Fig. 4: Clu specifically interacts with Mfn2 to enhance mitochondrial fusion.
From: Clusterin drives myeloid bias in aged hematopoietic stem cells by regulating mitochondrial function
a, Immunoblot (left) and quantification (right) of cytoplasmic Clu (cClu) and secreted Clu (sClu) in yHSC and oHSC lysates (n = 3 per group). b, Co-immunoprecipitation (co-IP) analyses of cClu-v5 with Drp1-10 × myc, Mfn1-10 × myc or Mfn2-10 × myc overexpressed in HPC7 cells (n = 3). Immunoblots (IBs) were performed using automated ProteinSimple WES capillary electrophoresis system. c, Co-IP analysis of endogenous Clu with Mfn1 and Mfn2 in lineage negative (lineage−) bone marrow cells (n = 3). Immunoblots were performed using the ProteinSimple WES. d, Immunofluorescence images (left) and quantification (right) of mitochondria (MitoGreen) in WT, Mfn1 KO and Mfn2 KO MEFs without (Ctrl) or with cClu overexpression (Clu OE). MEFs were transduced with mCherry control or cClu-T2A-mCherry lentivirus (n = 4 per group). T2A was used to separate Clu and mCherry into two proteins. Scale bar, 10 μm. e, Immunofluorescence images of mitochondria (MitoRed), cClu-v5, and Mfn2 in shCtrl and shMfn2 yHSCs overexpressing cClu (left). Fluorescence intensity profile plots in arbitrary units (a.u. along the solid lines (middle) and quantification (right) of mitochondria length by MitoRed (n = 10 imaging fields containing 20 cells per group). Scale bar, 5 μm. f, Cytoplasmic Clu (UniProt: Q06890) and Mfn2 (UniProt: Q80U63) interacting surfaces predicted by AlphaFold 3. g, The amino acid sequences and their corresponding missense and deletion mutations (mut1 and mut2) on Clu surfaces predicted to be involved in Mfn2 interaction. h, Co-IP analyses of WT-cClu-v5, cClu-mut1-v5, or cClu-mut2-v5 with Mfn2-10 × myc. Clu mutants were separately overexpressed with Mfn2 in HPC7 cells (n = 3). Immunoblots were performed using the ProteinSimple WES. i, Immunofluorescence images of mitochondria (MitoRed) in WT-cClu, cClu-mut1, and cClu-mut2 overexpressed yHSCs (left). Fluorescence intensity profile plots in arbitrary units (a.u.) along the solid lines (middle) and quantification (right) of mitochondria length by MitoRed (n = 10 imaging fields containing 20 cells per group). Scale bar, 5 μm. Unpaired two-tailed Student’s t-test (a,d). One-way ANOVA with Tukey’s multiple comparison test (e,i). Error bars represent mean ± s.e.m. (a,d,e,i).
