Actinin BioID reveals sarcomere crosstalk with oxidative metabolism through interactions with IGF2BP2

Figures

• 

  Figure 1.. BioID to identify actinin proximity partners through sarcomere assembly (A) Overview of actinin localization in cardiomyocytes including sarcomere Z-disk, focal adhesion, and cortical actin cytoskeleton. (B) Overview of the BioID method using Actinin-BirA* fusion to study actinin-proximal protein networks. (C) Actinin-BirA* and control non-BirA* iPSC-CM lysates were probed with anti-actinin and anti-GAPDH antibodies to identify (left) and quantify (right) Actinin-BirA* fusion (~130 kD) and endogenous actinin (~100 kD). (D) Actinin-BirA* cardiac microtissues have similar twitch force compared to non-BirA* controls (n = 12–15) (scale bar, 150 μm). (E) Confocal micrograph of Actinin-BirA* iPSC-CMs decorated with antibodies to actinin (red), streptavidin-AF488 (green), and DAPI DNA co-stain (blue) (scale bar, 10 μm). (F) Overview of BioID experimental methods. (G) iPSC-CM lysates were probed with streptavidin-horseradish peroxidase (HRP) to examine Actinin-BirA*-biotinylated proteins. (H) Heatmap and hierarchical clustering of log2-transformed intensity values for the 324 Actinin-BirA* hits (L2FC ≥ 1 and FDR < 0.05 relative to control non-BirA*) from combined TMT experiments. (I)…

• 

  Figure 2.. Identification of transcripts bound to RNA-binding proteins in actinin neighborhoods (A) Overview of modified RIP-seq protocol to study RNA transcripts bound to RNA-binding proteins in actinin neighborhoods by streptavidin affinity purification of RNA-binding proteins followed by RNA isolation and sequencing. (B) PCA plot shows clustering of Actinin-BirA* RIP-seq samples relative to non-BirA* controls (n = 4). (C) Volcano plot of RIP-seq data shaded red for 945 enriched transcripts (FDR < 0.05, L2FC ≥ 0.5) or blue for 1,113 depleted transcripts (FDR < 0.05, L2FC ≤ −0.5). Differentially enriched transcripts were analyzed by GO and enrichment terms are listed for enriched and depleted transcripts. (D) ETC gene components spanning respiratory chain complex I-V proteins are enriched on average. Each circle represents a single ETC gene; black circle for statistically significant transcript. (E) Glycolysis gene components are not enriched on average. Each circle represents a single glycolysis gene. (F) Thin and thick filament sarcomere are depleted on average. Each circle represents a single sarcomere gene. (G) qPCR validation of candidate RIP-seq hits (n = 4). (H) Representative…

• 

  Figure 3.. Fine mapping actinin interactions with the RNA-binding protein IGF2BP2 (A) Representative immunoblot probed with antibodies to IGF2BP2, PCPB1, PCPB2, and SERBP1 in streptavidin affinity-purified iPSC-CM lysates. Double band represents two IGF2BP2 splice products. (B) Representative immunoblot probed with antibodies to IGF2BP2, PCPB1, PCPB2, and SERBP1 in anti-HA immunoprecipitated iPSC-CM lysates. (C) M2H (conducted in HEK293T cells) results demonstrating that IGF2BP2 interacts with full-length actinin, but not PCBP1, PCPB2, or SERBP1. (D) Representative confocal micrograph demonstrating partial colocalization of actinin (anti-actinin; red) with IGF2BP2 (anti-IGF2BP2; green) with DAPI DNA (blue) co-stain in iPSC-CMs (scale bars: main image, 10 μm; inset, 5 μm). (E) M2H study conducted to fine map the direct interaction between actinin and IGF2BP2. IGF2BP2 was divided into RRM (residues 1–160) and KH (residues 161–600) domains to fine map the interaction. (F) M2H results to map the direct interaction between the KH domain of IGF2BP2 and Actinin. Actinin was divided into AB (residues 1–275), rod (residues 276–750), and CaM domains (residues 751–895). (G) M2H results to map…

• 

  Figure 4.. Actinin-IGF2BP2 interactions regulate ETC transcript localization to actinin neighborhoods and metabolic adaptation to hypercontractility in an HCM model (A) Representative immunoblot of lysates from iPSC-CMs treated with shRNA targeting IGF2BP2 or scramble control probed with antibodies to IGF2BP2 and GAPDH. (B) Streptavidin affinity purification of transcripts bound to RNA-binding proteins in actinin neighborhoods after IGF2BP2 knockdown, followed by qPCR of ETC transcripts NDUFA1, NDUFA8, NDUFA11, and COX6B1. (C) qPCR analysis of global transcript levels of NDUFA1, NDUFA8, NDUFA11, and COX6B1 after IGF2BP2 knockdown. (D) Streptavidin affinity purification of transcripts bound to actinin-proximal RNA-binding proteins with overexpressing lenti-Actinin-WT or lenti-Actinin-E445A, followed by qPCR of ETC transcripts NDUFA1, NDUFA8, NDUFA11, and COX6B1. (E) Oxygen consumption rates after co-overexpression of either lenti-Actinin-WT or lenti-Actinin-E445A with lenti-cTnT-WT or lenti-cTnT-R92Q in iPSC-CMs. (F) OCR parameter quantifications comparing lenti-Actinin-WT and lenti-Actinin-E445A with either lenti-cTnT-WT or lenti-cTnT-R92Q. (G) LDH activity in conditioned media collected…