Numb and Numblike regulate sarcomere assembly and maintenance

Figures

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  Figure 1. Numb and Numblike are required for sarcomere assembly. (A) Electron microscopy of the myocardial sarcomere of CTL and MLC-2v-cre DKO mice on E14.5 (original magnification, ×12,0000). (B) Electrocardiogram of CTL and MLC-2v-cre DKO mice on P60.5. (C) Electrophysiology of gastrocnemius of CTL and CKMM-cre DKO mice on P60.5. (D) Statistic data of heart rate of CTL and MLC-2v-cre DKO mouse on P60.5 (n = 5). (E) Statistic data of heart rate of CTL and α-MHC-cre DKO mice on P90.5 (n = 4). (F) Stimulus voltage (threshold and plateau) on gastrocnemius of CTL and CKMM-cre DKO mice on P60.5 (n = 6). (G) Maximum, minimum, and average tension of gastrocnemius of CTL and CKMM-cre DKO mice on P60.5 (n = 6). Scale bars: 1 μm. Two-tailed Student’s t test with error bars showing mean ± SD.

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  Figure 2. Numb is a sarcomeric molecule. (A) Western blots for Numb and other ABPs in the developing heart of WT (CTL) mice. (B) Western blots for Numb and other ABPs in the developing SKMs of CTL mice. (C) The protein expression level of Numb and other ABPs in the developing heart of CTL mice (n = 4) quantitated by densitometric analysis of Western blot. (D) The protein expression level of Numb and other ABPs in developing SKMs of CTL mice (n = 4) quantitated by densitometric analysis of Western blot. (E) Double immunofluorescence staining of Numb with Desmin in the CTL mouse heart on E9.5 and P60.5 (Desmin, green; Numb, red; original magnification, ×630, zoom in ×2). (F) Double immunofluorescence staining of Numb with Desmin in the mouse SKM on P5.5 and P60.5 (Desmin, green; Numb, red; original magnification, ×630, zoom in ×2). Data are mean ± SD. Scale bars: 10 μm.

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  Figure 3. Numb and Numblike interact with sarcomeric α-Actin. (A) Double immunofluorescence staining of Numb with phalloidin in CMs and SKMs of mice on P60.5 (phalloidin, green; Numb, red; original magnification, ×630, zoom in ×4). (B) Coimmunoprecipitation of Numb with other sarcomeric ABPs in heart and SKM lysates. (C) Coimmunoprecipitation of the transfected Myc-Numb65, Myc-Numb66, Myc-Numblike with EGFP–α-Actin (ACTC1) and EGFP–β-Actin in AD293 cells. (D) Coimmunoprecipitation of the transfected Myc-Numb65, Myc-Numb66, and Myc-Numblike with EGFP–α-Actin (ACTA) and EGFP–β-actin in the conducted AD293 cells. (E) Schematic of Myc-Numb and Myc-Numblike truncation. (F) Coimmunoprecipitation of the transfected truncated Myc-Numb and Myc-Numblike with EGFP–α-Actin (ACTC1) in AD293 cells. Scale bars: 5 μm.

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  Figure 4. Numb and Numblike regulate sarcomeric α-Actin filament formation. (A) TIRFM micrographs of Actin (ACTA) filaments 10 minutes after initiating polymerization of Mg-ATP-Actin (1 mM) with 100 nM His-Numb65, His-Numb66, and His-Numblike (original magnification, ×1000). (B) Time course of fluorescence increase upon polymerization of 2 mM Mg-ATP-Actin (6% pyrene-labeled) with 100 nM His-Numb65, His-Numb66, and His-Numblike (n = 3). Data are mean ± SD. (C) TIRF micrographs of Actin (ACTA) filaments 10 minutes after initiating polymerization of 1 mM Mg-ATP-Actin with 100 nM ACTN2 and 100 nM His-Numb65, His-Numb66, and His-Numblike, respectively (original magnification, ×1000). (D) Double immunofluorescence staining of Numb and phalloidin in the SKMs of CTL and CKMM-cre DKO mice on P60.5 (phalloidin, green; Numb, red; original magnification, ×630, zoom in ×2). (E) Electron microscopy of the gastrocnemius muscle of CKMM-cre DKO mice electroporated with EGFP-tagged Numb65, Numb66, and Numblike (original magnification, ×20,000). Scale bars: 10 μm (A, C, D) or 1 μm (E).

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  Figure 5. Numb and Numblike regulate Z-disc assembly and integrity through ACTN. (A) Double immunofluorescence staining of Numb and ACTN in the CMs of E14.5 CTL and MLC-2v-cre DKO mice after 72-hour culture in vitro (ACTN, green; Numb, red; original magnification, ×630, zoom in ×2; amplified images: original magnification, ×630, zoom in ×5). (B) Double immunofluorescence staining of Numb and ACTN in the SKMs of CKMM-cre DKO mice on P60 (ACTN, green; Numb, red; original magnification, ×630, zoom in ×2). (C) Coimmunoprecipitation of N-WASP with other ABPs in heart and SKM lysates of CTL and CKMM-cre DKO mice on P60. (D and E) Relative protein level of ABPs immunoprecipitated by N-WASP in heart and SKM lysates of CTL and CKMM-cre DKO mice on P60 (n = 3). (F) Coimmunoprecipitation of α-Actin (ACTC1) with ACTN in heart lysates of CTL and α-MHC-cre DKO mice on P60. (G) Relative ACTN protein level immunoprecipitated by ACTC1 in heart lysates of CTL and α-MHC-Cre DKO mice on P60 (n = 4). Scale bars: 5 μm. Two-tailed Student’s t test with error bars showing mean ± SD.

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  Figure 6. A schematic illustration of how Numb and Numblike regulate sarcomere assembly and maintenance. Numb and Numblike bind to sarcomeric α-Actin to regulate sarcomeric ACTN alignment, branching, and striation. The loss of function of Numb and Numblike compromises thin filament assembly and Z-disc consolidation.