Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts
Introduction
Mitochondria of mammalian cells form a dynamic network of tubular and punctate organelles that continuously fuse and divide, the equilibrium between these antagonistic events resulting in the cell mitochondrial morphology [1], [2]. While its molecular factors are progressively identified, the role and relevance of mitochondrial morphology in the cell biology are still the matter of much debate [3], [4], [5]. Mammalian mitochondrial fission involves at least four proteins, including GDAP1 and dynamin-related protein 1 (Drp1), while mitochondrial fusion requires the ubiquitous GTPases Mitofusins (Mfn1, Mfn2) and optic atrophy 1 (OPA1). Mutations in genes encoding several of these proteins were found responsible for neurological human diseases, thus showing the major role of mitochondrial dynamics at least in neuronal cell life [6]. Mfn2 mutations are responsible for autosomal dominant forms of Charcot-Marie-Tooth neuropathy [7], [8] while GDAP1 mutations cause autosomal recessive forms of the disease [9], [10]; OPA1 mutations are one of the most frequent cause of autosomal dominant optic atrophy characterized by primary degeneration of the retinal ganglion cell layer and ascending atrophy of the optic nerve [11].
It clinically resembles Leber’s hereditary optic neuropathy, which is due to oxidative phosphorylation (OXPHOS) defect caused by mitochondrial DNA (mtDNA) mutations [12], thus suggesting intricate relationship of mitochondrial dynamics and OXPHOS. Indeed, modulation of OXPHOS by mitochondrial dynamics has been demonstrated in immortalized cultured cells where the perturbation of mitochondrial fusion (by knock down of OPA1 or knockout of Mfn1 or Mfn2) or fission (by knockdown of Drp1) induced OXPHOS defect [13], [14]. Conversely, OXPHOS has been proposed to influence mitochondrial dynamics. In cancer cell lines stimulation of respiration by nutritional modulation induced increased length and connection of mitochondria [15]. Human cybrid cells carrying deleterious mtDNA mutations as well as mouse embryonic fibroblasts harbouring an error-prone mtDNA polymerase gamma displayed fragmented mitochondria [14], [16], [17]. These results suggested that actively respiring mitochondria retain a filamentous morphology and that, on the other hand, mitochondrial dysfunction results in mitochondrial fragmentation. However human cells devoid of mtDNA and therefore of a functional respiratory chain have been shown to be fusion competent and efficiently exchange proteins and mtDNA [1], [18]. Furthermore mitochondrial fusion was shown to depend on the ΔΨm, but not on the intramitochondrial synthesis of ATP [1], [2].
The goal of this study is to clarify the impact of defective cellular bioenergetics on mitochondrial membrane dynamics. To this end, we first modulated OXPHOS of fibroblasts derived from control healthy subjects and analyzed the consequences on the dynamics of the mitochondrial compartment. We show that pharmacological modulation of OXPHOS, but not of glycolysis, influence mitochondrial dynamics of control cells. We then characterized mitochondrial dynamics in primary fibroblasts harbouring diverse genetic alterations of OXPHOS. Although their mitochondrial morphology appeared essentially normal under basal conditions, massive mitochondrial fragmentation was provoked when drastic decrease of the cell energetic supply was obtained by the co-existence of severe genetic OXPHOS defect and glycolysis inhibition. The influence of deoxyglucose treatment on mitochondrial morphology may therefore be considered an indicator of severe OXPHOS defects.
Section snippets
Fibroblasts lines
Control cell lines were obtained from the tissue repository of AFM (Association Française contre les Myopathies). They were anonymous samples derived from a 47-year-old female (L1), a 10-year-old female (L2), a 1-year-old male (L3), a 9-year-old female (L4) and a 23-year-old male (L5). Patients with a mitochondrial disease were recruited through the mitochondrial diagnostic activity of La Salpêtrière hospital, to which author PF and author AL directly participate. Patients with an
Control fibroblasts maintain filamentous mitochondria under basal culture conditions
Mitochondria may appear as long branched mitochondrial filaments, shorter mitochondrial tubules and bean-shaped or punctate structures. Their overall appearance vary between cell types [1], [13], [26]. In order to study the impact of cellular bioenergetics on mitochondrial morphology and dynamics, we first characterized mitochondrial morphology in primary human skin fibroblasts derived from five healthy subjects. Immunofluorescence of fixed cells revealed highly elongated branched mitochondrial
Discussion
In order to clarify the impact of cellular bioenergetics on the morphology of the mitochondrial compartment, we analyzed both aspects in human skin fibroblasts, which provide an accessible source of human primary cells, easily grown in monolayers where their mitochondrial compartment may be readily observed. As most cultured cells, skin fibroblasts derive most their energy from glycolysis. They may therefore be considered disputable models for OXPHOS analysis. However they have demonstrated
Acknowledgements
This work has been supported by INSERM and by grants from the French Ministry of Science and Technology and from AFM (Association Française contre les Myopathies). MR is a CNRS researcher. We wish to thank the AFM tissue repository for providing the control cells and Mélanie Pucelle for excellent technical assistance.
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These two authors equally contributed to the work.