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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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Redistribution of wild-type and mutant Fas-YFP fusion proteins after anti-Fas treatment. (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. The top panels show vertical maximum intensity projections of the stacks and the bottom panels show a mid-cell section of the same stack rotated 90°. The numbers underneath each panel are the percentage of cells exhibiting receptor SPOTS after 30 min of anti-Fas mAb treatment. (B) Comparison of SPOTS induced by FasL and anti-Fas. SKW6.4 cells were transiently transfected with the indicated Fas-YFP fusion protein constructs and treated with either anti-Fas mAb or FasL-FLAG with anti-FLAG cross-linking as described in the Patients, cell lines, plasmids, and reagents section in Materials and methods. After 60 min at 37°C, live cells were scored for the presence of SPOTS or caps by a blinded observer in duplicate. The numbers are the average ± SEM for each condition. Without anti-FLAG cross-linking <10% SPOTS or caps were observed (not depicted). (C) Cell death induced in SKW6.4 the same preparations of FasL and anti-Fas as in B. Cell death was measured at 6 h by annexin/PI staining.
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fig4: Redistribution of wild-type and mutant Fas-YFP fusion proteins after anti-Fas treatment. (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. The top panels show vertical maximum intensity projections of the stacks and the bottom panels show a mid-cell section of the same stack rotated 90°. The numbers underneath each panel are the percentage of cells exhibiting receptor SPOTS after 30 min of anti-Fas mAb treatment. (B) Comparison of SPOTS induced by FasL and anti-Fas. SKW6.4 cells were transiently transfected with the indicated Fas-YFP fusion protein constructs and treated with either anti-Fas mAb or FasL-FLAG with anti-FLAG cross-linking as described in the Patients, cell lines, plasmids, and reagents section in Materials and methods. After 60 min at 37°C, live cells were scored for the presence of SPOTS or caps by a blinded observer in duplicate. The numbers are the average ± SEM for each condition. Without anti-FLAG cross-linking <10% SPOTS or caps were observed (not depicted). (C) Cell death induced in SKW6.4 the same preparations of FasL and anti-Fas as in B. Cell death was measured at 6 h by annexin/PI staining.

Mentions: To examine if the changes we observed in the subcellular localization of Fas occurred during signaling in live cells, we used confocal microscopy to study the localization of Fas-YFP fusion proteins before and after receptor triggering by FasL or agonistic antibodies. In transiently transfected Cos cells, Fas-YFP fusion proteins were distributed diffusely on the plasma membrane in most cells. Some cells exhibited additional intracellular fluorescence in patterns consistent with Golgi complex localization. After addition of agonistic anti-Fas antibody or FasL, intense foci of Fas-YFP fluorescence appeared at the plasma membrane within 8–15 min, peaking at 60 min (Fig. 4 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200406101/DC1). These foci appeared before changes in cell shape or nuclear morphology characteristic of apoptosis. Three-dimensional reconstruction of confocal z-sections of these cells confirmed the surface localization of SPOTS (Fig. 4 A). As with endogenous Fas, caspase inhibition did not block formation of SPOTS. In contrast, deletion of the Fas DD almost completely blocked the formation of SPOTS. We observed quantitatively similar results in SKW6.4 cells transfected with Fas-YFP, including the dependence of SPOTS formation on the Fas DD (Fig. 4 B). FasL was somewhat less efficient at inducing SPOTS despite equivalent maximal levels of apoptosis induced by FasL and anti-Fas mAb (Fig. 4, B and C). These data indicate that Fas SPOTS form in living cells after antibody and ligand stimulation and are not simply the result of passive antibody-mediated receptor clustering.


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)

Redistribution of wild-type and mutant Fas-YFP fusion proteins after anti-Fas treatment. (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. The top panels show vertical maximum intensity projections of the stacks and the bottom panels show a mid-cell section of the same stack rotated 90°. The numbers underneath each panel are the percentage of cells exhibiting receptor SPOTS after 30 min of anti-Fas mAb treatment. (B) Comparison of SPOTS induced by FasL and anti-Fas. SKW6.4 cells were transiently transfected with the indicated Fas-YFP fusion protein constructs and treated with either anti-Fas mAb or FasL-FLAG with anti-FLAG cross-linking as described in the Patients, cell lines, plasmids, and reagents section in Materials and methods. After 60 min at 37°C, live cells were scored for the presence of SPOTS or caps by a blinded observer in duplicate. The numbers are the average ± SEM for each condition. Without anti-FLAG cross-linking <10% SPOTS or caps were observed (not depicted). (C) Cell death induced in SKW6.4 the same preparations of FasL and anti-Fas as in B. Cell death was measured at 6 h by annexin/PI staining.
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Related In: Results  -  Collection

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fig4: Redistribution of wild-type and mutant Fas-YFP fusion proteins after anti-Fas treatment. (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. The top panels show vertical maximum intensity projections of the stacks and the bottom panels show a mid-cell section of the same stack rotated 90°. The numbers underneath each panel are the percentage of cells exhibiting receptor SPOTS after 30 min of anti-Fas mAb treatment. (B) Comparison of SPOTS induced by FasL and anti-Fas. SKW6.4 cells were transiently transfected with the indicated Fas-YFP fusion protein constructs and treated with either anti-Fas mAb or FasL-FLAG with anti-FLAG cross-linking as described in the Patients, cell lines, plasmids, and reagents section in Materials and methods. After 60 min at 37°C, live cells were scored for the presence of SPOTS or caps by a blinded observer in duplicate. The numbers are the average ± SEM for each condition. Without anti-FLAG cross-linking <10% SPOTS or caps were observed (not depicted). (C) Cell death induced in SKW6.4 the same preparations of FasL and anti-Fas as in B. Cell death was measured at 6 h by annexin/PI staining.
Mentions: To examine if the changes we observed in the subcellular localization of Fas occurred during signaling in live cells, we used confocal microscopy to study the localization of Fas-YFP fusion proteins before and after receptor triggering by FasL or agonistic antibodies. In transiently transfected Cos cells, Fas-YFP fusion proteins were distributed diffusely on the plasma membrane in most cells. Some cells exhibited additional intracellular fluorescence in patterns consistent with Golgi complex localization. After addition of agonistic anti-Fas antibody or FasL, intense foci of Fas-YFP fluorescence appeared at the plasma membrane within 8–15 min, peaking at 60 min (Fig. 4 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200406101/DC1). These foci appeared before changes in cell shape or nuclear morphology characteristic of apoptosis. Three-dimensional reconstruction of confocal z-sections of these cells confirmed the surface localization of SPOTS (Fig. 4 A). As with endogenous Fas, caspase inhibition did not block formation of SPOTS. In contrast, deletion of the Fas DD almost completely blocked the formation of SPOTS. We observed quantitatively similar results in SKW6.4 cells transfected with Fas-YFP, including the dependence of SPOTS formation on the Fas DD (Fig. 4 B). FasL was somewhat less efficient at inducing SPOTS despite equivalent maximal levels of apoptosis induced by FasL and anti-Fas mAb (Fig. 4, B and C). These data indicate that Fas SPOTS form in living cells after antibody and ligand stimulation and are not simply the result of passive antibody-mediated receptor clustering.

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