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We developed a new method using tetracysteine (TC)-tagging for live-cell imaging of voltage-dependent anion channel 1 (VDAC1). This technique reveals VDAC1 dynamics at mitochondria, ER-mitochondria contact sites, and during apoptosis.

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Area of Science:

  • Cell Biology
  • Mitochondrial Biology
  • Molecular Imaging

Background:

  • Voltage-dependent anion channel 1 (VDAC1) regulates mitochondrial function and cellular energy. Existing imaging methods lack precision for visualizing VDAC1 in living cells. Accurate VDAC1 visualization is critical for understanding its role in cellular processes.
  • The outer mitochondrial membrane houses VDAC1, a key regulator of metabolic and energy homeostasis. Current visualization techniques are insufficient for studying VDAC1 dynamics in real-time within living cells.

Purpose of the Study:

  • To develop and validate a novel live-cell imaging technique for VDAC1 using tetracysteine (TC)-tagging. To investigate the distribution and dynamics of VDAC1 in living cells, particularly at mitochondrial contact sites and during apoptosis.
  • To establish a precise method for visualizing voltage-dependent anion channel 1 (VDAC1) in living cells. To explore the localization and dynamic behavior of VDAC1, especially in relation to the endoplasmic reticulum and apoptotic pathways.

Main Methods:

  • Utilized tetracysteine (TC)-tagging for precise VDAC1 imaging in living cells. Validated TC-tagged VDAC1 localization using immunofluorescence. Observed VDAC1 dynamics at mitochondrial fission sites and ER-mitochondria contact sites.
  • Employed tetracysteine (TC)-tagging to enable live-cell visualization of VDAC1. Confirmed the specificity of TC-VDAC1 labeling via immunofluorescence. Analyzed VDAC1 distribution at ER-mitochondria contact sites and its colocalization with BAK during apoptosis.

Main Results:

  • TC-tagged VDAC1 exhibited a cluster-like distribution on mitochondria. The majority of VDAC1-TC clusters were found at endoplasmic reticulum (ER)-mitochondria contact sites. VDAC1 colocalized with BCL-2 Antagonist/Killer (BAK)-clusters during apoptotic stimulation.
  • Live-cell imaging revealed a clustered distribution of TC-tagged VDAC1 on mitochondria. VDAC1-TC clusters predominantly localized to ER-mitochondria contact sites. Upon apoptotic stimuli, VDAC1 was observed to colocalize with BAK clusters. VDAC1 was also visualized at mitochondrial fission sites.

Conclusions:

  • The TC-tag is a suitable tool for live-cell imaging of VDAC1, providing new insights into its cellular roles. This method facilitates the study of VDAC1 dynamics at ER-mitochondria contact sites and during apoptosis. Findings highlight VDAC1's involvement in mitochondrial dynamics and cellular stress responses.
  • Tetracysteine (TC)-tagging enables accurate live-cell visualization of VDAC1 dynamics. This technique reveals VDAC1's localization at ER-mitochondria contact sites and its association with BAK during apoptosis. The study underscores the utility of TC-tagging for exploring VDAC1's function in cellular processes.