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SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics

Nature Cell Biology volume 15, pages 1445–1454 (2013)Cite this article

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Abstract

Actin’s polymerization properties are markedly altered by oxidation of its conserved Met 44 residue. Mediating this effect is a specific oxidation–reduction (redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met 44. We now find that this actin-regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase (MsrB) enzyme SelR that opposes Mical redox activity and Semaphorin–Plexin repulsion to direct multiple actin-dependent cellular behaviours in vivo. SelR specifically catalyses the reduction of the R isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereospecifically to generate actin Met-44-R-sulfoxide (actinMet(R)O−44), and also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible redox actin regulatory system that controls cell and developmental biology.

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Figure 1: SelR counteracts Mical-mediated actin-dependent changes in vivo.
Figure 2: SelR restores the polymerization properties of Mical-treated actin.
Figure 3: SelR/MsrB reverses Mical-mediated actinMet 44 oxidation.
Figure 4: SelR opposes Mical-mediated effects in different cell types.
Figure 5: SelR is required in vivo for normal actin organization and cellular morphology.
Figure 6: SelR counteracts Semaphorin–Plexin–Mical repulsive signalling.

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Acknowledgements

We thank C. Cowan, E. Kavalali, H. Kramer, C. Pak, E. Reisler and M. Rosen for comments and FlyBase, J. Yoon (UT Southwestern, USA), H. Aberle (University of Münster, Germany), Kyoto Stock Center, Vienna Drosophila RNAi Center and the Bloomington Stock Center for reagents. Mass spectrometry was performed by Y. Li (UT Southwestern Protein Chemistry Technology Center). Supported by Cancer Prevention Research Institute of Texas (CPRIT) predoctoral (R-J.H.), NIDA T-32-DA7290 postdoctoral (C.S.S.) and NIH (NS073968; MH085923) and Welch Foundation (I-1749) (J.R.T.) grants.

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  1. Departments of Neuroscience and Pharmacology and Neuroscience Graduate Program, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    Ruei-Jiun Hung, Christopher S. Spaeth, Hunkar Gizem Yesilyurt & Jonathan R. Terman

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R-J.H., C.S.S., H.G.Y. and J.R.T. designed/performed experiments and analysed data, R-J.H., C.S.S. and J.R.T. prepared the manuscript, R-J.H. wrote the paper, and C.S.S. and J.R.T. assisted in the writing of the paper.

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Supplementary Figure 1 Additional genetic analyses of SelR and Semaphorin/Plexin/Mical signalling.

a–b, Elevating the levels of SelR in a wild-type background generates abnormally bent bristles (a) that resemble Mical loss-of-function homozygous (Mical−/−) mutant and Mical dominant-negative mutant bristles (b; 1). c, Bristle specific expression of high levels of SelR in a Mical bristle overexpression background generates bristles that resemble Mical−/− mutant bristles (b). d, The bristle defects that result from 1 copy overexpression of SelR in bristles is strongly enhanced by loss of 1 copy of Mical (Mical heterozygous [Mical+/−] mutants). MicalDf(3R)swp2 allele. Chi-Square Test; ***P<0.0001. n = 20 animals per genotype. Replicated in at least 2 independent experiments (separate crosses) per genotype. e-g, SelR counteracts Semaphorin/Plexin A (PlexA) signaling. e, Overexpression of PlexA in bristles (x2 Bristle PlexA) results in bristle branching 1. Overexpression of SelR in bristles strongly suppresses these PlexA-dependent defects (x2 Bristle PlexA + x1 Bristle SelR). t-test; ***P<0.0001. n = 20 animals per genotype. Mean + standard error of the mean (SEM). Replicated in at least 2 independent experiments (separate crosses) per genotype. f, A loss-of-function/dominant negative mutant of PlexA (PlexA without its cytoplasmic region [PlexAΔCyto]; 1,2,3,4) generates bristles defects when it is expressed in bristles (x1 Bristle PlexAΔCyto; 1). Bristle overexpression of 1 copy of SelR strongly enhances these PlexAΔCyto bristle defects (x1 Bristle PlexAΔCyto+x1 Bristle SelR). In contrast, the catalytically dead SelRC124S does not enhance these PlexAΔCyto bristle defects (x1 Bristle PlexAΔCyto+x1 Bristle SelRC124S)but suppresses them. Chi-Square Test; ***P<0.0001. n = 8 animals per genotype. Replicated in at least 2 independent experiments (separate crosses) per genotype. g, A SelR heterozygous loss-of-function mutant (SelRDelta3 mutant/+) enhances PlexA-dependent bristle defects. n = 20 animals per genotype. Mean ± standard error of the mean (SEM). Replicated in at least 2 independent experiments (separate crosses) per genotype.

Supplementary Figure 2 Purity of Drosophila SelR, SelRC124S, and Eip71CD/MsrA proteins.

Standard Nickel affinity (HisTrap FF) and ion-exchange (UNO Q6 and HiTrap Q HP) chromatography procedures were employed for each protein purification. a, Purification of Drosophila SelR protein (similar to 5). Coomassie stained gels are shown and the arrowheads point to the SelR protein in both gels. The asterisk (*) indicates the SelR protein that was collected and dialyzed against SelR storage buffer prior to storage and use. b, Purification of Drosophila SelRC124S protein. Coomassie stained gels are shown and the arrowheads point to the SelRC124S protein in both gels. The asterisk (*) indicates the SelRC124S protein that was collected and dialyzed against SelRC124S storage buffer prior to storage and use. c, Purification of Drosophila Eip71CD/MsrA protein (similar to 6). Coomassie stained gels are shown and the arrowheads point to the MsrA protein in both gels. The purest eluted fraction of MsrA (*) was collected and dialyzed against MsrA storage buffer prior to storage and use. The molecular weight marker (M) is indicated in each gel.

Supplementary Figure 3 Further characterization of SelR effects on Mical-modified actin.

a, Schematic of SelR’s reducing activity and catalytic action– and how SelR can be subsequently converted from an oxidized state to a reduced state. SelR converts Met(R)-O in proteins to Met and this reduction reaction generates oxidized SelR. In particular, previous results characterizing SelR have revealed that SelR employs a catalytic cysteine (Cys)-124 thiolate, which directly interacts with methionine sulfoxide, resulting in a methionine–SelRCys−124 sulfenic acid intermediate (SelR-S-OH; 6). A subsequent reaction of this intermediate with SelRCys−69 generates an intramolecular disulfide (SelR-S-S; 6). In vitro (1), DTT can serve as a reducing agent to reduce the oxidized state of SelR 6. In vivo (2), one possible means to regenerate the oxidized state of SelR to a reduced state is through the thioredoxin (Trx)/thioredoxin reductase (TrxR) system 6,7. Thioredoxins/thioredoxin reductases are also reducing enzymes that have been implicated in regulating the properties of actin in response to oxidation 8,9,10 and also as being involved in Semaphorin/Plexin signaling 11. Other enzymes also exist that may reduce SelR 7,12,13. b, SelR, but not DTT nor thioredoxin (Trx)/thioredoxin reductase (TrxR)/NADPH alone, restores Mical-treated actin polymerization. Consistent with a catalytic requirement for SelR in restoring the polymerization properties of Mical-modified actin, it should also be noted as seen on this gel that SelR utilizes both DTT and thioredoxin/thioredoxin reductase to restore the polymerization properties of Mical-modified actin. Actin monomers/G-actin in supernatant (S); actin polymers/F-actin in pellet (P). Sedimentation/Coomassie staining assay. See also Figure S3g for uncropped gel. c, H2O2 alters actin polymerization over time when added in high concentrations (40 mM; 14,15,16) but neither SelR (2.4 μM; purple dots) nor MsrA (2.4 μM; blue dots) nor both together (1.2 μM of SelR and 1.2 μM of MsrA; grey dots) restores normal polymerization to H2O2-treated actin. d, SelR (green dots) but not SelRC124S (blue dots) induces polymerization of pyrene actin that has been treated with Mical/NADPH and purified. Pyrene actin assay. e-g, Uncropped Coomassie-stained gels for Figure 2b (e [red box]), Figure 3d (f), and Figure S3b (g).

Supplementary Figure 4 Mical and SelR Redox requirement and the Met-44 residue of actin in Mical/SelR-mediated bristle/actin reorganization.

a, Bristle-specific expression of a Mical transgene with point mutations disrupting Mical’s monooxygenase (Redox) domain (MicalΔredox) in a wild-type background generates bristle defects similar to when the levels of active SelR are increased in wild-type bristles1. b-c, Bristle overexpression of high levels of SelR (x2 bristle SelR) in a wild-type background generates severe bristle defects with multiple bends (b) that resemble bristles present in flies homozygous for a point mutation17 disrupting the Redox domain of Mical (c; Mical redox mutant [MicalI1367]; 1). d-e, Elevating the levels of wild-type SelR in bristles markedly suppresses the severe bristle/F-actin alterations 1 that result from hyperactive Mical Redox signaling (MicalredoxCH). f, In contrast to its enhancing effects on wild-type Mical in bristles, the SelR mutant (SelRDelta3/+) suppresses the effects of a loss of Mical activity (MicalΔredox) in bristles (x1 Bristle MicalΔredox+SelRDelta3/+). n = 20 animals per genotype. Replicated in at least 2 independent experiments (separate crosses) per genotype. g, Expressing a non-Mical oxidizable version of actin in bristles (x1 Bristle ActinM44L) generates bristle defects that are enhanced by SelR (x1 Bristle ActinM44L+x1Bristle SelR). Chi-Square Test; ***P<0.0001. n = 10 animals per genotype. Replicated in at least 2 independent experiments (separate crosses) per genotype.

Supplementary Figure 5 SelR expression patterns and localization.

a, Embryonic and larval patterns of SelR expression. a, In situ hybridization using an antisense probe that is specific to SelR reveals staining in Drosophila embryos including in the nervous system (brain and cord). A YFP protein fusion trap Drosophila transgenic line, SelRCPTI001015 (FlAnnotater [http://www.flyprot.org/stock_report.php?stock_id=17068#]; 18), reveals expression in the embryonic and larval nervous systems in a pattern that is similar to Mical17,19 including, as shown here, in the brain and cord, and including, in neurons and axons. Expression in embryonic muscles is also seen in this image (e.g., areas between the brackets). The higher-power inset (from the outlined region) shows SelRCPTI001015 expression in motor axons. b, SelR (CPTI001015) is present in bristles where it is distributed in small puntae (green). SelR overlaps in localization with Mical (Mical antibody 19 staining). The tip of the bristle is indicated with an arrow. c, SelR (CPTI001015), like Mical1, also localizes along the striped pattern of bundled actin filaments (phalloidin staining) in the bristle. d, SelR protein localizes to axons as seen in the embryonic (left) and larval (right) CNS, following expression of UAS: GFPSelR in neurons using the ELAV-GAL4 driver.

Supplementary Figure 6 Characterization of SelR loss-of-function mutants and additional analyses of SelR axon guidance defects.

a, Loss-of-function SelR mutations. Several transposable element mutations are situated within SelR. In addition, transposable element mutations that contained FRT sites, e00293 and d04974, were identified as situated in genes that flanked the SelR locus. Using a FLP-FRT strategy20, we employed these two transposable element alleles to delete the region between d04974 and e00293 and generate the SelRDelta3 allele. Crosses between SelRDelta3 and DfExel6159 and DfExel7305 are semi-lethal. Crosses between SelRDelta3 and DfExel7306 (which does not remove SelR; see diagram) exhibit mendelian ratios. b-d, Further presentation of axon guidance defects found in SelR neuronal overexpression and SelR−/− mutants. For reference, the nerves projecting into each hemisegment are numbered (1, 2, 3) as are the intersegmental nerve (ISN) axons. b, Further characterization of neuronal overexpression of SelR and the guidance of model axons including those within the Drosophila intersegmental nerve b (ISNb) and segmental nerve a (SNa). In wild-type embryos, ISNb axons correctly innervate their muscle targets (open arrows). In contrast, neuronal overexpression of SelR (Neuronal SelR) generates highly penetrant axon guidance defects in which ISNb axons often fail to innervate their muscle targets (closed arrows). We also observe similar highly penetrant SNa axon guidance defects in which 69% of the hemisegments were affected and 81% of these defects resemble Semaphorin−1a−/− 21, PlexinA−/− 22, and Mical−/−1,19,23 mutants. Replicated in at least 2 independent experiments (separate crosses) per genotype. Scale bar in b applies to both images. Genotype of the SelR neuronal overexpression embryos was: Elav-GAL4/+; UAS: SelR/+. c, Further characterization of SelR−/− mutant axon guidance defects. c1, Multiple different axon guidance defects are seen in SelR−/− mutants including a lack of ISNb innervation of muscles 6/7 and 12/13 (arrows), abnormal fasciculation/clumping of ISNb axons (arrowhead), and a paucity of fasciculated/bundled ISNb axons (axons in hemisegment 2). c2, SelR−/− mutants also exhibit CNS axon guidance defects including abnormal midline crossing (open arrowheads). SelR−/− = SelRDelta3/DfExel7305 for these images. d, Increasing the levels of Mical in neurons generates motor (closed arrowheads) and CNS (open arrows and open arrowheads) axon guidance defects (Neuronal Mical image and graph; 1). The CNS from Figure S6d (Neuronal Mical) is a portion of the same CNS as that presented in Figure 6c. Expressing SelR in neurons in combination with Mical significantly rescues these axon guidance defects (Neuronal Mical + Neuronal SelR image [open arrows] and graph). Chi-Square Test; ***P<0.0001. n = 80 hemisegments assessed in 8 animals per genotype. Replicated in at least 2 independent experiments (separate crosses) per genotype. Scale bar in d applies to both images.

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Hung, RJ., Spaeth, C., Yesilyurt, H. et al. SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics. Nat Cell Biol 15, 1445–1454 (2013). https://doi.org/10.1038/ncb2871

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