Quantitative optical nanoscopy of mitochondrial-derived vesicles in neurons classifies pre-peroxisomal and clearing organelles.

Healthy mitochondria are crucial for maintaining neuronal homeostasis. Their activity depends on a dynamic lipid and protein exchange through fusion, fission, and vesicular trafficking. Studying vesicles in neurons is challenging with conventional microscopy due to their small size, heterogeneity, and dynamics. We use multicolour stimulated emission depletion nanoscopy to uncover the ultrastructure of mitochondrial-derived vesicles (MDVs) in live neurons, biosensors to define their functional state, and a pulse-chase strategy to identify their turnover in situ. We identified three populations of vesicular structures: one transporting degradation products originating from oxidative stress, one shuttling cargo and newly translated proteins for local organelle biogenesis and one consisting of small, functional mitochondria. Furthermore, we provide evidence supporting that de novo peroxisomes biogenesis occurs via the fusion of endoplasmic reticulum and MDVs at mitochondrial sites. Our data provide mechanistic insight into organelle biogenesis driven by significant diversity in MDV morphology, functional state, and molecular composition.