Volume 24, Issue 7, Pages 1880-1889 (August 2018) The C. elegans BRCA2-ALP/Enigma Complex Regulates Axon Regeneration via a Rho GTPase-ROCK-MLC Phosphorylation Pathway  Tatsuhiro Shimizu, Strahil Iv. Pastuhov, Hiroshi Hanafusa, Kunihiro Matsumoto, Naoki Hisamoto  Cell Reports  Volume 24, Issue 7, Pages 1880-1889 (August 2018) DOI: 10.1016/j.celrep.2018.07.049 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 24, 1880-1889DOI: (10.1016/j.celrep.2018.07.049) Copyright © 2018 The Authors Terms and Conditions

Figure 1 BRC-2 Is Required for Efficient Axon Regeneration (A) Structure of BRC-2. Schematic diagrams of BRC-2 and its mammalian counterpart BRCA2 are shown. Domains are shown as follows: a BRC motif (red) and an OB fold (green). The bold line underneath denotes the extent of the deleted region in the tm1086 mutant. (B) Representative D-type motor neurons in wild-type and brc-2 mutant animals 24 hr after laser surgery. In wild-type animals, a severed axon has regenerated a growth cone (arrow). In brc-2 mutants, proximal ends of axons failed to regenerate (arrowhead). Scale bar, 10 μm. (C) Percentages of axons that initiated regeneration 24 hr after laser surgery. Error bars indicate 95% confidence intervals (CIs). ∗p < 0.05, ∗∗p < 0.01, as determined by Fisher’s exact test. Cell Reports 2018 24, 1880-1889DOI: (10.1016/j.celrep.2018.07.049) Copyright © 2018 The Authors Terms and Conditions

Figure 2 ALP-1 Is Required for Efficient Axon Regeneration (A) Structure of ALP-1. Schematic diagrams of ALP-1 isoforms are shown. The alp-1 gene encodes one ALP (ALP-1a) and three Enigma (ALP-1b, -1c, and -1d) isoforms. Domains are shown as follows: a PDZ domain (P; yellow) and a LIM domain (L; blue). The bold underline denotes the extent of the deleted region in the ok820 mutant. The Enigma isoforms have four LIM domains. (B) Percentages of axons that initiated regeneration 24 hr after laser surgery. Error bars indicate 95% CIs. ∗p < 0.05, as determined by Fisher’s exact test. NS, not significant. (C) Interaction between BRC-2 and ALP-1. HEK293 cells were transfected with plasmids encoding FLAG-ALP-1 and T7-BRC-2, as indicated. Whole-cell extracts (WCEs) and immunoprecipitated complexes obtained with anti-T7 antibody (immunoprecipitation [IP]: T7) were analyzed by immunoblotting (IB). Cell Reports 2018 24, 1880-1889DOI: (10.1016/j.celrep.2018.07.049) Copyright © 2018 The Authors Terms and Conditions

Figure 3 ALP-1 Interacts with LET-502 (A) RHO-1–LET-502 signaling pathway. RHO-1-GTP activates LET-502. (B) Yeast two-hybrid assays for the interactions of LET-502 with BRC-2 and ALP-1. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-ALP-1, GAL4 DBD-BRC-2, and GAL4 AD-LET-502, as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 10 mM 5-aminotriazole (AT) for 4 days. (C) Interaction between ALP-1 and LET-502. HEK293 cells were transfected with plasmids encoding FLAG-ALP-1 and HA-LET-502 (Full or C2), as indicated. WCEs and immunoprecipitated complexes obtained with anti-FLAG antibody (IP: FLAG) were analyzed by IB. Cell Reports 2018 24, 1880-1889DOI: (10.1016/j.celrep.2018.07.049) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Effects of BRC-2 on the Interaction between ALP-1 and LET-502 (A) Structure of LET-502. Schematic diagrams of LET-502 are shown. Domains are shown as follows: a kinase domain (kinase; red), a RBD (R; green), and a PH domain (PH; orange). (B and C) Yeast two-hybrid assays for the interactions of ALP-1 with LET-502-N and LET-502-C in the absence (B) or presence (C) of BRC-2. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-ALP-1, GAL4 AD-LET-502-N, GAL4 AD-LET-502-C, and BRC-2 (C) as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and tryptophan and containing 1 mM (B) or 10 mM (C) 5-AT for 4 days. Cell Reports 2018 24, 1880-1889DOI: (10.1016/j.celrep.2018.07.049) Copyright © 2018 The Authors Terms and Conditions

Figure 5 BRC-2 and ALP-1 Are Associated with the Rho–ROCK Pathway in the Regulation of Axon Regeneration (A and B) Percentages of axons that initiated regeneration 24 hr after laser surgery. The percentages in wild-type and mutant animals (A) and the effects of transgenes in brc-2 and alp-1 mutants (B) are shown. Error bars indicate 95% CIs. ∗p < 0.05, ∗∗p < 0.01, as determined by Fisher’s exact test. NS, not significant. (C) Comparison of ROCK and LET-502 structures. Schematic diagrams of human ROCK1, LET-502, and LET-502ΔC are shown. Domains are shown as follows: a kinase domain (kinase; red), a RBD (R; green), and a PH domain (PH; orange). Cell Reports 2018 24, 1880-1889DOI: (10.1016/j.celrep.2018.07.049) Copyright © 2018 The Authors Terms and Conditions

Figure 6 LET-502, BRC-2, and ALP-1 Promote Axon Regeneration through Phosphorylation of the MLC (A) Structure of MLC-4. Schematic diagrams of MLC-4 and its mammalian counterpart MLC2 are shown. Asterisks indicate regulatory residues Thr-17 and Ser-18. EF-hand domains (EFh; black) are shown. (B) Percentages of axons that initiated regeneration 24 hr after laser surgery. Error bars indicate 95% CIs. ∗p < 0.05, as determined by Fisher’s exact test. NS, not significant. (C) Yeast two-hybrid assays for the interaction of ALP-1 with MLC-4. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-ALP-1 and GAL4 AD-MLC-4 (wild-type [WT], DD, or AA), as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 1 mM 5-AT for 4 days. Cell Reports 2018 24, 1880-1889DOI: (10.1016/j.celrep.2018.07.049) Copyright © 2018 The Authors Terms and Conditions

Figure 7 Effects of LET-502 on the Phosphorylation of MLC-4 (A) Phosphorylation of MLC-4 in D-type motor neurons after axon injury. Immunofluorescent staining of severed axons in animals carrying Punc-25::venus::mlc-4 (WT) or Punc-25::venus::mlc-4(AA) (AA) are shown. Each image was taken 6 hr after laser surgery. Arrowheads indicate the ends of proximal axons. The numbers (n) of axons examined are shown below. All of them showed the same pattern of P-MLC-4 fluorescence. Swelled tip formation: wild-type + MLC-4(WT), 7 out of 7; wild-type + MLC-4(AA), 4 out of 4; let-502ts + MLC-4(WT), 1 out of 4. Scale bar, 2 μm. (B) Morphology of severed ends in D-type motor neurons after axon injury. Fluorescent images of severed axons in wild-type, let-502 and MLC-4(DD)-expressing let-502 animals are shown. D-type motor neurons were visualized by expressing GFP from the D neuron-specific promoter Punc-25. Each image was taken 6 hr after laser surgery. Scale bars, 2 μm. See also Figure S4. (C) Regenerating growth cones of D-type motor neurons. Severed axons of D neurons in wild-type and MLC-4(DD)-expressing wild-type animals were visualized by GFP. Time-lapse imaging was performed as described in the Experimental Procedures. The images of each severed axon taken at the indicated times after growth cone formation are shown. The images from the merged time series are shown to the right, with time information displayed as a gradual transition from green to red. Scale bar, 5 μm. See also Figure S5. Cell Reports 2018 24, 1880-1889DOI: (10.1016/j.celrep.2018.07.049) Copyright © 2018 The Authors Terms and Conditions