BioArchitecture, 6:22–27, 2016 Ó 2016 Taylor and Francis Group, LLC ISSN: 1949-0992 print / 1949-100X online DOI: 10.1080/19490992.2015.1131891
Mechanoprotection by skeletal muscle caveolae Harriet P Lo,1 Thomas E Hall,1 and Robert G Parton1,2,* 1
The University of Queensland; Institute for Molecular Bioscience; St. Lucia, Queensland, Australia 2 Centre for Microscopy and Microanalysis; St. Lucia, Queensland, Australia
ABSTRACT. Caveolae, small bulb-like pits, are the most abundant surface feature of many vertebrate cell types. The relationship of the structure of caveolae to their function has been a subject of considerable scientific interest in view of the association of caveolar dysfunction with human disease. In a recent study Lo et al.1 investigated the organization and function of caveolae in skeletal muscle. Using quantitative 3D electron microscopy caveolae were shown to be predominantly organized into multilobed structures which provide a large reservoir of surface-connected membrane underlying the sarcolemma. These structures were preferentially disassembled in response to changes in membrane tension. Perturbation or loss of caveolae in mouse and zebrafish models suggested that caveolae can protect the muscle sarcolemma against damage in response to excessive membrane activity. Flattening of caveolae to release membrane into the bulk plasma membrane in response to increased membrane tension can allow cell shape changes and prevent membrane rupture. In addition, disassembly of caveolae can have widespread effects on lipid-based plasma membrane organization. These findings suggest that the ability of the caveolar membrane system to respond to mechanical forces is a crucial evolutionarily-conserved process which is compromised in disease conditions associated with mutations in key caveolar components. KEYWORDS. caveolae, mechanoprotection, skeletal muscle, T-tubule ABBREVIATIONS. PS, phosphatidylserine; PI(4,5)P2, phosphatidylinositol 4,5 bisphosphate
The sarcolemma of the skeletal muscle fiber is a highly specialized structure, which must cope with the physical demands of muscle contraction, respond to extracellular signals, and transmit action potentials from the surface of the muscle to the interior. These functions are facilitated by differentiation of the muscle surface into distinct macrodomains; the sarcolemma, with associated microdomains such as
caveolae and clathrin coated pits, and a second extensive domain, the T-tubule system, itself possessing distinct microdomains termed triads specialized for electrical coupling to the sarcoplasmic muscle to allow synchronized calcium release. Understanding how these membrane systems are generated, maintained, and their role in muscle function is crucial as numerous human disease conditions are associated with
*Correspondence to: Robert G Parton; Email: [email protected] Received December 2, 2015; Accepted December 9, 2015. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/kbia. Commentary to: Lo HP, Nixon SJ, Hall TE, Cowling BS, Ferguson C, Morgan GP, Schieber NL, Fernandez-Rojo MA, Bastiani M, Floetenmeyer M, Martel N, Laporte J, Pilch PF, Parton RG. The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle. J Cell Biol 2015; 210:833-49; PMID:26323694; http://dx.doi.org/10.1083/jcb.201501046 22
MECHANOPROTECTION BY SKELETAL MUSCLE CAVEOLAE
perturbation of the sarcolemma, caveolae, and the T-tubule system.2-5 This highly specialized membrane organization is a feature of mature muscle fibers and is difficult to recapitulate in cell culture models. The recent paper by Lo et al.1 used mature muscle fibers isolated from the flexor digitorum brevis of adult mice to quantitatively characterize the 3 dimensional architecture of the surface of skeletal muscle by electron microscopy. The major findings of this analysis include a highly extensive system of caveolae which comprise over 50% of the sarcolemmal area, organization of caveolae as multilobed clustered structures (also termed rosettes) connected to the sarcolemma usually by a single neck, and T-tubules joined to the sarcolemma via the clusters of caveolae. This striking organization prompted a functional dissection of the role of caveolae in the skeletal muscle of mice and the zebrafish, Danio rerio. Caveolae are characterized by their size (6– 80 nm) and morphology (‘uncoated’ bulbshaped pits), and by the presence of the structural proteins, caveolins and cavins.6-8 Caveolins are small oligomeric cholesterol-binding integral membrane proteins whereas cavins are peripheral membrane proteins thought to form extended filament-like striations on the inner face of the caveolae.9 Both a caveolin (caveolin-110,11 and caveolin-312 in non-muscle and muscle cells, respectively) and PTRF/cavin1 are required for caveola formation in vertebrate cells.13,14 The functions of caveolae have been the subject of considerable debate. Caveolae have been proposed to act as signaling centers with caveolin playing a crucial role in binding to, and inactivating, a wide range of signaling proteins (reviewed in Okamoto et al15 but see also Collins et al16). Caveolae have also been implicated in endocytosis, lipid regulation, and mechanosensation,6 particularly in endothelia.17 Early studies of muscle18 and endothelial cells19 also provided the first indications that caveolae may form plastic domains, which are sensitive to membrane tension. Stretching of muscle caused a loss of caveolae, apparently by their flattening,18 and a similar effect, ‘unfolding’ of caveolae, was proposed in
23
endothelial cells upon changes in capillary volume.19 With the discovery of the cavin proteins and their characterization as peripheral membrane proteins of caveolae this was further characterized using a combination of cell biological and biophysical approaches.20 Caveolae were shown to flatten in response to changes in membrane tension, both upon cell swelling or with stretch. This process was energy-independent and caused release of cavins from the caveolae, raising the possibility that cavins can act as cytosolic signals for changes in membrane tension. These findings prompted experimental testing of the role of caveolae in skeletal muscle.1 Swelling of WT muscle fibers caused loss of morphological caveolae due to their flattening into the sarcolemma. This was particularly noticeable in areas of the sarcolemma that formed blebs. These hemi-spherical structures several microns in diameter lacked caveolae completely and showed a complete separation of CAV3 and cavin-1. Electron tomography revealed that multilobed caveolar clusters were preferentially lost as the fibers swelled suggesting that these structures may be particularly sensitive to membrane tension changes (Fig. 1). We interpret the loss of these structures as indicating their flattening into the sarcolemma as no evidence for scission from the surface was obtained. Skeletal muscle fibers isolated in parallel from cavin1-null mice, a model of caveolar disease, showed a highly aberrant sarcolemmal architecture. These fibers lacked caveolae, showed a ruffled surface with irregular invaginations, and a disrupted Ttubule network. The caveola-null fibers showed increased rupture of the sarcolemma in response to hypotonic medium, as compared to the wild type fibers, and this could be rescued by expression of cavin1-GFP to generate caveolae.1 The finding that the membrane contained within caveolae, both within single pits at the cell surface and in the multilobed clusters of caveolae and their associated tubules, can constitute such a large reservoir of membrane suggests that flattening of caveolae, and particularly the groups of caveolae, can provide a highly significant area of
24
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FIGURE 1. Model for proposed organizational changes in response to changes in membrane tension. Caveolae (middle panel) can be flattened in response to increased membrane tension (left) or form multilobed clustered structures in response to decreased membrane tension (right).
membrane allowing the cell to change shape and cope with membrane force changes. In non-muscle cells direct membrane force measurements showed that in cells lacking caveolae a significant change in membrane force was observed upon cell swelling.20 This increase was not observed in the wild type cells that contained abundant caveolae. As caveolae are flattening under these conditions a logical conclusion is that caveola flattening allows the cell to swell without altering overall membrane tension and without compromising plasma membrane integrity. In cells lacking caveolae this eventually causes cell rupture as seen both in cultured cells20 and in muscle fibers1 upon cell swelling. Consistent with this hypothesis and extending the analysis to a more physiological situation, Lo et al.1 showed that zebrafish models of caveolar disease also showed rupture of the skeletal muscle sarcolemma when swimming in a viscous medium, a system which stimulates high muscle activity. A simple model would suggest that caveolae provide a reservoir of membrane which is used as the cell changes shape to protect the cell. While these physical properties of the caveolar system undoubtedly contribute to the protective role of caveolae this may only be part of the
protective mechanism. Our observations in fibroblasts21 and in isolated muscle fibers1 suggest that there may be additional processes involved which rely on more specific features of the caveolar system. Isolated muscle fibers from cavin1¡/¡ mice show a highly ruffled sarcolemma as compared to the sarcolemma of WT mouse fibers. In the caveola-null fibers there are also abundant relatively large irregular surface-connected invaginations of the surface.1 The ruffled surface plus surfaceconnected vacuoles actually contribute a similar surface area to the combined sarcolemma plus caveolae in the WT mice. Thus, a similar area of membrane might be available when the cell changes shape. Flattening of any folded membrane may therefore not be sufficient to explain the lower fragility of the WT caveolarich muscle surface. The molecular features of the plasma membrane pits which distinguish caveolae from bulk plasma membrane may well play a role in this process. Both caveolins and cavins bind lipids and are likely to generate unique domains with specific lipid composition, changing the way these regions of the membrane respond to plasma membrane forces as compared to the bulk plasma membrane. Caveolins are small integral membrane proteins and so the study of
MECHANOPROTECTION BY SKELETAL MUSCLE CAVEOLAE
lipid binding is not straightforward. Nevertheless, caveolins have been shown to bind cholesterol and fatty acids, and a polypeptide corresponding to a conserved region of caveolin can induce domains of phosphatidylserine (PS) and phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) in model liposome systems.22 Cavins also bind both PS and PI(4,5)P2 in vitro.9,13,23 It has been proposed that low affinity interactions with caveolins and with specific lipids clustered by caveolin might contribute to the formation of caveolae driven by cavins and caveolins.24 This network of low affinity interactions presumably allows disassembly of caveolae in response to increased membrane tension as cavins are released from caveolae as they flatten. In addition, with an estimated 140–150 caveolin molecules25 and 50 cavin1 molecules26 in a single caveolae the lipid domain generated within caveolae may be quite distinct to that of the bulk plasma membrane. The biophysical properties of the caveolar domain generated by the combination of caveolin insertion into the membrane, the cytoplasmic coat of cavin proteins, and their distinct lipid composition might be very different to that of the bulk membrane but at present the precise quantitative parameters are unknown. What then happens when a caveola flattens into the membrane? First, caveolin becomes more mobile and can diffuse more freely in the plane of the membrane.13,20 Thus, caveolin may be released into the bulk membrane to interact with non-caveolar components specifically under these conditions. Secondly, caveolar flattening can cause changes in bulk plasma membrane lipid organization. In fibroblasts loss of caveolin-1, cavin-1, or disruption of caveolae as cells swell cause increased clustering of PS in distant domains of the plasma membrane.21 This has impacts on Ras signaling as specific Ras isoforms are dependent on the lipid organization of the plasma membrane. In particular, K-ras nanoclustering and signaling activity, that are dependent on PS, are all increased upon caveolar disruption. Although these studies were conducted in model fibroblast systems similar processes may be occurring in the muscle sarcolemma.
25
Caveolar flattening and release of specific lipids could induce changes in the plasma membrane, protecting the cell through the alteration of the physical properties of the sarcolemma. For example, caveolin-1 expression changes membrane fluidity as judged by examining the dynamics of specific plasma membrane lipids. 27,28 Caveolar flattening could also theoretically modulate specific signal transduction pathways. Intriguingly recent studies showed that electrical activity can alter PS clustering and K-ras signaling.29 Third, the cavin coat proteins are released from caveolae. Rather than simply a passive structural component we suggest that cavins can interact with intracellular targets as a signaling mechanism. Intriguingly, cavin4, the muscle-specific isoform of the cavin family, accumulates within the nucleus of skeletal muscle fibers lacking cavin1.1 In this case loss of cavin1, which would normally recruit cavin4 to caveolae, may in some ways mimic the release of cavins upon caveolar flattening. Release of cavins from caveolae may play a role in regulating vital cellular processes such as muscle hypertrophy.30 In conclusion, as well as providing a reservoir of membrane that can flatten into the sarcolemma, we suggest that a number of features of the caveolar system may be important in the protection of the muscle surface. Naturally, numerous questions remain. Do different combinations of cavins alter the stability of caveolae? How do the multilobed clusters of caveolae respond to membrane tension in comparison to individual pits and how is their formation induced? There is a need to understand the composition of these structures and to understand the biophysics of their formation and disassembly. How does the unique lipid composition of caveolae relate to their function? And what are the cellular targets for released cavins? What is the role of caveolae at the neck of the T-tubule? Could this organization help buffer membrane tension changes as a muscle fiber changes shape to prevent T-tubule rupture, analogous to the postulated role of caveolae on the sarcolemma? A multidisciplinary approach involving a range of specialties including biophysics, cell biology, and
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physiology, is needed to help solve the puzzle of the role of caveolae in health and dysfunction in human disease.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST No potential conflicts of interest were disclosed.
FUNDING The authors acknowledge funding from the National Health and Medical Research Council of Australia (grant numbers APP1037320, APP1045092, APP1058565 and APP569542 to R.G. Parton). REFERENCES 1. Lo HP, Nixon SJ, Hall TE, Cowling BS, Ferguson C, Morgan GP, Schieber NL, Fernandez-Rojo MA, Bastiani M, Floetenmeyer M, et al. The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle. J Cell Biol 2015; 210:833-49; PMID:26323694; http://dx.doi.org/ 10.1083/jcb.201501046 2. Al-Qusairi L, Laporte J. T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases. Skelet Muscle 2011; 1:26; PMID:21797990; http://dx.doi.org/10.1186/20445040-1-26 3. Engelman JA, Zhang X, Galbiati F, Volonte D, Sotgia F, Pestell RG, Minetti C, Scherer PE, Okamoto T, Lisanti MP. Molecular genetics of the caveolin gene family: implications for human cancers, diabetes, Alzheimer disease, and muscular dystrophy. Am J Hum Genet 1998; 63:1578-87; PMID:9837809; http://dx.doi.org/10.1086/302172 4. Bansal D, Campbell KP. Dysferlin and the plasma membrane repair in muscular dystrophy. Trends Cell Biol 2004; 14:206-13; PMID:15066638; http://dx. doi.org/10.1016/j.tcb.2004.03.001 5. Ervasti JM, Campbell KP. Membrane organization of the dystrophin-glycoprotein complex. Cell 1991; 66:1121-31; PMID:1913804; http://dx.doi.org/ 10.1016/0092-8674(91)90035-W 6. Parton RG, del Pozo MA. Caveolae as plasma membrane sensors, protectors and organizers. Nat Rev Mol Cell Biol 2013; 14:98-112; PMID:23340574; http://dx.doi.org/10.1038/nrm3512
7. Ariotti N, Parton RG. SnapShot: caveolae, caveolins, and cavins. Cell 2013; 154:704-e1; PMID:23911330; http://dx.doi.org/10.1016/j.cell.2013.07.009 8. Hansen CG, Nichols BJ. Exploring the caves: cavins, caveolins and caveolae. Trends Cell Biol 2010; 20:177-86; PMID:20153650; http://dx.doi.org/ 10.1016/j.tcb.2010.01.005 9. Kovtun O, Tillu VA, Jung W, Leneva N, Ariotti N, Chaudhary N, Mandyam RA, Ferguson C, Morgan GP, Johnston WA, et al. Structural insights into the organization of the cavin membrane coat complex. Dev Cell 2014; 31:405-19; PMID:25453557; http:// dx.doi.org/10.1016/j.devcel.2014.10.002 10. Drab M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, Menne J, Lindschau C, Mende F, Luft FC, et al. Loss of Caveolae, Vascular Dysfunction, and Pulmonary Defects in Caveolin-1 Gene-Disrupted Mice. Science 2001; 9:9. 11. Razani B, Combs TP, Wang XB, Frank PG, Park DS, Russell RG, Li M, Tang B, Jelicks LA, Scherer PE, et al. Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities. J Biol Chem 2002; 277:8635-47; PMID:11739396; http://dx.doi.org/ 10.1074/jbc.M110970200 12. Minetti C, Bado M, Broda P, Sotgia F, Bruno C, Galbiati F, Volonte D, Lucania G, Pavan A, Bonilla E, et al. Impairment of caveolae formation and T-system disorganization in human muscular dystrophy with caveolin-3 deficiency. Am J Pathol 2002; 160:265-70; PMID:11786420; http://dx.doi.org/ 10.1016/S0002-9440(10)64370-2 13. Hill MM, Bastiani M, Luetterforst R, Kirkham M, Kirkham A, Nixon SJ, Walser P, Abankwa D, Oorschot VM, Martin S, et al. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. Cell 2008; 132:113-24; PMID:18191225; http://dx.doi.org/ 10.1016/j.cell.2007.11.042 14. Liu L, Brown D, McKee M, Lebrasseur NK, Yang D, Albrecht KH, Ravid K, Pilch PF. Deletion of Cavin/ PTRF causes global loss of caveolae, dyslipidemia, and glucose intolerance. Cell Metab 2008; 8:310-7; PMID:18840361; http://dx.doi.org/10.1016/j.cmet. 2008.07.008 15. Okamoto T, Schlegel A, Scherer PE, Lisanti MP. Caveolins, a family of scaffolding proteins for organizing “preassembled signaling complexes” at the plasma membrane. J Biol Chem 1998; 273:5419-22; PMID:9488658; http://dx.doi.org/10.1074/jbc.273. 10.5419 16. Collins BM, Davis MJ, Hancock JF, Parton RG. Structure-Based Reassessment of the Caveolin Signaling Model: Do Caveolae Regulate Signaling through Caveolin-Protein Interactions? Dev Cell 2012; 23:11-20; PMID:22814599; http://dx.doi.org/ 10.1016/j.devcel.2012.06.012
MECHANOPROTECTION BY SKELETAL MUSCLE CAVEOLAE
17. Yu J, Bergaya S, Murata T, Alp IF, Bauer MP, Lin MI, Drab M, Kurzchalia TV, Stan RV, Sessa WC. Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels. J Clin Invest 2006; 116:1284-91; PMID:16670769; http://dx.doi.org/10.1172/JCI27100 18. Dulhunty AF, Franzini-Armstrong C. The relative contributions of the folds and caveolae to the surface membrane of frog skeletal muscle fibres at different sarcomere lengths. J Physiol 1975; 250:513-39; PMID:1080806; http://dx.doi.org/10.1113/jphysiol. 1975.sp011068 19. Lee J, Schmid-Schonbein GW. Biomechanics of skeletal muscle capillaries: hemodynamic resistance, endothelial distensibility, and pseudopod formation. Ann Biomed Eng 1995; 23:226-46; PMID:7631979; http://dx.doi.org/10.1007/BF02584425 20. Sinha B, Koster D, Ruez R, Gonnord P, Bastiani M, Abankwa D, Stan RV, Butler-Browne G, Vedie B, Johannes L, et al. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 2011; 144:402-13; PMID:21295700; http://dx.doi.org/ 10.1016/j.cell.2010.12.031 21. Ariotti N, Fernandez-Rojo MA, Zhou Y, Hill MM, Rodkey TL, Inder KL, Tanner LB, Wenk MR, Hancock JF, Parton RG. Caveolae regulate the nanoscale organization of the plasma membrane to remotely control Ras signaling. J Cell Biol 2014; 204:777-92; PMID:24567358; http://dx.doi.org/10.1083/jcb.201307055 22. Wanaski SP, Ng BK, Glaser M. Caveolin scaffolding region and the membrane binding region of SRC form lateral membrane domains. Biochemistry 2003; 42:42-56; PMID:12515538; http://dx.doi.org/10.1021/bi012097n 23. Bastiani M, Liu L, Hill MM, Jedrychowski MP, Nixon SJ, Lo HP, Abankwa D, Luetterforst R, Fernandez-Rojo M, Breen MR, et al. MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes. J Cell Biol 2009; 185:1259-73; PMID:19546242; http://dx.doi.org/ 10.1083/jcb.200903053 24. Kovtun O, Tillu VA, Ariotti N, Parton RG, Collins BM. Cavin family proteins and the assembly of
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caveolae. J Cell Sci 2015; 128:1269-78; PMID:25829513; http://dx.doi.org/10.1242/jcs. 167866 Pelkmans L, Zerial M. Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae. Nature 2005; 436:128-33; PMID:16001074; http:// dx.doi.org/10.1038/nature03866 Gambin Y, Ariotti N, McMahon KA, Bastiani M, Sierecki E, Kovtun O, Polinkovsky ME, Magenau A, Jung W, Okano S, et al. Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae. eLife 2014; 3:e01434 Chaudhary N, Gomez GA, Howes MT, Lo HP, McMahon KA, Rae JA, Schieber NL, Hill MM, Gaus K, Yap AS, et al. Endocytic crosstalk: cavins, caveolins, and caveolae regulate clathrin-independent endocytosis. PLoS Biol 2014; 12:e1001832; PMID:24714042; http://dx.doi.org/10.1371/journal. pbio.1001832 Hoffmann C, Berking A, Agerer F, Buntru A, Neske F, Chhatwal GS, Ohlsen K, Hauck CR. Caveolin limits membrane microdomain mobility and integrin-mediated uptake of fibronectinbinding pathogens. J Cell Sci 2010; 123: 4280-91; PMID:21098633; http://dx.doi.org/10. 1242/jcs.064006 Zhou Y, Wong CO, Cho KJ, van der Hoeven D, Liang H, Thakur DP, Luo J, Babic M, Zinsmaier KE, Zhu MX, et al. SIGNAL TRANSDUCTION. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling. Science 2015; 349:873-6; PMID:26293964; http://dx.doi.org/ 10.1126/science.aaa5619 Tagawa M, Ueyama T, Ogata T, Takehara N, Nakajima N, Isodono K, Asada S, Takahashi T, Matsubara H, Oh H. MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis. Am J Physiol Cell Physiol 2008; 295:C490-8; PMID:18508909; http://dx.doi.org/10.1152/ajpcell. 00188.2008
Mechanoprotection by skeletal muscle caveolae Harriet P Lo,1 Thomas E Hall,1 and Robert G Parton1,2,* 1
The University of Queensland; Institute for Molecular Bioscience; St. Lucia, Queensland, Australia 2 Centre for Microscopy and Microanalysis; St. Lucia, Queensland, Australia
ABSTRACT. Caveolae, small bulb-like pits, are the most abundant surface feature of many vertebrate cell types. The relationship of the structure of caveolae to their function has been a subject of considerable scientific interest in view of the association of caveolar dysfunction with human disease. In a recent study Lo et al.1 investigated the organization and function of caveolae in skeletal muscle. Using quantitative 3D electron microscopy caveolae were shown to be predominantly organized into multilobed structures which provide a large reservoir of surface-connected membrane underlying the sarcolemma. These structures were preferentially disassembled in response to changes in membrane tension. Perturbation or loss of caveolae in mouse and zebrafish models suggested that caveolae can protect the muscle sarcolemma against damage in response to excessive membrane activity. Flattening of caveolae to release membrane into the bulk plasma membrane in response to increased membrane tension can allow cell shape changes and prevent membrane rupture. In addition, disassembly of caveolae can have widespread effects on lipid-based plasma membrane organization. These findings suggest that the ability of the caveolar membrane system to respond to mechanical forces is a crucial evolutionarily-conserved process which is compromised in disease conditions associated with mutations in key caveolar components. KEYWORDS. caveolae, mechanoprotection, skeletal muscle, T-tubule ABBREVIATIONS. PS, phosphatidylserine; PI(4,5)P2, phosphatidylinositol 4,5 bisphosphate
The sarcolemma of the skeletal muscle fiber is a highly specialized structure, which must cope with the physical demands of muscle contraction, respond to extracellular signals, and transmit action potentials from the surface of the muscle to the interior. These functions are facilitated by differentiation of the muscle surface into distinct macrodomains; the sarcolemma, with associated microdomains such as
caveolae and clathrin coated pits, and a second extensive domain, the T-tubule system, itself possessing distinct microdomains termed triads specialized for electrical coupling to the sarcoplasmic muscle to allow synchronized calcium release. Understanding how these membrane systems are generated, maintained, and their role in muscle function is crucial as numerous human disease conditions are associated with
*Correspondence to: Robert G Parton; Email: [email protected] Received December 2, 2015; Accepted December 9, 2015. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/kbia. Commentary to: Lo HP, Nixon SJ, Hall TE, Cowling BS, Ferguson C, Morgan GP, Schieber NL, Fernandez-Rojo MA, Bastiani M, Floetenmeyer M, Martel N, Laporte J, Pilch PF, Parton RG. The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle. J Cell Biol 2015; 210:833-49; PMID:26323694; http://dx.doi.org/10.1083/jcb.201501046 22
MECHANOPROTECTION BY SKELETAL MUSCLE CAVEOLAE
perturbation of the sarcolemma, caveolae, and the T-tubule system.2-5 This highly specialized membrane organization is a feature of mature muscle fibers and is difficult to recapitulate in cell culture models. The recent paper by Lo et al.1 used mature muscle fibers isolated from the flexor digitorum brevis of adult mice to quantitatively characterize the 3 dimensional architecture of the surface of skeletal muscle by electron microscopy. The major findings of this analysis include a highly extensive system of caveolae which comprise over 50% of the sarcolemmal area, organization of caveolae as multilobed clustered structures (also termed rosettes) connected to the sarcolemma usually by a single neck, and T-tubules joined to the sarcolemma via the clusters of caveolae. This striking organization prompted a functional dissection of the role of caveolae in the skeletal muscle of mice and the zebrafish, Danio rerio. Caveolae are characterized by their size (6– 80 nm) and morphology (‘uncoated’ bulbshaped pits), and by the presence of the structural proteins, caveolins and cavins.6-8 Caveolins are small oligomeric cholesterol-binding integral membrane proteins whereas cavins are peripheral membrane proteins thought to form extended filament-like striations on the inner face of the caveolae.9 Both a caveolin (caveolin-110,11 and caveolin-312 in non-muscle and muscle cells, respectively) and PTRF/cavin1 are required for caveola formation in vertebrate cells.13,14 The functions of caveolae have been the subject of considerable debate. Caveolae have been proposed to act as signaling centers with caveolin playing a crucial role in binding to, and inactivating, a wide range of signaling proteins (reviewed in Okamoto et al15 but see also Collins et al16). Caveolae have also been implicated in endocytosis, lipid regulation, and mechanosensation,6 particularly in endothelia.17 Early studies of muscle18 and endothelial cells19 also provided the first indications that caveolae may form plastic domains, which are sensitive to membrane tension. Stretching of muscle caused a loss of caveolae, apparently by their flattening,18 and a similar effect, ‘unfolding’ of caveolae, was proposed in
23
endothelial cells upon changes in capillary volume.19 With the discovery of the cavin proteins and their characterization as peripheral membrane proteins of caveolae this was further characterized using a combination of cell biological and biophysical approaches.20 Caveolae were shown to flatten in response to changes in membrane tension, both upon cell swelling or with stretch. This process was energy-independent and caused release of cavins from the caveolae, raising the possibility that cavins can act as cytosolic signals for changes in membrane tension. These findings prompted experimental testing of the role of caveolae in skeletal muscle.1 Swelling of WT muscle fibers caused loss of morphological caveolae due to their flattening into the sarcolemma. This was particularly noticeable in areas of the sarcolemma that formed blebs. These hemi-spherical structures several microns in diameter lacked caveolae completely and showed a complete separation of CAV3 and cavin-1. Electron tomography revealed that multilobed caveolar clusters were preferentially lost as the fibers swelled suggesting that these structures may be particularly sensitive to membrane tension changes (Fig. 1). We interpret the loss of these structures as indicating their flattening into the sarcolemma as no evidence for scission from the surface was obtained. Skeletal muscle fibers isolated in parallel from cavin1-null mice, a model of caveolar disease, showed a highly aberrant sarcolemmal architecture. These fibers lacked caveolae, showed a ruffled surface with irregular invaginations, and a disrupted Ttubule network. The caveola-null fibers showed increased rupture of the sarcolemma in response to hypotonic medium, as compared to the wild type fibers, and this could be rescued by expression of cavin1-GFP to generate caveolae.1 The finding that the membrane contained within caveolae, both within single pits at the cell surface and in the multilobed clusters of caveolae and their associated tubules, can constitute such a large reservoir of membrane suggests that flattening of caveolae, and particularly the groups of caveolae, can provide a highly significant area of
24
Lo et al.
FIGURE 1. Model for proposed organizational changes in response to changes in membrane tension. Caveolae (middle panel) can be flattened in response to increased membrane tension (left) or form multilobed clustered structures in response to decreased membrane tension (right).
membrane allowing the cell to change shape and cope with membrane force changes. In non-muscle cells direct membrane force measurements showed that in cells lacking caveolae a significant change in membrane force was observed upon cell swelling.20 This increase was not observed in the wild type cells that contained abundant caveolae. As caveolae are flattening under these conditions a logical conclusion is that caveola flattening allows the cell to swell without altering overall membrane tension and without compromising plasma membrane integrity. In cells lacking caveolae this eventually causes cell rupture as seen both in cultured cells20 and in muscle fibers1 upon cell swelling. Consistent with this hypothesis and extending the analysis to a more physiological situation, Lo et al.1 showed that zebrafish models of caveolar disease also showed rupture of the skeletal muscle sarcolemma when swimming in a viscous medium, a system which stimulates high muscle activity. A simple model would suggest that caveolae provide a reservoir of membrane which is used as the cell changes shape to protect the cell. While these physical properties of the caveolar system undoubtedly contribute to the protective role of caveolae this may only be part of the
protective mechanism. Our observations in fibroblasts21 and in isolated muscle fibers1 suggest that there may be additional processes involved which rely on more specific features of the caveolar system. Isolated muscle fibers from cavin1¡/¡ mice show a highly ruffled sarcolemma as compared to the sarcolemma of WT mouse fibers. In the caveola-null fibers there are also abundant relatively large irregular surface-connected invaginations of the surface.1 The ruffled surface plus surfaceconnected vacuoles actually contribute a similar surface area to the combined sarcolemma plus caveolae in the WT mice. Thus, a similar area of membrane might be available when the cell changes shape. Flattening of any folded membrane may therefore not be sufficient to explain the lower fragility of the WT caveolarich muscle surface. The molecular features of the plasma membrane pits which distinguish caveolae from bulk plasma membrane may well play a role in this process. Both caveolins and cavins bind lipids and are likely to generate unique domains with specific lipid composition, changing the way these regions of the membrane respond to plasma membrane forces as compared to the bulk plasma membrane. Caveolins are small integral membrane proteins and so the study of
MECHANOPROTECTION BY SKELETAL MUSCLE CAVEOLAE
lipid binding is not straightforward. Nevertheless, caveolins have been shown to bind cholesterol and fatty acids, and a polypeptide corresponding to a conserved region of caveolin can induce domains of phosphatidylserine (PS) and phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) in model liposome systems.22 Cavins also bind both PS and PI(4,5)P2 in vitro.9,13,23 It has been proposed that low affinity interactions with caveolins and with specific lipids clustered by caveolin might contribute to the formation of caveolae driven by cavins and caveolins.24 This network of low affinity interactions presumably allows disassembly of caveolae in response to increased membrane tension as cavins are released from caveolae as they flatten. In addition, with an estimated 140–150 caveolin molecules25 and 50 cavin1 molecules26 in a single caveolae the lipid domain generated within caveolae may be quite distinct to that of the bulk plasma membrane. The biophysical properties of the caveolar domain generated by the combination of caveolin insertion into the membrane, the cytoplasmic coat of cavin proteins, and their distinct lipid composition might be very different to that of the bulk membrane but at present the precise quantitative parameters are unknown. What then happens when a caveola flattens into the membrane? First, caveolin becomes more mobile and can diffuse more freely in the plane of the membrane.13,20 Thus, caveolin may be released into the bulk membrane to interact with non-caveolar components specifically under these conditions. Secondly, caveolar flattening can cause changes in bulk plasma membrane lipid organization. In fibroblasts loss of caveolin-1, cavin-1, or disruption of caveolae as cells swell cause increased clustering of PS in distant domains of the plasma membrane.21 This has impacts on Ras signaling as specific Ras isoforms are dependent on the lipid organization of the plasma membrane. In particular, K-ras nanoclustering and signaling activity, that are dependent on PS, are all increased upon caveolar disruption. Although these studies were conducted in model fibroblast systems similar processes may be occurring in the muscle sarcolemma.
25
Caveolar flattening and release of specific lipids could induce changes in the plasma membrane, protecting the cell through the alteration of the physical properties of the sarcolemma. For example, caveolin-1 expression changes membrane fluidity as judged by examining the dynamics of specific plasma membrane lipids. 27,28 Caveolar flattening could also theoretically modulate specific signal transduction pathways. Intriguingly recent studies showed that electrical activity can alter PS clustering and K-ras signaling.29 Third, the cavin coat proteins are released from caveolae. Rather than simply a passive structural component we suggest that cavins can interact with intracellular targets as a signaling mechanism. Intriguingly, cavin4, the muscle-specific isoform of the cavin family, accumulates within the nucleus of skeletal muscle fibers lacking cavin1.1 In this case loss of cavin1, which would normally recruit cavin4 to caveolae, may in some ways mimic the release of cavins upon caveolar flattening. Release of cavins from caveolae may play a role in regulating vital cellular processes such as muscle hypertrophy.30 In conclusion, as well as providing a reservoir of membrane that can flatten into the sarcolemma, we suggest that a number of features of the caveolar system may be important in the protection of the muscle surface. Naturally, numerous questions remain. Do different combinations of cavins alter the stability of caveolae? How do the multilobed clusters of caveolae respond to membrane tension in comparison to individual pits and how is their formation induced? There is a need to understand the composition of these structures and to understand the biophysics of their formation and disassembly. How does the unique lipid composition of caveolae relate to their function? And what are the cellular targets for released cavins? What is the role of caveolae at the neck of the T-tubule? Could this organization help buffer membrane tension changes as a muscle fiber changes shape to prevent T-tubule rupture, analogous to the postulated role of caveolae on the sarcolemma? A multidisciplinary approach involving a range of specialties including biophysics, cell biology, and
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physiology, is needed to help solve the puzzle of the role of caveolae in health and dysfunction in human disease.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST No potential conflicts of interest were disclosed.
FUNDING The authors acknowledge funding from the National Health and Medical Research Council of Australia (grant numbers APP1037320, APP1045092, APP1058565 and APP569542 to R.G. Parton). REFERENCES 1. Lo HP, Nixon SJ, Hall TE, Cowling BS, Ferguson C, Morgan GP, Schieber NL, Fernandez-Rojo MA, Bastiani M, Floetenmeyer M, et al. The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle. J Cell Biol 2015; 210:833-49; PMID:26323694; http://dx.doi.org/ 10.1083/jcb.201501046 2. Al-Qusairi L, Laporte J. T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases. Skelet Muscle 2011; 1:26; PMID:21797990; http://dx.doi.org/10.1186/20445040-1-26 3. Engelman JA, Zhang X, Galbiati F, Volonte D, Sotgia F, Pestell RG, Minetti C, Scherer PE, Okamoto T, Lisanti MP. Molecular genetics of the caveolin gene family: implications for human cancers, diabetes, Alzheimer disease, and muscular dystrophy. Am J Hum Genet 1998; 63:1578-87; PMID:9837809; http://dx.doi.org/10.1086/302172 4. Bansal D, Campbell KP. Dysferlin and the plasma membrane repair in muscular dystrophy. Trends Cell Biol 2004; 14:206-13; PMID:15066638; http://dx. doi.org/10.1016/j.tcb.2004.03.001 5. Ervasti JM, Campbell KP. Membrane organization of the dystrophin-glycoprotein complex. Cell 1991; 66:1121-31; PMID:1913804; http://dx.doi.org/ 10.1016/0092-8674(91)90035-W 6. Parton RG, del Pozo MA. Caveolae as plasma membrane sensors, protectors and organizers. Nat Rev Mol Cell Biol 2013; 14:98-112; PMID:23340574; http://dx.doi.org/10.1038/nrm3512
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