t i c l e
i n f o
Article history: Received 18 December 2010 Received in revised form 21 March 2011 Accepted 8 April 2011 Available online 14 April 2011 Keywords: Sarcoplasmic reticulum Excitation–contraction coupling Striated muscle Calcium signalling Muscle cell biology
a b s t r a c t The sarcoplasmic reticulum is a unique organelle found in muscle cells that is dedicated to the regulation of Ca2+ homeostasis and activation of myofilament contraction. The functional requirement for an efficient and synchronous activation of Ca2+ release from the SR, following the depolarization of the plasma membrane, accounts for the complex three-dimensional organization of internal membranes observed in muscle cells and for the localization of proteins at specific sites of the SR. Recent advancements in understanding the molecular basis of SR structure and function have greatly increased our understanding of muscle cellular physiology and biology. Parallel work has revealed that several human diseases affecting skeletal and cardiac tissues are linked to either mutations or altered post-translational modifications of SR proteins. © 2011 Elsevier Ltd. All rights reserved.
Organelle facts • The SR is a dynamic Ca2+ storage organelle found in muscle cells. • The SR maintains specialized associations with the myofibrils and T tubules. • The longitudinal SR is dedicated to Ca2+ uptake. • The junctional SR is the site specialized for Ca2+ release. • Mutations in SR proteins are associated with several human diseases.
1. Introduction Muscle cells contain a unique form of endoplasmic reticulum called sarcoplasmic reticulum (SR) that is specialized in the regulation of Ca2+ homeostasis and control of muscle contraction (Kaisto and Metsikkö, 2003; Franzini-Armstrong, 2004; Rossi et al., 2008). In order to efficiently release Ca2+ close to the contractile apparatus, the SR is organized in a complex three-dimensional network that encircles each of the thousands of myofibrils that pack muscle cells. In this elaborate arrangement, the SR is accompanied by a second system of internal membranes formed by thousands of narrow tubules, called T tubules, which are continuous with
∗ Tel.: +39 0577 234 079; fax: +39 0577 234 191. E-mail address: [email protected] 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.04.004
the sarcolemma from where they spread into the cytoplasm of muscle cells. T tubules also surround each single myofibril with a very precise pattern whereby each T tubule is positioned close to the enlargements of the SR network called terminal cisternae (Franzini-Armstrong, 2004). This complex organization is required to provide the structural basis for the efficient transduction of the depolarization-induced signal from the plasma membrane to the SR to achieve synchronous activation of Ca2+ release in cells that may be up to several cm long and hundreds of m thick (Rios et al., 1991). In this review we will mainly address the molecular aspects underlying the organization of the SR with respect to myofibrils and T tubules. SR Ca2+ regulatory proteins and Ca2+ signalling in muscle cells will be only partially discussed here, but are the focus of excellent reviews (Franzini-Armstrong and Protasi, 1997; Rossi and Dirksen, 2006; Endo M, 2009).
2. SR function The SR appears as a network of tubules and cisternae that surrounds the myofibrils. The vast majority of SR tubules form the so-called longitudinal SR (L-SR), that covers most of the length of the sarcomere and regularly merge into larger membrane structures, known as terminal cisternae. The L-SR tubules and terminal cisternae are organized in a highly stereotyped pattern that is aligned with the underlying sarcomere (Fig. 1B). Regarding the organization of the SR, but also of other structures, skeletal and cardiac muscles may present several subtle differences: in this review, unless specified, we shall refer to skeletal muscle. In skeletal muscles, two terminal cisternae positioned on the opposite sides of one T tubule form the so-called “triad” (Franzini-Armstrong,
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Fig. 1. (A) Proteins of the SR are localized in distinct regions of the skeletal muscle fibers. Longitudinal cryosections of skeletal muscle fibers were stained with antibodies against SERCA, ank1.5, RyRs, JP1, DHPR and ␣-actinin. As shown, SERCA staining is mainly co-localized with ␣-actinin, which identifies the Z-disk region of the sarcomere. On the contrary, ank1.5 yields a strong band that alternates with ␣-actinin, in a staining compatible with M-band localization. Staining for RyRs identifies a distinct region, corresponding to the border between the A–I band, which also stains for all j-SR proteins (like JP1) and for the DPHR on T tubule. It should be mentioned that, at the level of resolution of confocal microscope, each of the single bands observed when staining for jSR proteins corresponds to a single triad, i.e. corresponds to two terminal cisternae. (B) Schematic representation of SR and myofilaments in a mammalian skeletal muscle fiber: longitudinal SR (L-SR) tubules, Terminal cisternae (TC) and T tubules (TT) are reported in parallel to specific sarcomere regions to indicate their relative localization. Bar = 5 m.
2004). The membrane of the terminal cisternae facing the T tubule is called the junctional SR (jSR). The jSR is the site where the Ca2+ release channels, also called ryanodine receptors (RyRs), and other SR proteins, like triadin, junctin and calsequestrin are localized (Franzini-Armstrong, 2004). Specialized voltage-sensitive Ca2+ channels, the dihydropyridine receptors (DHPRs), are located in the region of the T tubule membrane facing the jSR. Following the depolarization of the plasmamembrane, activation of DHPRs on the T tubule triggers the opening of the RyRs on the SR. Activation of RyRs releases massive amounts of Ca2+ from the SR that start muscle con-
traction, hence the entire process is called excitation–contraction (E–C) coupling (Rios et al., 1991; Lanner et al., 2010). Maintenance of Ca2+ homeostasis requires that following activation of Ca2+ release, Ca2+ is re-uptaken again and stored into the SR until the next events of release occur. A large number of proteins participate in these events; among them, we shall only mention the most known ones. After each cycle of Ca2+ release, the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) pumps lead to muscle relaxation, by restoring low levels of resting Ca2+ in the cytosol (Rossi and Dirksen, 2006). Storage of Ca2+ is facilitated by
V. Sorrentino / The International Journal of Biochemistry & Cell Biology 43 (2011) 1075–1078
the presence in the SR lumen of a high number of Ca2+ -binding proteins, including calsequestrin, sarcalumenin, histidine-rich protein and junctate (Franzini-Armstrong and Protasi, 1997; Lanner et al., 2010). Calsequestrin is the most abundant Ca2+ -binding protein of the SR. In the presence of high concentrations of Ca2+ , Calsequestrin forms large polymers localized near the jSR membrane of the terminal cisternae, where it assembles through its interaction with triadin and junctin (Franzini-Armstrong and Protasi, 1997). Since triadin and junctin can also interact with the RyRs, it has been proposed that these two proteins provide a bridge that keeps high concentrations of Calsequestrin, and hence of Ca2+ , close to the Ca2+ release channels (Györke et al., 2004; Goonasekera et al., 2007).
3. SR physiology 3.1. Assembly of the SR around myofibrils and organization of proteins in the longitudinal SR In spite of the evident high level of complexity of the threedimensional organization of the SR, only recently, two molecules, ank1.5 and obscurin, that appear to be involved in the association of the SR with the myofibrils, have been identified. Ank1.5 is a small muscle-specific isoform of ankyrin1 (Bagnato et al., 2003; Kontrogianni et al., 2003) that is situated on the SR. It has been demonstrated that ank1.5 can bind with its cytosolic tail the Cterminal region of obscurin, a large protein on the myofilaments (Young et al., 2001). In adult skeletal muscle, obscurin is localized at the M-band of the sarcomere. Interestingly, ank1.5 is found in the region of the L-SR that surrounds the M-band and this localization depends on ank1.5 ability to bind to obscurin (Bagnato et al., 2003; Cusimano et al., 2009). Mice knockout for the obscurin gene have normal sarcomeric structure and function but present signs of mild myopathy, altered localization of ank1.5, and a reduced amount of longitudinal SR around the myofibrils (Lange et al., 2009). Ank1.5 knockout mice have a reduced contractile performance accompanied by alterations in longitudinal SR structure and in the myofibrillar apparatus (Sorrentino et al., unpublished). Although the complete puzzle is not yet solved, we can envision a scheme where SR organization results from a process of membrane assembly and restructuring that ends in the formation of a network of tubules that is held close to the myofibrils thanks to specific contacts established between ank1.5 on the L-SR and obscurin at the M-band (Flucher et al., 1993; Giacomello and Sorrentino, 2009). In skeletal muscle fibers, the L-SR covers most of the A and I bands of the sarcomere, with a differential density of membranes that is higher in correspondence of the Z-disk (Fig. 1B). Accordingly, immunostaining of L-SR proteins with specific antibodies and analysis by fluorescence or confocal microscopes consistently yields a stronger signal in the region of the SR that overlaps the Z-disk, and a much weaker signal in correspondence of the M-band (Salanova et al., 2002). This contrasts with the preferential localization of ank1.5 at the M band (compare SERCA and ank1.5 signals in Fig. 1A). The dynamic properties of L-SR proteins have been further analysed by the Fluorescence Recovery After Photobleaching (FRAP) technique. These studies have shown that SERCA pumps are highly mobile while ank1.5 displays a relatively lower mobility within the L-SR membrane (Cusimano et al., 2009). The reduced mobility of ank1.5 appears to depend on its ability to bind to obscurin, since deletion of the obscurin binding site in ank1.5 resulted in an increased mobility and in re-localization of the mutant protein to the Z-disk (Cusimano et al., 2009). These data thus suggest that proteins that are free to move in the L-SR membrane, like SERCA or mutant ank1.5, are mainly observed around the Z-disk probably because of the higher density of SR membranes in this region, while alternative localizations, such as that of ank1.5 at
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the M-band, seem to be dependent on an active retention mechanism. 3.2. Organization of the jSR and of triads In mammalian skeletal muscle fibers, terminal cisternae and T-tubules are located at the junctions between A–I bands (FranziniArmstrong, 2004). The assembly of triad junctions has been demonstrated to be mediated by Junctophilins (JPs), a family of SR transmembrane proteins that extend from the surface of the SR to the sarcolemma and that play a central role in holding together these two membrane compartments (Ito et al., 2001). Overexpression of JPs cause the SR to move close to the plasmamembrane, while abrogation of the expression of JP1 and JP2, by means of gene targeting techniques, reduces the establishment of couplings between SR and T tubules in knockout mice (Takeshima et al., 2000). Several types of evidence indicate that jSR proteins are assembled in a stable macromolecular complex of high molecular mass organized around the RyR Ca2+ -release channels (Lanner et al., 2010). Interestingly, since JP1 has been shown to interact with RyR1 (Phimister et al., 2007) and RyR1 on its own can interact with other jSR proteins like including triadin and DHPR, it appears possible that the junctional SR proteins may be recruited at triads by sequential events mediated by protein-protein interaction (Lanner et al., 2010). This is also in agreement with data from FRAP-based experiments that indicated that junctional proteins, like JP1, RyR1, triadin and DHPR, all displayed a progressive reduction in their mobility following the assembly into junctional domains (Cusimano et al., 2009). However, although we can draw a working model for assembly of SR domains and for protein localization in these areas, we still have no clue about exactly how triads are positioned at the A–I border in skeletal muscle. Experiments following embryonic development and in vitro differentiation of skeletal muscle cells, have shown that DHPRs and RyRs co-localize and become functionally coupled a few days before triads are formed and aligned with the A–I band border (Flucher et al., 1993; Takekura et al., 2001; Cusimano et al., 2009). This means that the organization of mature triads follows the organization of junctional proteins and that the final localization of triads at the A–I border may require the establishment of additional interactions with the contractile apparatus. The identity of the proteins responsible for the positioning of triads in correspondence to specific regions of the sarcomere remains an important, yet unanswered, question. 4. SR in human pathology Mutations in SR proteins involved in Ca2+ homeostasis have been detected in several human diseases affecting skeletal and cardiac muscle tissues. Mutations in the RYR1 gene have been found in patients predisposed to Malignant Hyperthermia (MH), a life-threatening crisis commonly triggered by exposure to common anaesthetics or exertional/environmental heat stroke, and in patients with central core disease (CCD), a rare myopathy with hypotonia and proximal muscle weakness. Identification of a subset of MH patients with mutations in the DHPR but not in the RYR1 gene, clearly points to MH as a dysfunction of the skeletal muscle e–c coupling machinery (Betzenhauser and Marks, 2010). Mutations in either RYR2 or Calsequestrin2 genes have been found in patients with severe stress-induced ventricular tachycardia, where an aberrant Ca2+ release from the SR during diastole triggers ventricular arrhythmia resulting in cardiac arrest and sudden death (Györke et al., 2009; Kushnir and Marks, 2010). Mutations in the SERCA1a gene, resulting in a slow-down in the reuptake of Ca2+ from the cytosol with delayed relaxation and skeletal muscle
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cramping, have been linked to Brody’s disease (Brini and Carafoli, 2009). In addition to genetic diseases, multiple evidence indicates that -adrenergic stimulation regulates the activity of several SR proteins, including SERCA and RyRs. Among these, a direct phosphorylation of RyR2 by PKA, while playing a positive effect on cardiac performance in healthy individuals, seems to contribute to further deterioration in the cardiac performance in failing heart (Wehrens et al., 2003; Shan et al., 2010). Such studies have opened the way to search for new therapeutic approaches to treating cardiac diseases (Watanabe et al., 2009; Kushnir and Marks, 2010). In conclusion, the identification of mutations in the obscurin-binding domain of titin suggests that alterations in proteins involved in the organization of the SR may also cause muscular diseases (Fukuzawa et al., 2008). 5. Future outlook The past years have seen a major advancement in our understanding of the role of SR proteins involved in regulation of Ca2+ signalling in both physiological and pathological conditions. More recently, new data have started to describe how the threedimensional network of SR membranes is organized with respect to T tubules and to the myofilaments. However, many details are still missing. Identification of additional proteins responsible for the organization of the three-dimensional arrangement of the SR and T tubules, and of proteins, either present in the contractile apparatus or the extra-sarcomeric cytoskeleton, that organize the stereotyped pattern of triads at the A–I border may provide novel information in muscle physiology. Acknowledgements I thank Daniela Rossi and Emiliana Giacomello for comments on the manuscript and for help with Fig. 1. This work was supported in part by a grant from Telethon (GGP08153). References Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V. Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles. J Cell Biol 2003;160:245–53. Betzenhauser MJ, Marks AR. Ryanodine receptor channelopathies. Pflugers Arch 2010;460:467–80. Brini M, Carafoli E. Calcium pumps in health and disease. Physiol Rev 2009;89:1341–78. Cusimano V, Pampinella F, Giacomello E, Sorrentino V. Assembly and dynamics of proteins of the longitudinal and junctional sarcoplasmic reticulum in skeletal muscle cells. Proc Natl Acad Sci U S A 2009;106:4695–700. Endo M. Calcium induced calcium release in skeletal muscle. Physiol Rev 2009;89:1153–76. Flucher BE, Takekura H, Franzini-Armstrong C. Development of the excitation–contraction coupling apparatus in skeletal muscle: association of sarcoplasmic reticulum and transverse tubules with myofibrils. Dev Biol 1993;160:135–47.
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