Mitochondria-derived nuclear ATP surge protects against mechanical confinement-induced proliferation defects

The physical microenvironment regulates cell behaviour. Whether physical space constraints reshape the subcellular localisation of organelles and affect metabolism is, however, unknown. Proteomics analysis revealed that cellular confinement induces a strong enrichment of mitochondrial proteins within the nuclear compartment. High-resolution microscopy confirmed that mechanical cell confinement leads to a rapid re-localisation of mitochondria to the nuclear periphery. This nucleus-mitochondria proximity is mediated by an endoplasmic reticulum-based net that entraps the mitochondria in an actin-dependent manner. Functionally, the mitochondrial proximity results in a nuclear ATP surge, which can be reverted by the pharmacological inhibition of mitochondrial ATP production or the reduction of proximity via actin depolymerisation. Inhibition of this nuclear ATP surge reveals long-term effects on cell fitness and proliferation from DNA damage repair delays and cell cycle defects. Together, our data indicate that physical confinement triggers a metabolic rewiring that enables timely nuclear DNA damage repair and cell cycle progression. HeLa cells were treated with or without Oligomycin A (1 µM) for 30 min in glucose-free complete media. Cells were then trypsinized and either left in suspension (controls) or confined using a hybrid confiner. Briefly, cells were confined in glass-bottom 6-well plates (adapted from 4D Cell, CSOW 610) under coverslips with 3µm height microspacers. Both, coverslips and the glass-bottom plate were plasma-cleaned and treated with 0.5 mg/ml PLL(20)-g[3,5]-PEG(2) (Susos) to prevent attachment. Following an acute 15 minute confinement, ATAC seq was perfomed.