Journal: Developmental cell

Article Title: Arginine Methylation by PRMT2 Controls the Functions of the Actin Nucleator Cobl.

doi: 10.1016/j.devcel.2018.03.007

Figure Lengend Snippet: Figure 1. PRMT2 Is a Binding Partner of the Actin Nucleator Cobl (A) Yeast-2-hybrid analysis using BD-Cobl10011337 as bait identified PRMT2 as Cobl binding partner (for example, prey AD-3456 encoding for aa 40–445 of PRMT2). Growth on quadruple dropout media (-Ade/-His/-Leu/-Trp) and b-Gal-activity (blue) indicate reporter gene activities. Successful transfection with both plasmids is seen by growth on -Leu/-Trp medium. (B) Scheme of mouse PRMT2 domain structure with SH3 and catalytic domain (GI: 117938280). Also see Figures S1A and S1B for further information and high- sequence conservation between mouse, rat, and human PRMT2. (C) Coprecipitation analyses using immobilized GST-PRMT2 and deletion mutants thereof, respectively, and GFP-Cobl overexpressed in HEK293 cells (for GFP controls see Figure S1C). Cobl interacts with PRMT2 in an SH3 domain-dependent manner (blue arrowhead). (D–G) Reconstitutions of Cobl/PRMT2 protein complexes in intact COS-7 cells. Mitochondrially anchored human PRMT2 (Mito-mCherry-PRMT2) (E) and PRMT2 SH3 domain (Mito-mCherry-PRMT2SH3) (G), but neither the deletion mutant lacking the SH3 domain (Mito-mCherry-PRMT2DSH3) (F) nor Mito-mCherry (D) re- sulted in a specific recruitment of GFP-Cobl to mitochondria (stained with MitoTracker). Arrowheads mark examples of mitochondrial colocalization (appearing white in merge). For GFP controls see Figures S1D–S1G. Scale bar, 10 mm.

Article Snippet: For analyzing the argininemethylation of GFP-Cobl and its mutants, HEK293 cells were lyzed in lysis or RIPA buffer and GFP-Cobl fusion proteins were immunoprecipitated by 5 mg antiGFP antibodies (Abcam, ab290).

Techniques: Binding Assay, Activity Assay, Transfection, Sequencing, Mutagenesis, Staining

Journal: Developmental cell

Article Title: Arginine Methylation by PRMT2 Controls the Functions of the Actin Nucleator Cobl.

doi: 10.1016/j.devcel.2018.03.007

Figure Lengend Snippet: Figure 5. Cobl is Arginine-Methylated, and Arginine Methylation Is a Crucial Requirement for Cobl-Mediated Dendrite and Dendritic Branch Formation (A–D) Representative images (anti-MAP2 channels) of rat primary hippocampal neurons transfected with GFP-Cobl and incubated with methylation inhibitors. Scale bar, 10 mm. (E and F) Quantitative analyses unveiling a suppression of Cobl-mediated dendrite and dendritic branch formation on incubation with methylation inhibitors. GFP/ DMSO, n = 107; GFP-Cobl/DMSO, n = 102; GFP-Cobl/MTA, n = 58; GFP-Cobl/Adox, n = 70; GFP-Cobl/AMI-1, n = 73 cells (24 hr inhibitor incubations; relatively mild conditions not leading to significant negative effects in control cells [Figure S3]). Data are mean ± SEM normalized to internal GFP/DMSO control. Statistical significances, one-way ANOVA and Tukey’s post-hoc test. *p < 0.05, ***p < 0.001. (G) Immunoblotting with anti-Me-Arg antibodies revealing that endogenous Cobl precipitated from rat brain extracts is arginine methylated. Arrowheads mark full- length Cobl. (H) Immunoprecipitation analyses revealing that GFP-Cobl expressed in HEK293 cells and immunoprecipitated with anti-GFP antibodies is arginine methylated (arrowhead). White lines indicate lanes omitted from the blots.

Article Snippet: For analyzing the argininemethylation of GFP-Cobl and its mutants, HEK293 cells were lyzed in lysis or RIPA buffer and GFP-Cobl fusion proteins were immunoprecipitated by 5 mg antiGFP antibodies (Abcam, ab290).

Techniques: Methylation, Transfection, Incubation, Control, Western Blot, Immunoprecipitation

Journal: Developmental cell

Article Title: Arginine Methylation by PRMT2 Controls the Functions of the Actin Nucleator Cobl.

doi: 10.1016/j.devcel.2018.03.007

Figure Lengend Snippet: Figure 6. Arginine Methylation Occurs in Cobl’s C-Terminal Actin Nucleating Part and Is Promoted by PRMT2 SH3 Domain Association with the Cobl Homology Domain (A and B) Mapping of PRMT2 SH3 domain binding sites on Cobl. (A) Scheme of (murine) Cobl summarizing the experimental data shown in (B). Yellow boxes within Cobl Homology domain, KrRAPpPP motifs; orange box, further, multiple PxxP motifs. Red boxes, G-actin-binding WH2 domains. Lines depict GFP-tagged Cobl deletion mutants (expressed in HEK293 cells) used to map the PRMT2 binding site coarsely by coprecipitation experiments with immobilized GST-PRMT2SH3 and anti-GFP immunoblotting (B) (green, binding; red, not binding). For controls see Figure S5.

Article Snippet: For analyzing the argininemethylation of GFP-Cobl and its mutants, HEK293 cells were lyzed in lysis or RIPA buffer and GFP-Cobl fusion proteins were immunoprecipitated by 5 mg antiGFP antibodies (Abcam, ab290).

Techniques: Methylation, Binding Assay, Western Blot