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Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)
Published on: April 20, 2015
Perforin rapidly induces plasma membrane phospholipid flip-flop
Sunil S Metkar1, Baikun Wang, Elena Catalan
1Department of Medicine, NorthShore University HealthSystems Research Institute, Evanston, Illinois, United States of America.
Plos One
|September 21, 2011
Summary
Cytotoxic cell granule secretion uses perforin (PFN) to deliver granzymes into target cells. Unexpectedly, PFN forms structures favoring phosphatidylserine flip-flop, facilitating granzyme B (GzmB) entry for apoptosis.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- The cytotoxic cell granule secretory pathway is crucial for host defense, involving the delivery of granzymes into target cells.
- Perforin (PFN) forms pores in target cell membranes, facilitating granzyme entry and mediating apoptosis.
- Previous models suggested PFN forms large, barrel-stave pores, but experimental evidence in target cells remains inconclusive.
Purpose of the Study:
- To investigate the mechanism of perforin-mediated cell death and granzyme delivery.
- To reconcile structural data of PFN pores with functional observations in target cells.
- To elucidate the role of membrane properties in PFN pore formation and function.
Main Methods:
- Utilized Annexin-V and Lactadherin staining to measure phosphatidylserine flip-flop.
- Assessed propidium iodide uptake to evaluate membrane integrity.
- Investigated the effects of calcium influx, lipids, and cholesterol depletion on PFN function.
- Observed antigen-specific mouse CD8 T cells and their interaction with target cells.
Main Results:
- PFN concentrations inducing granzyme-mediated apoptosis correlated with phosphatidylserine flip-flop, not large pore formation.
- Reduced calcium influx did not affect Annexin-V reactivity.
- Mouse CD8 T cells induced rapid phosphatidylserine flip-flop in target cells.
- Lipids enhancing membrane curvature and cholesterol depletion increased phosphatidylserine flip-flop.
Conclusions:
- PFN oligomers may form unusual arc-like structures in the plasma membrane, rather than classical pores.
- These structures, a mixture of lipids and PFN, act as flexible gateways for granzyme B (GzmB) translocation.
- This mechanism explains how GzmB accesses the target cell cytosol to induce apoptosis.
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