List of plasmids used in this study.

The voltage-dependent anion channel 1 (VDAC1) is a crucial gatekeeper in the outer mitochondrial membrane, controlling metabolic and energy homeostasis. The available methodological approaches fell short of accurate visualization of VDAC1 in living cells. To permit precise VDAC1 imaging, we utilized the tetracysteine (TC)-tag and visualized VDAC1 dynamics in living cells. TC-tagged VDAC1 had a cluster-like distribution on mitochondria. The labeling of TC-tagged VDAC1 was validated with immunofluorescence. The majority of VDAC1-TC-clusters were localized at endoplasmic reticulum (ER)-mitochondria contact sites. Notably, VDAC1 colocalized with BCL-2 Antagonist/Killer (BAK)-clusters upon apoptotic stimulation. Using this new tool, we were able to observe VDAC1-TC at mitochondrial fission sites. These findings highlight the suitability of the TC-tag for live-cell imaging of VDAC1, shedding light on the roles of VDAC1 in cellular processes.