Super-resolution live imaging reveals spatiotemporal uncoupling of mitochondria-ER contacts and mitochondrial fission in stem cell aging

Cellular senescence is a major driver of tissue aging and functional decline. Although mitochondrial dysfunction is a well-established hallmark of senescence, how inter-organelle communication regulates mitochondrial dynamics during stem cell aging remains poorly understood. In particular, whether age-associated alterations in mitochondria–endoplasmic reticulum contacts (MERCs) disrupt mitochondrial homeostasis and contribute to cellular senescence remains unclear.

Here, using grazing incidence structured illumination microscopy (GI-SIM), we characterized the spatiotemporal coordination between MERCs and mitochondrial fission in two independent models of human mesenchymal stem cell (hMSC) aging: replicative senescence and Hutchinson–Gilford progeria syndrome (HGPS). In both models, senescent hMSCs displayed reduced mitochondrial fission, mitochondrial elongation, altered ER architecture and dynamics, and decreased MERC coverage. Live-cell super-resolution imaging further revealed that the spatial coupling of MERCs to mitochondrial fission sites was impaired during hMSC senescence.

Mechanistically, we identified downregulation of the ER-resident tethering protein VAPB as a key contributor to MERC dysfunction in senescent hMSCs. CRISPR/Cas9-mediated depletion of VAPB in young hMSCs recapitulated multiple senescence-associated defects, including disrupted ER architecture and dynamics, reduced MERC coverage, impaired coupling between MERCs and mitochondrial fission sites, decreased mitochondrial fission, mitochondrial elongation, and the acquisition of senescence-associated phenotypes. Conversely, CRISPR activation (CRISPRa) of endogenous VAPB expression in senescent hMSCs partially restored MERC coverage, enhanced MERC-associated mitochondrial fission, improved mitochondrial dynamics, and attenuated senescence-associated phenotypes.

Together, these findings establish VAPB-dependent mitochondria–ER tethering as an important regulator of mitochondrial fission and hMSC senescence. Our study identifies the coordination between MERCs and mitochondrial fission as a previously underappreciated layer of stem cell aging and suggests that preserving this coordination may help maintain stem cell homeostasis and function during aging.