Membranes arrest the coarsening of mitochondrial condensates in human cells.

Mitochondria contain double membranes that enclose their contents. Within their interior, the mitochondrial genome and its RNA products are condensed into ~100 nm sized (ribo)nucleoprotein complexes. How these endogenous condensates maintain their roughly uniform size and spatial distributions within mitochondria remains unclear. Here, we engineer optogenetic tools (mt-optoIDR) that enable controlled formation of synthetic condensates within live mitochondria upon light activation in HeLa cells. Using high-resolution microscopy, we visualize the nucleation of small, yet elongated condensates (mt-opto-condensates), which recapitulate the morphologies of endogenous mt-condensates. These narrow size distributions are independent of mt-optoIDR sequence features, suggesting the mitochondrial environment influences condensate formation. Consistently, mt-opto-condensates fluctuate within voids in between cristae in tubular mitochondria. To directly isolate the contribution of the mitochondrial membranes, we overexpress the dominant negative membrane fusion mutant (Drp1K38A), which results in the formation of bulbous mitochondria with restructured cristae. Based on quantitative particle tracking, bulbous mitochondria support significantly increased dynamics and rapid coarsening of mt-opto-condensates into a single, prominent droplet-in contrast to the membrane confinement observed in tubular mitochondria. Together, these observations inform how membranes can constrain the growth and dynamics of the condensates they enclose, without the need for additional regulatory mechanisms.