NEWS AND VIEWS
Redox switch for actin Hermann Aberle Oxidation of actin methionine residues by the oxidation–reduction enzyme Mical is known to lead to actin filament depolymerization. SelR enzymes are now shown to reduce these oxidized actin methionines, revealing a regulated redox reaction mechanism through which cells control the assembly and disassembly of actin filaments. Controlled polymerization and depolymerization of actin is essential for establishing and maintaining cell shape and function. Previous studies demonstrated that Mical (molecule interacting with CasL) can oxidize specific methionine residues in polymerized actin filaments, leading to filament breakdown1,2. Mical-mediated oxidation and depolymerization of actin filaments was shown to be linked to extracellular Semaphorin signals that are transduced through Plexins1,2. Two studies by Lee et al.3 and Hung et al.4 now report that this process is reversible through the action of SelR enzymes, effectively identifying a redox switch that controls assembly and disassembly of actin filaments. Mical proteins consist of a N-terminal monooxygenase (MO) domain followed by several protein–protein interaction domains, including a calponin homology (CH) domain, a Lin11–Isl1–Mec3 (LIM) domain and a C-terminal coiled-coil domain5–7. The coiled-coil domain of Drosophila melanogaster Mical interacts with the cytoplasmic domain of the axonal guidance receptor Plexin A (ref. 6). Plexins are a family of transmembrane proteins that bind to secreted or membrane-bound Semaphorins and transduce repulsive guidance information into developing axons, turning growth cones away from Semaphorin-expressing tissues. The direct binding to Plexin A together with the presence of a CH domain, which is typically found in actin-binding proteins, indicated that Mical might be involved in the transduction of extracellular signals to the underlying cytoskeleton, possibly by oxidation of a number of substrates6. To investigate this possibility, Hung et al. used Drosophila bristles, a simple yet highly sensitive genetic system that faithfully reflects even small changes in actin organization4. Dozens of bristle mutations Hermann Aberle is at Heinrich-Heine-Universität Düsseldorf, AG Funktionelle Zellmorphologie, Universitätsstraße 1, Geb. 26-12-00, 40223 Düsseldorf, Germany. e-mail: [email protected]
have been identified in the past, with classic bristle mutants such as singed, forked or stubble affecting crosslinking and bundling of actin filaments, leading to twisted, kinked or shorter bristles, respectively 8. Visualization of actin organization in bristles demonstrated that Mical could enzymatically destabilize actin filaments downstream of Semaphorin signals1. Overexpression of full-length or truncated Mical led to strongly kinked or branched bristles, and the presence of the MO and CH domains were both necessary and sufficient to induce these phenotypes1. Mical was further shown biochemically to interact with polymerized actin filaments and depolymerize them in an NADPH-dependent manner, leading to shorter and thinner actin bundles1. Monooxygenases usually introduce covalently bound oxygen atoms into their substrates. The mass of Mical-treated actin was found to increase by merely 32 daltons, which reflected the oxidation of methionine 44 (Met 44) and methionine 47 (Met 47) located at the pointed end of actin, at the critical interface between two successive actin monomers2. Thus, oxidation of lipophilic methionines to hydrophilic sulfoxides probably destabilizes filaments by interfering with the interaction of adjacent actin molecules. Intriguingly, the exchange of Met 44 with leucine (M44L) prevented Micalinduced oxidation but not polymerization. To investigate the fate of oxidized actin molecules, Hung et al.4 screened transposable element insertion lines for suppression of Mical-induced bristle phenotypes4. They identified Drosophila Selenoprotein R (SelR) as the enzyme that reduces the methionine sulfoxides to methionines4. SelR encodes a methionine sulfoxide reductase (Msr)9, and interestingly its homologue MsrB1 had been recently shown to antagonize the action of Mical in mammalian cells3. Hung et al. demonstrated that purified recombinant SelR protein was able to erase the inhibitory modifications introduced by Mical as well as to induce re-polymerization of Mical-treated actin (Fig. 1)4. They further ruled out unspecific effects such as general redox
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 12 | DECEMBER 2013 © 2013 Macmillan Publishers Limited. All rights reserved
reactions, and showed that related sulfoxide reductases of the MsrA family failed to reduce the methionines. The specificity of SelR was established by demonstrating the inactivity of enzymatically dead SelR towards actin and by showing that the two oxygen atoms in Met 44 and Met 47 were no longer detectable after SelR treatment. The authors further showed that the Mical–SelR-mediated redox mechanism was not limited to actin-rich bristles but occurred also in other actin-rich tissues. Overexpression of SelR in muscles severely disorganized actin filaments, resembling Mical mutant actin phenotypes in muscles. Similarly, SelR mutants phenocopied Mical overexpression phenotypes, indicating that SelR antagonizes the effects of Mical. To investigate whether SelR also plays a role in Semaphorin–Plexin signalling, they examined migrating motor axons in Drosophila embryos (Fig. 1). If SelR plays a role in axon guidance, it should counteract Mical function. As predicted, overexpression of SelR in neurons led to diminished repulsion, characterized by increased axonal fasciculation and a failure of motor axons to reach their target muscles. Similar guidance defects were observed in Mical loss-of-function mutants, as well as in Semaphorin-1a and Plexin A mutants. Furthermore, guidance defects induced by increased repulsion, for example by overexpression of Plexin A, were efficiently rescued by simultaneous overexpression of SelR, demonstrating that SelR is able to neutralize repulsive signals. Thus, besides their general monitoring role in repairing oxidatively damaged methionines, SelR (MsrB) enzymes have a functional role in signalling 4. The Drosophila Mical–SelR system seems to be highly conserved in mammals. Human Mical proteins also bind to Plexins and to another protein in the Semaphorin pathway, CRMP (collapsin response mediator protein)10,11. Loss of human Mical1 dramatically alters the actin cytoskeleton in a variety of cell lines, leading to a disappearance of actin stress fibres and accumulation of actin at the cell cortex and its protrusions12. The high conservation 1403
NEWS AND VIEWS
Growth cone SelR
Sema-1a Plexin A
ox ox
ox x o
Reduced actin Oxidized actin
Mical
Depolymerization
Actin pool
Actin filament
Polymerization
Figure 1 Possible function of the Mical–SelR redox system in a Drosophila growth cone. Membranebound Semaphorin-1a (red) signals through Plexin A (orange) to induce Mical (yellow)-mediated depolymerization of actin filaments (pink), followed by retraction of filopodia. Oxidized (ox) actin molecules are reduced by SelR (green) and recycled into cytoplasmic pools for re-polymerization. Polymerization and depolymerization reactions on opposite sides of the growth cone effectively turn it away from the repulsive source.
of this mechanism across different species is highlighted by the recent findings of Lee et al.3, which demonstrated that the human selenoprotein MsrB1 stereospecifically reverses the oxidation of the methionines at the pointed end of actin and functions as a re-assembly factor for Mical-disassembled actin3. The function of MsrB1 was assessed in macrophages, highly motile cells that depend on a dynamic actin cytoskeleton. MsrB1 expression was dramatically upregulated during activation of the innate immune response. Consequently, MsrB1-deficient macrophages showed defects in processes dependent on actin polymerization (such as reduced numbers of filopodia, less pinocytosis and lower cytokine release), possibly suppressing the innate immune response — which was, however, not directly tested by Lee et al. Nevertheless, high selenoprotein expression is believed to promote anti-inflammatory responses and benefit patients with low immune status3.
1404
Notably, several mutations in the human actin gene ACTA1 alter the methionines at the pointed end and have been associated with a form of congenital myopathy called intermediate nemaline myopathy 13. These patients suffer from muscle weakness and hypotonia, caused by abnormal accumulations of rod-like aggregates in the sarcoplasm. Nemaline myopathy shows genetic heterogeneity, and mutations in tropomyosin, nebulin, troponin and other muscle-specific genes have been identified, suggesting that one of the human Micals could be a further candidate gene. Interestingly, Mical mutations cause dramatic rearrangements of contractile muscle filaments and loss of sarcomeric organization in Drosophila, accompanied by accumulations of detached thin and thick filaments in the sarcoplasm and at postsynaptic densities14. Myosin filaments are therefore also affected in Mical mutants, and future experiments have to show if this is just a secondary effect or if Mical activity plays a
more prominent role in the redox regulation of contractile filaments and muscle sarcomeres. After a decade of Mical research, the finding that Mical and SelR (MsrB) reversibly control actin polymerization opens up new research avenues. Future studies would need to address the precise role of these proteins in mammals, for example by generating knockout animals and transgenic lines. Given that a number of findings point to the fact that actin-rich muscles are especially vulnerable, it would be interesting to examine the role of the Mical–SelR redox system in late-onset myopathies and other diseases affecting the formation, maturation and/or maintenance of sarcomeres. Another question arising from these findings is the potential interplay of this redox system with other actin regulators such as cofilins or the Arp2/3 complex, which could reveal fundamental similarities or differences in the organization and dynamics of the actin cytoskeleton in growth cones, muscles and bristles. There seem to be a myriad of bristle mutants and other model systems waiting to address these questions. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Hung, R. J. et al. Nature 463, 823–827 (2010). 2. Hung, R. J., Pak, C. W. & Terman, J. R. Science 334, 1710–1713 (2011). 3. Lee, B. C. et al. Mol. Cell 51, 397–404 (2013). 4. Hung, R. J., Spaeth, C. S., Yesilyurt, H. G. & Terman, J. R. Nat. Cell Biol. 15, 1445–1454 (2013). 5. Suzuki, T. et al. J. Biol. Chem. 277, 14933–14941 (2002). 6. Terman, J. R., Mao, T., Pasterkamp, R. J., Yu, H. H. & Kolodkin, A. L. Cell 109, 887–900 (2002). 7. Weide, T., Teuber, J., Bayer, M. & Barnekow, A. Biochem. Biophys. Res. Comm. 306, 79–86 (2003). 8. Tilney, L. G. & DeRosier, D. J. Proc. Natl Acad. Sci. USA 102, 18785–18792 (2005). 9. Kryukov, G. V., Kumar, R. A., Koc, A., Sun, Z. & Gladyshev, V. N. Proc. Natl Acad. Sci. USA 99, 4245– 4250 (2002). 10. Zhou, Y., Gunput, R. A., Adolfs, Y. & Pasterkamp, R. J. Cell. Mol. Life Sci. 68, 4033–4044 (2011). 11. Schmidt, E. F., Shim, S. O. & Strittmatter, S. M. J. Neurosci. 28, 2287–2297 (2008). 12. Giridharan, S. S., Rohn, J. L., Naslavsky, N. & Caplan, S. J. Cell Sci. 125, 614–624 (2012). 13. Laing, N. G. et al. Hum. Mutat. 30, 1267–1277 (2009). 14. Beuchle, D., Schwarz, H., Langegger, M., Koch, I. & Aberle, H. Mech. Dev. 124, 390–406 (2007).
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 12 | DECEMBER 2013 © 2013 Macmillan Publishers Limited. All rights reserved
Redox switch for actin Hermann Aberle Oxidation of actin methionine residues by the oxidation–reduction enzyme Mical is known to lead to actin filament depolymerization. SelR enzymes are now shown to reduce these oxidized actin methionines, revealing a regulated redox reaction mechanism through which cells control the assembly and disassembly of actin filaments. Controlled polymerization and depolymerization of actin is essential for establishing and maintaining cell shape and function. Previous studies demonstrated that Mical (molecule interacting with CasL) can oxidize specific methionine residues in polymerized actin filaments, leading to filament breakdown1,2. Mical-mediated oxidation and depolymerization of actin filaments was shown to be linked to extracellular Semaphorin signals that are transduced through Plexins1,2. Two studies by Lee et al.3 and Hung et al.4 now report that this process is reversible through the action of SelR enzymes, effectively identifying a redox switch that controls assembly and disassembly of actin filaments. Mical proteins consist of a N-terminal monooxygenase (MO) domain followed by several protein–protein interaction domains, including a calponin homology (CH) domain, a Lin11–Isl1–Mec3 (LIM) domain and a C-terminal coiled-coil domain5–7. The coiled-coil domain of Drosophila melanogaster Mical interacts with the cytoplasmic domain of the axonal guidance receptor Plexin A (ref. 6). Plexins are a family of transmembrane proteins that bind to secreted or membrane-bound Semaphorins and transduce repulsive guidance information into developing axons, turning growth cones away from Semaphorin-expressing tissues. The direct binding to Plexin A together with the presence of a CH domain, which is typically found in actin-binding proteins, indicated that Mical might be involved in the transduction of extracellular signals to the underlying cytoskeleton, possibly by oxidation of a number of substrates6. To investigate this possibility, Hung et al. used Drosophila bristles, a simple yet highly sensitive genetic system that faithfully reflects even small changes in actin organization4. Dozens of bristle mutations Hermann Aberle is at Heinrich-Heine-Universität Düsseldorf, AG Funktionelle Zellmorphologie, Universitätsstraße 1, Geb. 26-12-00, 40223 Düsseldorf, Germany. e-mail: [email protected]
have been identified in the past, with classic bristle mutants such as singed, forked or stubble affecting crosslinking and bundling of actin filaments, leading to twisted, kinked or shorter bristles, respectively 8. Visualization of actin organization in bristles demonstrated that Mical could enzymatically destabilize actin filaments downstream of Semaphorin signals1. Overexpression of full-length or truncated Mical led to strongly kinked or branched bristles, and the presence of the MO and CH domains were both necessary and sufficient to induce these phenotypes1. Mical was further shown biochemically to interact with polymerized actin filaments and depolymerize them in an NADPH-dependent manner, leading to shorter and thinner actin bundles1. Monooxygenases usually introduce covalently bound oxygen atoms into their substrates. The mass of Mical-treated actin was found to increase by merely 32 daltons, which reflected the oxidation of methionine 44 (Met 44) and methionine 47 (Met 47) located at the pointed end of actin, at the critical interface between two successive actin monomers2. Thus, oxidation of lipophilic methionines to hydrophilic sulfoxides probably destabilizes filaments by interfering with the interaction of adjacent actin molecules. Intriguingly, the exchange of Met 44 with leucine (M44L) prevented Micalinduced oxidation but not polymerization. To investigate the fate of oxidized actin molecules, Hung et al.4 screened transposable element insertion lines for suppression of Mical-induced bristle phenotypes4. They identified Drosophila Selenoprotein R (SelR) as the enzyme that reduces the methionine sulfoxides to methionines4. SelR encodes a methionine sulfoxide reductase (Msr)9, and interestingly its homologue MsrB1 had been recently shown to antagonize the action of Mical in mammalian cells3. Hung et al. demonstrated that purified recombinant SelR protein was able to erase the inhibitory modifications introduced by Mical as well as to induce re-polymerization of Mical-treated actin (Fig. 1)4. They further ruled out unspecific effects such as general redox
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 12 | DECEMBER 2013 © 2013 Macmillan Publishers Limited. All rights reserved
reactions, and showed that related sulfoxide reductases of the MsrA family failed to reduce the methionines. The specificity of SelR was established by demonstrating the inactivity of enzymatically dead SelR towards actin and by showing that the two oxygen atoms in Met 44 and Met 47 were no longer detectable after SelR treatment. The authors further showed that the Mical–SelR-mediated redox mechanism was not limited to actin-rich bristles but occurred also in other actin-rich tissues. Overexpression of SelR in muscles severely disorganized actin filaments, resembling Mical mutant actin phenotypes in muscles. Similarly, SelR mutants phenocopied Mical overexpression phenotypes, indicating that SelR antagonizes the effects of Mical. To investigate whether SelR also plays a role in Semaphorin–Plexin signalling, they examined migrating motor axons in Drosophila embryos (Fig. 1). If SelR plays a role in axon guidance, it should counteract Mical function. As predicted, overexpression of SelR in neurons led to diminished repulsion, characterized by increased axonal fasciculation and a failure of motor axons to reach their target muscles. Similar guidance defects were observed in Mical loss-of-function mutants, as well as in Semaphorin-1a and Plexin A mutants. Furthermore, guidance defects induced by increased repulsion, for example by overexpression of Plexin A, were efficiently rescued by simultaneous overexpression of SelR, demonstrating that SelR is able to neutralize repulsive signals. Thus, besides their general monitoring role in repairing oxidatively damaged methionines, SelR (MsrB) enzymes have a functional role in signalling 4. The Drosophila Mical–SelR system seems to be highly conserved in mammals. Human Mical proteins also bind to Plexins and to another protein in the Semaphorin pathway, CRMP (collapsin response mediator protein)10,11. Loss of human Mical1 dramatically alters the actin cytoskeleton in a variety of cell lines, leading to a disappearance of actin stress fibres and accumulation of actin at the cell cortex and its protrusions12. The high conservation 1403
NEWS AND VIEWS
Growth cone SelR
Sema-1a Plexin A
ox ox
ox x o
Reduced actin Oxidized actin
Mical
Depolymerization
Actin pool
Actin filament
Polymerization
Figure 1 Possible function of the Mical–SelR redox system in a Drosophila growth cone. Membranebound Semaphorin-1a (red) signals through Plexin A (orange) to induce Mical (yellow)-mediated depolymerization of actin filaments (pink), followed by retraction of filopodia. Oxidized (ox) actin molecules are reduced by SelR (green) and recycled into cytoplasmic pools for re-polymerization. Polymerization and depolymerization reactions on opposite sides of the growth cone effectively turn it away from the repulsive source.
of this mechanism across different species is highlighted by the recent findings of Lee et al.3, which demonstrated that the human selenoprotein MsrB1 stereospecifically reverses the oxidation of the methionines at the pointed end of actin and functions as a re-assembly factor for Mical-disassembled actin3. The function of MsrB1 was assessed in macrophages, highly motile cells that depend on a dynamic actin cytoskeleton. MsrB1 expression was dramatically upregulated during activation of the innate immune response. Consequently, MsrB1-deficient macrophages showed defects in processes dependent on actin polymerization (such as reduced numbers of filopodia, less pinocytosis and lower cytokine release), possibly suppressing the innate immune response — which was, however, not directly tested by Lee et al. Nevertheless, high selenoprotein expression is believed to promote anti-inflammatory responses and benefit patients with low immune status3.
1404
Notably, several mutations in the human actin gene ACTA1 alter the methionines at the pointed end and have been associated with a form of congenital myopathy called intermediate nemaline myopathy 13. These patients suffer from muscle weakness and hypotonia, caused by abnormal accumulations of rod-like aggregates in the sarcoplasm. Nemaline myopathy shows genetic heterogeneity, and mutations in tropomyosin, nebulin, troponin and other muscle-specific genes have been identified, suggesting that one of the human Micals could be a further candidate gene. Interestingly, Mical mutations cause dramatic rearrangements of contractile muscle filaments and loss of sarcomeric organization in Drosophila, accompanied by accumulations of detached thin and thick filaments in the sarcoplasm and at postsynaptic densities14. Myosin filaments are therefore also affected in Mical mutants, and future experiments have to show if this is just a secondary effect or if Mical activity plays a
more prominent role in the redox regulation of contractile filaments and muscle sarcomeres. After a decade of Mical research, the finding that Mical and SelR (MsrB) reversibly control actin polymerization opens up new research avenues. Future studies would need to address the precise role of these proteins in mammals, for example by generating knockout animals and transgenic lines. Given that a number of findings point to the fact that actin-rich muscles are especially vulnerable, it would be interesting to examine the role of the Mical–SelR redox system in late-onset myopathies and other diseases affecting the formation, maturation and/or maintenance of sarcomeres. Another question arising from these findings is the potential interplay of this redox system with other actin regulators such as cofilins or the Arp2/3 complex, which could reveal fundamental similarities or differences in the organization and dynamics of the actin cytoskeleton in growth cones, muscles and bristles. There seem to be a myriad of bristle mutants and other model systems waiting to address these questions. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Hung, R. J. et al. Nature 463, 823–827 (2010). 2. Hung, R. J., Pak, C. W. & Terman, J. R. Science 334, 1710–1713 (2011). 3. Lee, B. C. et al. Mol. Cell 51, 397–404 (2013). 4. Hung, R. J., Spaeth, C. S., Yesilyurt, H. G. & Terman, J. R. Nat. Cell Biol. 15, 1445–1454 (2013). 5. Suzuki, T. et al. J. Biol. Chem. 277, 14933–14941 (2002). 6. Terman, J. R., Mao, T., Pasterkamp, R. J., Yu, H. H. & Kolodkin, A. L. Cell 109, 887–900 (2002). 7. Weide, T., Teuber, J., Bayer, M. & Barnekow, A. Biochem. Biophys. Res. Comm. 306, 79–86 (2003). 8. Tilney, L. G. & DeRosier, D. J. Proc. Natl Acad. Sci. USA 102, 18785–18792 (2005). 9. Kryukov, G. V., Kumar, R. A., Koc, A., Sun, Z. & Gladyshev, V. N. Proc. Natl Acad. Sci. USA 99, 4245– 4250 (2002). 10. Zhou, Y., Gunput, R. A., Adolfs, Y. & Pasterkamp, R. J. Cell. Mol. Life Sci. 68, 4033–4044 (2011). 11. Schmidt, E. F., Shim, S. O. & Strittmatter, S. M. J. Neurosci. 28, 2287–2297 (2008). 12. Giridharan, S. S., Rohn, J. L., Naslavsky, N. & Caplan, S. J. Cell Sci. 125, 614–624 (2012). 13. Laing, N. G. et al. Hum. Mutat. 30, 1267–1277 (2009). 14. Beuchle, D., Schwarz, H., Langegger, M., Koch, I. & Aberle, H. Mech. Dev. 124, 390–406 (2007).
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 12 | DECEMBER 2013 © 2013 Macmillan Publishers Limited. All rights reserved
