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Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment

Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggere...

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Autores principales: Frazzette, Nicholas, Cruz, Anthony C., Wu, Xufeng, Hammer, John A., Lippincott-Schwartz, Jennifer, Siegel, Richard M., Sengupta, Prabuddha
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9221696/
https://www.ncbi.nlm.nih.gov/pubmed/35741037
http://dx.doi.org/10.3390/cells11121908
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author Frazzette, Nicholas
Cruz, Anthony C.
Wu, Xufeng
Hammer, John A.
Lippincott-Schwartz, Jennifer
Siegel, Richard M.
Sengupta, Prabuddha
author_facet Frazzette, Nicholas
Cruz, Anthony C.
Wu, Xufeng
Hammer, John A.
Lippincott-Schwartz, Jennifer
Siegel, Richard M.
Sengupta, Prabuddha
author_sort Frazzette, Nicholas
collection PubMed
description Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas–Fas and Fas–FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.
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spelling pubmed-92216962022-06-24 Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment Frazzette, Nicholas Cruz, Anthony C. Wu, Xufeng Hammer, John A. Lippincott-Schwartz, Jennifer Siegel, Richard M. Sengupta, Prabuddha Cells Article Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas–Fas and Fas–FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD. MDPI 2022-06-12 /pmc/articles/PMC9221696/ /pubmed/35741037 http://dx.doi.org/10.3390/cells11121908 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Frazzette, Nicholas
Cruz, Anthony C.
Wu, Xufeng
Hammer, John A.
Lippincott-Schwartz, Jennifer
Siegel, Richard M.
Sengupta, Prabuddha
Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment
title Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment
title_full Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment
title_fullStr Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment
title_full_unstemmed Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment
title_short Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment
title_sort super-resolution imaging of fas/cd95 reorganization induced by membrane-bound fas ligand reveals nanoscale clustering upstream of fadd recruitment
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9221696/
https://www.ncbi.nlm.nih.gov/pubmed/35741037
http://dx.doi.org/10.3390/cells11121908
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