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Related Experiment Video

Updated: Jun 17, 2026

A Fluorescence-based Assay of Phospholipid Scramblase Activity
09:52

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Published on: September 20, 2016

A single-vesicle fluorescence microscopy platform to quantify phospholipid scrambling.

Sarina Veit1, Grace I Dearden2, Kartikeya M Menon3

  • 1Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany.

Nature Structural & Molecular Biology
|June 15, 2026
PubMed
Summary

This study introduces a new method to measure single-protein lipid scrambling by voltage-dependent anion channel 1 (VDAC1) dimers. It reveals significant kinetic heterogeneity in scramblase activity, crucial for understanding membrane transport.

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Area of Science:

  • Membrane Biology
  • Biophysics
  • Protein Biochemistry

Background:

  • Scramblases are essential proteins that facilitate bidirectional phospholipid movement across cell membranes.
  • Voltage-dependent anion channel 1 (VDAC1) dimers are known to mediate phospholipid translocation from the endoplasmic reticulum to mitochondria.

Purpose of the Study:

  • To develop a high-throughput single-protein assay for quantifying lipid translocation rates of scramblases.
  • To investigate the kinetic heterogeneity of individual VDAC1 dimers in lipid scrambling.
  • To demonstrate the platform's versatility for studying other scramblases, such as bovine opsin.

Main Methods:

  • Reconstitution of vesicles with fluorescent phospholipids and VDAC1 dimers.
  • High-throughput imaging to quantify vesicle size and VDAC1 dimer content.
  • Development of a novel assay to measure lipid scrambling rates at the single-vesicle level.

Main Results:

  • Individual human VDAC1 dimers exhibit a wide range of lipid scrambling rates, from <100 to >10,000 translocations per second.
  • Kinetic heterogeneity in scramblase activity was observed, which is typically masked in ensemble measurements.
  • Specific VDAC1 dimers were identified as facilitating rapid lipid scrambling.

Conclusions:

  • The developed platform enables single-protein level analysis of scramblase activity, offering mechanistic insights.
  • Kinetic heterogeneity is a key feature of VDAC1-mediated lipid scrambling.
  • The assay is versatile and applicable to other scramblases, including G-protein-coupled receptors like bovine opsin.