mical directs the organization of actin in a number of different cell types   including within developing bristle processes , which are akin to mammalian mechanotransducing inner ear hair cells that detect sound . bristles have also long served as a simple , single cell model for characterizing actin dependent events in vivo . raising the levels of mical specifically in bristle cells using the gal4-uas   system ( bristle - specific gal4/uas : mical ) results in f - actin disassembly and bristle branching ( compare figure 1a and b ) that is dependent on mical 's redox activity and the met-44 residue of actin . thus , to better characterize mical - mediated f - actin alterations , we have initiated a large - scale genetic screen to look for enhancers and suppressors of mical - mediated bristle branching . one of the mutations that we identified in our genetic screen , the transposable element mutation ey22443 , strongly suppressed mical - induced actin - dependent bristle branching ( figure 1b - d ) . molecular analysis revealed that the ey22443 transposable element mutation was situated within the drosophila selr gene ( figure 1e ) . selr codes for a methionine sulfoxide reductase ( msrb ) family enzyme , that has been characterized for its ability to reduce oxidized methionine residues . in light of our observations that mical oxidizes methionine residues on actin , we wondered if selr might play a role in modulating mical 's effects on actin . the ey22443 transposable element mutation situated in selr contains a uas promoter ( figure 1e ) , thereby suggesting that this mutation might be abnormally inducing selr expression to suppress gal4/uas : mical - dependent bristle branching . to test this hypothesis consistent with our results with ey22443 ( figure 1c - d ) and another uas - containing mutation within selr , ep3340 ( figure 1d ) , multiple transgenic lines revealed that raising the levels of selr specifically in bristles strongly suppressed mical - induced bristle branching and even generated normal appearing bristles ( figure 1f ) . moreover , elevating the levels of selr in a wild - type background generated abnormally bent bristles that resembled mical mutant bristles ( figure s1 ; ) ; and these effects of selr were genetically enhanced by decreasing the levels of mical ( figure s1 ) . further analysis revealed that selr localized with mical at the tips of bristles and suppressed mical - mediated f - actin disassembly and reorganization ( figure 1f ) . therefore , selr counteracts the effects of mical on actin reorganization in vivo . to better understand the role of selr in counteracting mical - mediated actin reorganization , we purified recombinant drosophila selr protein ( figure s2 ) . using in vitro actin biochemical and imaging assays , we previously observed that purified mical protein in the presence of its coenzyme nadph disrupts actin polymerization and induces f - actin disassembly ( figure 2a ; ) . strikingly , we found that purified selr protein rescued the ability of mical - treated actin to polymerize ( figure 2a ) . this mical / selr - treated actin re - polymerized to an extent that was indistinguishable from normal untreated actin ( figure 2b ) . moreover , while mical - treated actin failed to polymerize even after removal of mical and nadph ( figure 2c ; ) , selr induced the polymerization of this purified mical - treated actin in a dosage - dependent manner ( figure 2c ) . selr converts methionine sulfoxide ( meto ) to methionine , requiring a redox active cysteine ( cys124 ) residue ( figure 2d - e ; ) and also utilizing reducing agents to cycle back to its reduced form ( figures 2d , s3 ; ) . in some cases methionine oxidation is also reversed by general reducing agents , so we wondered if mical - treated actin was specifically reversed by selr . in contrast to selr , neither chemical reducing agents such as dtt ( figures 2a [ buffer only contains dtt ] ; s3 ) nor other reducing enzymes including thioredoxins / thioredoxin reductases altered mical - mediated effects on actin in vitro ( figure s3 ) or in vivo ( figure 1d ) . furthermore , selr did not restore the normal polymerization properties of other oxidized forms of actin ( e.g. , h2o2-treated actin ; figure s3 ) , indicating that selr selectively affects mical- modified actin . mutating selr 's critical catalytic cysteine ( cys124 ) to generate an enzymatically dead selr ( selr ; figure 2e ; ) , abolished selr 's effects on mical - treated actin in vitro ( figures 2b , f ) and in vivo ( figure 2 g ) . moreover , consistent with such a role for selr 's reductase activity in counteracting mical 's oxidative effects on actin , elevating the levels of wild - type selr not only phenocopied the in vivo effects of disrupting mical 's monooxygenase ( redox ) domain ( figures s1 , s4 ) , but it also rescued the severe bristle / f - actin alterations that result from hyperactive mical redox signaling ( figure s4 ; mical ; ) . thus , selr specifically employs its catalytic activity to restore mical - treated actin polymerization and counteract the in vivo effects of mical . in many organisms , including drosophila and mammals , two main types of methionine sulfoxide reductases have been identified : selr ( msrb family proteins ) and drosophila eip71cd ( msra ) ( figure 3a ; ) . interestingly , selr and msra / eip71cd are both methionine sulfoxide reductases , but they do not exhibit similarity in their sequence , domain organization , or substrate specificity ( figure 3a - b ; 
 ) . in particular , methionine has a unique oxidation pattern in that two stereoisomers can be produced by oxidation . selr / msrb family proteins catalyze the reduction of the r - isomer of methionine sulfoxide ( methionine - r - sulfoxide ; figure 3b , top ) to methionine , while msra / eip71cd catalyzes the reduction of the s - isomer of methionine sulfoxide ( methionine - s - sulfoxide ; figure 3b , bottom ) to methionine . therefore , to further test the specificity of selr in restoring the polymerization properties of mical - treated actin , we purified recombinant msra / eip71cd protein ( figure s2 ; ) . unlike selr , msra / eip71cd did not restore the polymerization properties of mical - treated actin in vitro ( figure 3c ) , nor did it counteract mical - mediated actin reorganization / bristle branching in vivo ( figure 1d ) . these results further reveal that mical - treated actin polymerization is specifically restored by selr . moreover , in light of the isomer - specific nature of the methionine sulfoxide enzymes selr and msra / eip71cd , these results also indicate that mical oxidizes actin in a stereo - specific manner . mical oxidizes actin on its met-44 and met-47 residues , although it is the oxidation of the met-44 residue through which mical induces f - actin disassembly . previously , we determined the conditions to purify mical - treated actin , which is polymerization impaired and exhibits a mass increase of two oxygens ( 32 daltons ) . selr , but not the enzymatically dead selr protein , restored the polymerization properties of purified mical - treated actin ( figure s3 ) , an effect that was maintained even after removal of selr ( figure 3d ) . subjecting both purified mical / selr - treated and mical / selr - treated actin to mass spectrometry revealed that selr , but not the enzymatically dead selr protein , eliminated the mical - catalyzed two oxygen ( 32 dalton ) mass increase on actin ( figure 3e ) . mical 's ability to effect actin in vitro and in vivo is dependent on the presence of the methionine ( m ) 44 residue of actin . to further examine a physiological role for selr in reducing mical - mediated oxidation of met-44 , we turned to in vivo assays . we first noted that overexpression of either a non - mical oxidizable met44leu ( m44l ) version of actin   or wild - type selr generated the same effects : suppression of mical - mediated actin / bristle morphology and mical loss - of - function - like defects ( figures 1f , 2 g , s1 , s4 ; ) . furthermore , we found that actin worked in combination with selr to generate mical loss - of - function - like bristle defects ( figure s4 ) . moreover , actin prevented the enhanced mical - mediated bristle branching / actin reorganization that occured with expression of the reductase dead selr ( figure 2 g ) . thus , selr reverses mical - mediated oxidation of actin , including using its catalytic activity to directly reduce mical - induced meto-44 actin to met-44 actin ( figure 3f )  and these observations with purified proteins are supported by our in vivo genetic assays . in addition to bristle cells , mical regulates the organization of actin in multiple other cell types including mammalian cells in vitro and muscles in vivo . thus , we wondered if selr could also counteract the effects of mical on actin in these other cellular systems . our initial examination revealed that as in bristle cells , selr rescued mical - dependent changes in morphology and actin organization in cultured cells ( figure 4a ) . further examination revealed that overexpression of selr in muscles in vivo phenocopied the muscle actin defects found in mical mutants ( figure 4b ; ) . moreover , selr could even rescue the lethality and changes in actin organization associated with overexpression of mical in muscles ( figure 4c )  as well as the lethality that results when mical is broadly expressed using an actin promoter ( figure 4c ) . drosophila selr , like mical , is broadly expressed ( figure s5 ; ) and thus to better examine these mical - selr interactions and their physiological effects on actin , we characterized selr mutants ( figure s6 ) . strikingly , loss of selr generated bristle and muscle defects that resembled overexpression of mical ( figure 5a and 5e ) . moreover , loss of selr specifically enhanced mical - mediated effects on actin organization / bristle morphology ( figures 5b - c , s4 ) and phenocopied overexpression of the selr reductase mutant protein ( figure 5d ) . thus , selr , like mical , plays both important and selective roles in regulating actin organization in vivo in different cell types . likewise , an equilibrium between mical and selr activities underlies normal actin - directed cell biology . besides its redox region that mical uses to oxidize actin , mical has several other domains and protein interaction motifs including a region that interacts with the cytoplasmic portion of plexin ( figure 6a ; ) . plexins are receptors for semaphorin guidance cues and play critical roles in regulating multiple actin - dependent events in vivo . semaphorins / plexins signal through mical to induce changes in bristle morphology and f - actin disassembly , so we wondered if selr also counteracted the effects of semaphorin / plexin / mical signaling . employing loss and gain - of - function genetics in the bristle system , we found that similar to our results with mical , selr counteracted semaphorin / plexin effects on actin - dependent bristle morphology ( figure s1 ) . next , we turned to in vivo axon guidance assays , where semaphorins / plexins have been characterized as repulsive axon guidance molecules   and were first linked to mical family proteins . interestingly , one of the selr mutants that we found in our screen ( ep3340 , figure 1d - e ) recently emerged from a genetic screen as an uncharacterized regulator of axon guidance . employing our selr transgenic lines , we found that overexpression of selr generated axon guidance and synaptogenic defects that phenocopy mical mutants ( figures 6b , s6 ; ) . furthermore , selr mutants generated axon guidance defects that phenocopy increased semaphorin / plexin / mical - mediated repulsive axon guidance ( figures 6c , s6 ; ) . moreover , increasing the levels of selr rescued these semaphorin / plexin / mical - triggered repulsive axon guidance defects ( figures 6d , s6 ) . thus , selr also plays critical roles in axon guidance and synaptogenesis and counteracts the effects of semaphorin / plexin / mical repulsive signaling in vivo . mical directs the organization of actin in a number of different cell types   including within developing bristle processes , which are akin to mammalian mechanotransducing inner ear hair cells that detect sound . bristles have also long served as a simple , single cell model for characterizing actin dependent events in vivo . raising the levels of mical specifically in bristle cells using the gal4-uas   system ( bristle - specific gal4/uas : mical ) results in f - actin disassembly and bristle branching ( compare figure 1a and b ) that is dependent on mical 's redox activity and the met-44 residue of actin . thus , to better characterize mical - mediated f - actin alterations , we have initiated a large - scale genetic screen to look for enhancers and suppressors of mical - mediated bristle branching . one of the mutations that we identified in our genetic screen , the transposable element mutation ey22443 , strongly suppressed mical - induced actin - dependent bristle branching ( figure 1b - d ) . molecular analysis revealed that the ey22443 transposable element mutation was situated within the drosophila selr gene ( figure 1e ) . selr codes for a methionine sulfoxide reductase ( msrb ) family enzyme , that has been characterized for its ability to reduce oxidized methionine residues . in light of our observations that mical oxidizes methionine residues on actin , we wondered if selr might play a role in modulating mical 's effects on actin . the ey22443 transposable element mutation situated in selr contains a uas promoter ( figure 1e ) , thereby suggesting that this mutation might be abnormally inducing selr expression to suppress gal4/uas : mical - dependent bristle branching . to test this hypothesis consistent with our results with ey22443 ( figure 1c - d ) and another uas - containing mutation within selr , ep3340 ( figure 1d ) , multiple transgenic lines revealed that raising the levels of selr specifically in bristles strongly suppressed mical - induced bristle branching and even generated normal appearing bristles ( figure 1f ) . moreover , elevating the levels of selr in a wild - type background generated abnormally bent bristles that resembled mical mutant bristles ( figure s1 ; ) ; and these effects of selr were genetically enhanced by decreasing the levels of mical ( figure s1 ) . further analysis revealed that selr localized with mical at the tips of bristles and suppressed mical - mediated f - actin disassembly and reorganization ( figure 1f ) . to better understand the role of selr in counteracting mical - mediated actin reorganization , we purified recombinant drosophila selr protein ( figure s2 ) . using in vitro actin biochemical and imaging assays , we previously observed that purified mical protein in the presence of its coenzyme nadph disrupts actin polymerization and induces f - actin disassembly ( figure 2a ; ) . strikingly , we found that purified selr protein rescued the ability of mical - treated actin to polymerize ( figure 2a ) . this mical / selr - treated actin re - polymerized to an extent that was indistinguishable from normal untreated actin ( figure 2b ) . moreover , while mical - treated actin failed to polymerize even after removal of mical and nadph ( figure 2c ; ) , selr induced the polymerization of this purified mical - treated actin in a dosage - dependent manner ( figure 2c ) . selr converts methionine sulfoxide ( meto ) to methionine , requiring a redox active cysteine ( cys124 ) residue ( figure 2d - e ; ) and also utilizing reducing agents to cycle back to its reduced form ( figures 2d , s3 ; ) . in some cases methionine oxidation is also reversed by general reducing agents , so we wondered if mical - treated actin was specifically reversed by selr . in contrast to selr , neither chemical reducing agents such as dtt ( figures 2a [ buffer only contains dtt ] ; s3 ) nor other reducing enzymes including thioredoxins / thioredoxin reductases altered mical - mediated effects on actin in vitro ( figure s3 ) or in vivo ( figure 1d ) . furthermore , selr did not restore the normal polymerization properties of other oxidized forms of actin ( e.g. , h2o2-treated actin ; figure s3 ) , indicating that selr selectively affects mical- modified actin . mutating selr 's critical catalytic cysteine ( cys124 ) to generate an enzymatically dead selr ( selr ; figure 2e ; ) , abolished selr 's effects on mical - treated actin in vitro ( figures 2b , f ) and in vivo ( figure 2 g ) . moreover , consistent with such a role for selr 's reductase activity in counteracting mical 's oxidative effects on actin , elevating the levels of wild - type selr not only phenocopied the in vivo effects of disrupting mical 's monooxygenase ( redox ) domain ( figures s1 , s4 ) , but it also rescued the severe bristle / f - actin alterations that result from hyperactive mical redox signaling ( figure s4 ; mical ; ) . thus , selr specifically employs its catalytic activity to restore mical - treated actin polymerization and counteract the in vivo effects of mical . in many organisms , including drosophila and mammals , two main types of methionine sulfoxide reductases have been identified : selr ( msrb family proteins ) and drosophila eip71cd ( msra ) ( figure 3a ; ) . interestingly , selr and msra / eip71cd are both methionine sulfoxide reductases , but they do not exhibit similarity in their sequence , domain organization , or substrate specificity ( figure 3a - b ; 
 ) . in particular , methionine has a unique oxidation pattern in that two stereoisomers can be produced by oxidation . selr / msrb family proteins catalyze the reduction of the r - isomer of methionine sulfoxide ( methionine - r - sulfoxide ; figure 3b , top ) to methionine , while msra / eip71cd catalyzes the reduction of the s - isomer of methionine sulfoxide ( methionine - s - sulfoxide ; figure 3b , bottom ) to methionine . therefore , to further test the specificity of selr in restoring the polymerization properties of mical - treated actin , we purified recombinant msra / eip71cd protein ( figure s2 ; ) . unlike selr , msra / eip71cd did not restore the polymerization properties of mical - treated actin in vitro ( figure 3c ) , nor did it counteract mical - mediated actin reorganization / bristle branching in vivo ( figure 1d ) . these results further reveal that mical - treated actin polymerization is specifically restored by selr . moreover , in light of the isomer - specific nature of the methionine sulfoxide enzymes selr and msra / eip71cd , these results also indicate that mical oxidizes actin in a stereo - specific manner . mical oxidizes actin on its met-44 and met-47 residues , although it is the oxidation of the met-44 residue through which mical induces f - actin disassembly . previously , we determined the conditions to purify mical - treated actin , which is polymerization impaired and exhibits a mass increase of two oxygens ( 32 daltons ) . selr , but not the enzymatically dead selr protein , restored the polymerization properties of purified mical - treated actin ( figure s3 ) , an effect that was maintained even after removal of selr ( figure 3d ) . subjecting both purified mical / selr - treated and mical / selr - treated actin to mass spectrometry revealed that selr , but not the enzymatically dead selr protein , eliminated the mical - catalyzed two oxygen ( 32 dalton ) mass increase on actin ( figure 3e ) . mical 's ability to effect actin in vitro and in vivo is dependent on the presence of the methionine ( m ) 44 residue of actin . to further examine a physiological role for selr in reducing mical - mediated oxidation of met-44 , we turned to in vivo assays . we first noted that overexpression of either a non - mical oxidizable met44leu ( m44l ) version of actin   or wild - type selr generated the same effects : suppression of mical - mediated actin / bristle morphology and mical loss - of - function - like defects ( figures 1f , 2 g , s1 , s4 ; ) . furthermore , we found that actin worked in combination with selr to generate mical loss - of - function - like bristle defects ( figure s4 ) . moreover , actin prevented the enhanced mical - mediated bristle branching / actin reorganization that occured with expression of the reductase dead selr ( figure 2 g ) . thus , selr reverses mical - mediated oxidation of actin , including using its catalytic activity to directly reduce mical - induced meto-44 actin to met-44 actin ( figure 3f )  and these observations with purified proteins are supported by our in vivo genetic assays . in addition to bristle cells , mical regulates the organization of actin in multiple other cell types including mammalian cells in vitro and muscles in vivo . thus , we wondered if selr could also counteract the effects of mical on actin in these other cellular systems . our initial examination revealed that as in bristle cells , selr rescued mical - dependent changes in morphology and actin organization in cultured cells ( figure 4a ) . further examination revealed that overexpression of selr in muscles in vivo phenocopied the muscle actin defects found in mical mutants ( figure 4b ; ) . moreover , selr could even rescue the lethality and changes in actin organization associated with overexpression of mical in muscles ( figure 4c )  as well as the lethality that results when mical is broadly expressed using an actin promoter ( figure 4c ) . drosophila selr , like mical , is broadly expressed ( figure s5 ; ) and thus to better examine these mical - selr interactions and their physiological effects on actin , we characterized selr mutants ( figure s6 ) . strikingly , loss of selr generated bristle and muscle defects that resembled overexpression of mical ( figure 5a and 5e ) . moreover , loss of selr specifically enhanced mical - mediated effects on actin organization / bristle morphology ( figures 5b - c , s4 ) and phenocopied overexpression of the selr reductase mutant protein ( figure 5d ) . thus , selr , like mical , plays both important and selective roles in regulating actin organization in vivo in different cell types . likewise , an equilibrium between mical and selr activities underlies normal actin - directed cell biology . besides its redox region that mical uses to oxidize actin , mical has several other domains and protein interaction motifs including a region that interacts with the cytoplasmic portion of plexin ( figure 6a ; ) . plexins are receptors for semaphorin guidance cues and play critical roles in regulating multiple actin - dependent events in vivo . semaphorins / plexins signal through mical to induce changes in bristle morphology and f - actin disassembly , so we wondered if selr also counteracted the effects of semaphorin / plexin / mical signaling . employing loss and gain - of - function genetics in the bristle system , we found that similar to our results with mical , selr counteracted semaphorin / plexin effects on actin - dependent bristle morphology ( figure s1 ) . next , we turned to in vivo axon guidance assays , where semaphorins / plexins have been characterized as repulsive axon guidance molecules   and were first linked to mical family proteins . interestingly , one of the selr mutants that we found in our screen ( ep3340 , figure 1d - e ) recently emerged from a genetic screen as an uncharacterized regulator of axon guidance . employing our selr transgenic lines , we found that overexpression of selr generated axon guidance and synaptogenic defects that phenocopy mical mutants ( figures 6b , s6 ; ) . furthermore , selr mutants generated axon guidance defects that phenocopy increased semaphorin / plexin / mical - mediated repulsive axon guidance ( figures 6c , s6 ; ) . moreover , increasing the levels of selr rescued these semaphorin / plexin / mical - triggered repulsive axon guidance defects ( figures 6d , s6 ) . thus , selr also plays critical roles in axon guidance and synaptogenesis and counteracts the effects of semaphorin / plexin / mical repulsive signaling in vivo . our results reveal that mical - mediated actin alterations  a selective means to post - translationally regulate f - actin dynamics and cellular behaviors  are reversible . this micalcatalyzed reaction is directly reversed by a specific methionine sulfoxide reductase enzyme , selr / msrb , which we also find selectively controls actin - dependent cellular events in vivo and regulates specific neuronal , muscular , and mechanosensory developmental processes . we also find that selr counteracts semaphorins , which are one of the largest families of extracellular guidance cues and play a critical role in the formation and function of multiple tissues . thus , our results demonstrate an important role for these methionine sulfoxide reductases  enzymes thought to function primarily in the repair of oxidatively  damaged  methionine residues    in modulating normal signaling events . moreover , our genetic data , which reveals that selr and mical loss and gain - of - function phenotypes are opposite in appearance , indicate that selr has a specific , primary , and regulated role in counteracting mical during development . the mical substrate met-44 residue of actin is conserved in all actin family members from yeast to humans   and a dominant ( hetereozygous ) mutation in the met-44 residue ( m44 t ) of skeletal muscle actin underlies a human musculoskeletal disease associated with actin accumulation and aggregation ( nemaline myopathy ) . this met-44 mutant version of human skeletal muscle actin would be predicted to prevent mical from having effects on skeletal muscle actin  and generally phenocopies both mical mutants and selr muscle overexpression . however , the met-44 residue is well - conserved and is at a subunit interface in filaments   and thus mutating it may influence f - actin organization for reasons other than that it is non oxidizable . it should be noted however , that our previous results indicate that met-44 mutant actin ( m44l ) appears to polymerize normally in vitro and in vivo , but is resistant to mical - mediated f - actin disassembly . it is also interesting to note the differences in the cellular localization we see between selr and different forms of mical . for example , in bristles , selr shows overlapping localization with mical , but is more broadly distributed than full - length mical , which strongly localizes to bristle tips ( figure 1f ; ) . the broader cellular localization of selr is similar to that seen when the hyperactive mical is expressed in bristles and other cells ( figure 4a ; ) . one of the differences between full - length mical and the hyperactive mical is the presence of the plexin - interacting region ( figure 6a ; ) . our results indicate that full - length mical is susceptible to regulation by plexin , whereas the mical protein ( which does not have the plexin - interacting region ) is not regulated by plexin   ( see also ) . interestingly , the micals express multiple different transcripts , including versions that may be similar to mical . thus , there may be roles for both endogenous sema / plexin - regulated and perhaps , non sema / plexin - regulated forms of mical ( which appear to be more generally localized in cells ) . in any case , it should be noted that we find that selr rescues both the lethality and f - actin defects associated with overexpression of either full - length mical or mical . our results herein , coupled with our previous observations , also indicate that unlike diffusible oxidants that induce random protein modifications , mical - mediated oxidation is substrate , residue , and stereo - specific . our results indicate that mical oxidizes the methioinine-44 residue of actin stereospecifically to generate actin methionine-44-r - sulfoxide ( actin ) to alter f - actin dynamics . these observations contend that the enzyme - driven interconversion of specific met / met(r)o residues , similar to the reversible phosphorylation of specific serine , threonine , and tyrosine residues , provides a selective means to precisely modulate protein function . moreover , in contrast to a view that oxidation simply plays a destructive role in cell health and protein function , our results indicate that the site specific and reversible oxidation of proteins is critical for proper cellular physiology . thus , together , our results uncover a specific reversible redox cellular signaling system that dynamically regulates multiple actin cytoskeletal - mediated events and controls semaphorin / plexin repulsion .