Fig. 5: RIPK4 interacts with MFN2 to regulate mitochondrial dynamics. | Nature Communications

Fig. 5: RIPK4 interacts with MFN2 to regulate mitochondrial dynamics.

From: RIPK4-mediated MFN2 degradation drives osteogenesis through mitochondrial fragmentation and restricts myelopoiesis by blocking mitochondrial transfer

Fig. 5: RIPK4 interacts with MFN2 to regulate mitochondrial dynamics.

a, b TEM detected long extended tubules of mitochondria in muscle tissues (a) and osteoprogenitor cells (b) in 16-week-old Ripk4fl/fl, UbcCre/ERT2 Ripk4fl/fl and UbcCre/ERT2 Ripk4fl/flMfn2fl/fl mice (n = 5 per group). Scale bar, 500 nm. The right arrow defines the mitochondria. c Co-immunoprecipitation of endogenous MFN2 and FLAG-RIPK4 in HEK293T cells. d Endogenous immunoprecipitation of RIPK4 and MFN2 was performed in hBMSC cells. e Representative images PLA signal (in situ close association between FLAG and MFN2) in U2OS cells. The nuclear marker DAPI is in blue, and the PLA signal is in red. Scale bar, 10 μm. f HEK293T cells were transfected with plasmids expressing 3×FLAG-RIPK4 (WT, 3×FLAG-RIPK4; K51R, 3×FLAG-RIPK4-K51R, kinase-dead mutant; T184I, 3×FLAG RIPK4-T184I, ATP binding mutant), RIPK4 was immunoprecipitated using FLAG-M2 affinity gel. g Myc-MFN2 and 3×FLAG-RIPK4 expressing plasmids (WT, 3×FLAG-RIPK4; K51R, 3×FLAG-RIPK4-K51R) were transfected in HEK293T cells. Cells were harvested and cell lysates were incubated with or without lambda protein phosphatase (λ-PPase). Phosphorylated and non-phosphorylated MFN2 proteins were separated by Phos-Tag SDS-PAGE. h HA-MFN2 and 3×FLAG-RIPK4 or 3×FLAG-RIPK4 K51R expressing plasmids were co-transfected in HEK293T cells. HA-MFN2 was immunoprecipitated using anti-HA-agarose. i Immunoblots analysis of MFN2 and RIPK4 proteins in RIPK4 knockout HBMSC cells. j, k WT and K51R RIPK4 were inducibly expressed in U2OS cells with 50 ng/ml doxycycline for 24 h. MFN2, MFN1, DRP1, and OPA1 proteins were detected by immunoblots. l WT RIPK4 was inducibly expressed in U2OS cells with 50 ng/ml doxycycline for 24 h, and mitochondria were extracted according to the manufacturer’s protocol. MFN2, MFN1, and DRP1 proteins were detected by immunoblots and quantified by ImageJ. m RIPK4 was inducibly expressed in U2OS cells with 50 ng/ml doxycycline for 24 h, then cells were treated with MG132 or Bafilomycin A1 (BafA) for 4 h. MFN2 protein was detected by immunoblots. n FLAG-RIPK4 or FLAG-RIPK4-K51R with dsRed-mito expressing plasmids were co-transfected in U2OS cells for 24 h, then immune-stained for RIPK4 (green). dsRed-mito (red) was utilized to visualize the morphology of mitochondria. DAPI staining indicated the nuclei. Scale bar, 10 μm. Source data were provided as a Source Data file.

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