Novel localisation and possible function of LIN7 and IRSp53 in mitochondria of HeLa cells.
By means of immunofluorescence and subcellular fractionation experiments, we here demonstrate mitochondrial distribution of LIN7 and IRSp53 in HeLa cells. These peripheral proteins displayed a tight association with mitochondria and coimmunoprecipitated from mitochondrial fractions. In line with a role for LIN7 in the regulation of IRSp53 activity on actin dynamics, the morphology of mitochondria was similarly altered by changing the expression levels of either each protein or both, whereas mitochondrial morphology was preserved in cells overexpressing IRSp53 deleted of its binding domains for LIN7 (IRSp53Δ5) or for actin polymerisation modulators (IRSp53ΔSH3). In particular, the overexpression of full length LIN7 and/or IRSp53 increased the percentage of cells with short mitochondria, while downregulation of the endogenous proteins by shRNAs increased the amount of cells with elongated and perinuclear clustered mitochondria. These mitochondria were only partially resistant to fragmentation induced by dissipation of the mitochondrial membrane potential (i.e. treatment with sodium azide), whereas mitochondria were fully protected by the fission defective mutant Drp1 K38A. Overexpression of LIN7 or IRSp53 did not prevent the formation of hyperfused mitochondria in cells coexpressing the Drp1 K38A mutant, thus suggesting that LIN7-IRSp53 complex requires functional Drp1 to regulate mitochondrial morphology.
Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano and Institute of Neuroscience, Consiglio Nazionale delle Ricerche, Milan, Italy. Electronic address: ilaria.ferrari@biologo.onb.it.
Ilaria
I
Ferrari
Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano and Institute of Neuroscience, Consiglio Nazionale delle Ricerche, Milan, Italy. Electronic address: a.crespi@in.cnr.it.
Arianna
A
Crespi
Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano and Institute of Neuroscience, Consiglio Nazionale delle Ricerche, Milan, Italy. Electronic address: diego.fornasari@unimi.it.
Diego
D
Fornasari
Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano and Institute of Neuroscience, Consiglio Nazionale delle Ricerche, Milan, Italy. Electronic address: grazia.pietrini@unimi.it.
Grazia
G
Pietrini
D048868:Adaptor Proteins, Signal Transducing; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C119539:BAIAP2 protein, human; C486531:LIN7C protein, human; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins
Drp1-mediated fission; Hyperfused mitochondria; Mitochondrial fission