Related Experiment Video
Updated: May 2, 2026
Examining BCL-2 Family Function with Large Unilamellar Vesicles
Published on: October 5, 2012
Functional assessment and quantitative kinetic analysis of BAX activation using large unilamellar vesicles
Jesse D Gelles1, Thedoe Nyunt2, Md Abdullah Al Noman1
1Laboratory of Mitochondrial Biology in Human Health and Disease, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY, United States; Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY, United States; The Mount Sinai Tisch Cancer Center, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY, United States.
Abstract:
A myriad of diverse developmental and pro-death signals converge on the mitochondrial pathway of apoptosis, which is governed by the BCL‑2 family of proteins. Comprised of both pro- and anti-apoptotic family members, the BCL‑2 family functions to regulate mitochondrial outer membrane permeabilization (MOMP), often considered the "point of no return" in which a cell commits to an apoptotic outcome. Specifically, the effector BCL‑2 family proteins, BAX and BAK, are responsible for inducing MOMP and therefore investigations into their structural, cellular, and pharmacological regulation are critical to understanding the cellular commitment to apoptosis. A gold standard methodology for studying activation of BAX or BAK is the permeabilization of large unilamellar vesicles (LUVs), which are biochemically-defined model liposomes that mimic the major lipid composition of the outer mitochondrial membrane (OMM). Here, we provide a detailed protocol for generating LUVs containing a fluorescent dye/quencher pair to monitor real-time BAX activation and membrane permeabilization using a standard plate reader. Additionally, we detail example assay strategies to model interactions within the BCL‑2 family and provide a robust mathematical model for fitting and parameterizing kinetic LUV permeabilization data.
Insights
This study presents a new method to track BAX and BAK protein activation, key regulators of programmed cell death. The protocol uses fluorescent large unilamellar vesicles (LUVs) to monitor mitochondrial outer membrane permeabilization (MOMP) in real-time.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Apoptosis, or programmed cell death, is regulated by the BCL-2 protein family.
- Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis, controlled by BAX and BAK proteins.
- Studying BAX/BAK activation is crucial for understanding cell death pathways.
Purpose of the Study:
- To provide a detailed protocol for generating fluorescent large unilamellar vesicles (LUVs).
- To enable real-time monitoring of BAX activation and membrane permeabilization.
- To offer strategies for modeling BCL-2 family interactions and analyzing kinetic data.
Main Methods:
- Generation of LUVs mimicking mitochondrial outer membrane lipid composition.
- Incorporation of a fluorescent dye/quencher pair within LUVs.
- Real-time monitoring of BAX/BAK-induced LUV permeabilization using a plate reader.
Main Results:
- A robust protocol for LUV generation and BAX/BAK activity monitoring.
- Demonstrated utility in studying BCL-2 family interactions.
- A mathematical model for kinetic data analysis.
Conclusions:
- The developed LUV assay is a gold standard for studying BAX/BAK activation.
- This methodology facilitates research into the regulation of apoptosis.
- Enables quantitative analysis of mitochondrial pathway regulation.
