Visualizing interactions of VDAC1 in live cells using a tetracysteine tag

Summary 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 used the tetracysteine (TC)-tag approach and visualized VDAC1 dynamics in living cells. TC-tagged VDAC1 had a cluster-like distribution on mitochondria. The majority of VDAC1-clusters were localized at endoplasmic reticulum (ER)-mitochondria contact sites. Notably, VDAC1 colocalized with BCL-2 Antagonist/Killer (BAK)-clusters upon apoptotic stimulation. Additionally, VDAC1 was found at mitochondrial fission sites, likely promoting mitochondrial fragmentation. These findings highlight the suitability of the TC-tag for live-cell imaging of VDAC1, shedding light on the roles of VDAC1 in multiple cellular processes.