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SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane.

Siegel RM, Muppidi JR, Sarker M, Lobito A, Jen M, Martin D, Straus SE, Lenardo MJ - J. Cell Biol. (2004)

Bottom Line: Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex.We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling.Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. rsiegel@nih.gov

ABSTRACT
Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

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ALPS Fas mutations and patient cells display defective SPOTS formation and receptor downmodulation. (A) Cos-7 cells transiently transfected with the indicated Fas-YFP fusion proteins were treated with 1 μg/ml of APO-1 and 0.1 μg/ml of protein A for 30 min at 37°C and z-stack images of live cells were acquired by confocal microscopy and shown as in Fig. 2. (B) EBV-transformed B cell lines from healthy donors or patients with the indicated mutations were treated with anti-Fas for 1 h, stained for Fas as described in Materials and methods, and visually scored for receptor surface clustering or capping, with the total percentage of capped or clustered Fas given below each panel. Mid-cell confocal sections and three-dimensional reconstructions of representative cells are shown for anti-Fas–treated (bottom) or untreated (top) cells. Insets show three-dimensional reconstruction of z-stack images for a representative cell in each condition. The numbers below each panel represent the percentage of cells exhibiting SPOTS or caps after anti-Fas treatment. (C) Quantitation of surface Fas after treatment of the indicated EBV patient-derived cells with anti-Fas and normal cells treated with or without the zVAD-fmk caspase inhibitor.
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fig8: ALPS Fas mutations and patient cells display defective SPOTS formation and receptor downmodulation. (A) Cos-7 cells transiently transfected with the indicated Fas-YFP fusion proteins were treated with 1 μg/ml of APO-1 and 0.1 μg/ml of protein A for 30 min at 37°C and z-stack images of live cells were acquired by confocal microscopy and shown as in Fig. 2. (B) EBV-transformed B cell lines from healthy donors or patients with the indicated mutations were treated with anti-Fas for 1 h, stained for Fas as described in Materials and methods, and visually scored for receptor surface clustering or capping, with the total percentage of capped or clustered Fas given below each panel. Mid-cell confocal sections and three-dimensional reconstructions of representative cells are shown for anti-Fas–treated (bottom) or untreated (top) cells. Insets show three-dimensional reconstruction of z-stack images for a representative cell in each condition. The numbers below each panel represent the percentage of cells exhibiting SPOTS or caps after anti-Fas treatment. (C) Quantitation of surface Fas after treatment of the indicated EBV patient-derived cells with anti-Fas and normal cells treated with or without the zVAD-fmk caspase inhibitor.

Mentions: To correlate these biochemical abnormalities in Fas signaling with the subcellular localization of Fas, we transfected Fas-YFP fusion proteins containing non-FADD binding and the T225K FADD-binding Fas mutations into COS-7 cells and observed the morphology of receptor fluorescence in live cells after anti-Fas antibody treatment. Interestingly, cells transfected with either the non-FADD–binding D244V mutant or the T225K mutant failed to form SPOTS (Fig. 8 A). Similar results were seen with Jurkat cells transfected with FasT225K-YFP (Table I). Immunofluorescence analysis of endogenous Fas in EBV-transformed lymphoblastoid cell lines from ALPS patients bearing these same two mutations revealed an almost complete defect in formation of SPOTS in cells harboring heterozygous FasD244V mutations after treatment with anti-Fas mAb (Fig. 8 B). Cells heterozygous for the T225K Fas mutation exhibited a partial defect in formation of SPOTS as well as defects in receptor downmodulation and cap formation after receptor cross-linking (Fig. 8, B and C), although these defects were not as severe as in cells harboring non FADD-binding Fas mutations. Thus, the defect in caspase-8 processing in the DISC of heterozygous Fas T225K cells specifically impairs FAS SPOTS formation and receptor internalization downstream of FADD recruitment.


SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane.

Siegel RM, Muppidi JR, Sarker M, Lobito A, Jen M, Martin D, Straus SE, Lenardo MJ - J. Cell Biol. (2004)

ALPS Fas mutations and patient cells display defective SPOTS formation and receptor downmodulation. (A) Cos-7 cells transiently transfected with the indicated Fas-YFP fusion proteins were treated with 1 μg/ml of APO-1 and 0.1 μg/ml of protein A for 30 min at 37°C and z-stack images of live cells were acquired by confocal microscopy and shown as in Fig. 2. (B) EBV-transformed B cell lines from healthy donors or patients with the indicated mutations were treated with anti-Fas for 1 h, stained for Fas as described in Materials and methods, and visually scored for receptor surface clustering or capping, with the total percentage of capped or clustered Fas given below each panel. Mid-cell confocal sections and three-dimensional reconstructions of representative cells are shown for anti-Fas–treated (bottom) or untreated (top) cells. Insets show three-dimensional reconstruction of z-stack images for a representative cell in each condition. The numbers below each panel represent the percentage of cells exhibiting SPOTS or caps after anti-Fas treatment. (C) Quantitation of surface Fas after treatment of the indicated EBV patient-derived cells with anti-Fas and normal cells treated with or without the zVAD-fmk caspase inhibitor.
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fig8: ALPS Fas mutations and patient cells display defective SPOTS formation and receptor downmodulation. (A) Cos-7 cells transiently transfected with the indicated Fas-YFP fusion proteins were treated with 1 μg/ml of APO-1 and 0.1 μg/ml of protein A for 30 min at 37°C and z-stack images of live cells were acquired by confocal microscopy and shown as in Fig. 2. (B) EBV-transformed B cell lines from healthy donors or patients with the indicated mutations were treated with anti-Fas for 1 h, stained for Fas as described in Materials and methods, and visually scored for receptor surface clustering or capping, with the total percentage of capped or clustered Fas given below each panel. Mid-cell confocal sections and three-dimensional reconstructions of representative cells are shown for anti-Fas–treated (bottom) or untreated (top) cells. Insets show three-dimensional reconstruction of z-stack images for a representative cell in each condition. The numbers below each panel represent the percentage of cells exhibiting SPOTS or caps after anti-Fas treatment. (C) Quantitation of surface Fas after treatment of the indicated EBV patient-derived cells with anti-Fas and normal cells treated with or without the zVAD-fmk caspase inhibitor.
Mentions: To correlate these biochemical abnormalities in Fas signaling with the subcellular localization of Fas, we transfected Fas-YFP fusion proteins containing non-FADD binding and the T225K FADD-binding Fas mutations into COS-7 cells and observed the morphology of receptor fluorescence in live cells after anti-Fas antibody treatment. Interestingly, cells transfected with either the non-FADD–binding D244V mutant or the T225K mutant failed to form SPOTS (Fig. 8 A). Similar results were seen with Jurkat cells transfected with FasT225K-YFP (Table I). Immunofluorescence analysis of endogenous Fas in EBV-transformed lymphoblastoid cell lines from ALPS patients bearing these same two mutations revealed an almost complete defect in formation of SPOTS in cells harboring heterozygous FasD244V mutations after treatment with anti-Fas mAb (Fig. 8 B). Cells heterozygous for the T225K Fas mutation exhibited a partial defect in formation of SPOTS as well as defects in receptor downmodulation and cap formation after receptor cross-linking (Fig. 8, B and C), although these defects were not as severe as in cells harboring non FADD-binding Fas mutations. Thus, the defect in caspase-8 processing in the DISC of heterozygous Fas T225K cells specifically impairs FAS SPOTS formation and receptor internalization downstream of FADD recruitment.

Bottom Line: Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex.We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling.Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. rsiegel@nih.gov

ABSTRACT
Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

Show MeSH
Related in: MedlinePlus