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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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Related in: MedlinePlus

Dependence of Fas-YFP receptor internalization on the Fas DD and caspase activity. (A) FACS analysis of Fas-YFP internalization in transfected wild-type Jurkat cells after anti-Fas (top) or FasL (bottom) treatment for 2 h. The dashed histograms represent levels of Fas-YFP surface expression before treatment and the solid lines represent levels after treatment. The numbers are the change in geometric mean channel fluorescence between treated and untreated cells for each transfectant, with positive numbers indicating decreased fluorescence and negative numbers an increase after treatment. Populations were gated on viable cells expressing the Fas-YFP fusion protein. The fusion protein constructs are indicated above each panel. (B) FACS analysis of internalization of WT Fas-YFP fusion proteins in Jurkat clones stably overexpressing Bcl-x and v-FLIP and a control clone transfected with the drug-resistance plasmid alone. Results are shown as in A.
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fig6: Dependence of Fas-YFP receptor internalization on the Fas DD and caspase activity. (A) FACS analysis of Fas-YFP internalization in transfected wild-type Jurkat cells after anti-Fas (top) or FasL (bottom) treatment for 2 h. The dashed histograms represent levels of Fas-YFP surface expression before treatment and the solid lines represent levels after treatment. The numbers are the change in geometric mean channel fluorescence between treated and untreated cells for each transfectant, with positive numbers indicating decreased fluorescence and negative numbers an increase after treatment. Populations were gated on viable cells expressing the Fas-YFP fusion protein. The fusion protein constructs are indicated above each panel. (B) FACS analysis of internalization of WT Fas-YFP fusion proteins in Jurkat clones stably overexpressing Bcl-x and v-FLIP and a control clone transfected with the drug-resistance plasmid alone. Results are shown as in A.

Mentions: Cells were treated for 60 min with crosslinked anti-Fas APO-1-3 mAb and microscopically scored for receptor clustering with fluorescence microscopy by a blinded observer. Numbers represent the average and SD of at least two independent experiments. Specific Fas downmodulation was calculated from the median channel fluorescence data shown in Fig. 6. Fas-induced apoptosis was calculated from annexin and PI staining and normalized to the percent specific death in the appropriate control cells.


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)

Dependence of Fas-YFP receptor internalization on the Fas DD and caspase activity. (A) FACS analysis of Fas-YFP internalization in transfected wild-type Jurkat cells after anti-Fas (top) or FasL (bottom) treatment for 2 h. The dashed histograms represent levels of Fas-YFP surface expression before treatment and the solid lines represent levels after treatment. The numbers are the change in geometric mean channel fluorescence between treated and untreated cells for each transfectant, with positive numbers indicating decreased fluorescence and negative numbers an increase after treatment. Populations were gated on viable cells expressing the Fas-YFP fusion protein. The fusion protein constructs are indicated above each panel. (B) FACS analysis of internalization of WT Fas-YFP fusion proteins in Jurkat clones stably overexpressing Bcl-x and v-FLIP and a control clone transfected with the drug-resistance plasmid alone. Results are shown as in A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172594&req=5

fig6: Dependence of Fas-YFP receptor internalization on the Fas DD and caspase activity. (A) FACS analysis of Fas-YFP internalization in transfected wild-type Jurkat cells after anti-Fas (top) or FasL (bottom) treatment for 2 h. The dashed histograms represent levels of Fas-YFP surface expression before treatment and the solid lines represent levels after treatment. The numbers are the change in geometric mean channel fluorescence between treated and untreated cells for each transfectant, with positive numbers indicating decreased fluorescence and negative numbers an increase after treatment. Populations were gated on viable cells expressing the Fas-YFP fusion protein. The fusion protein constructs are indicated above each panel. (B) FACS analysis of internalization of WT Fas-YFP fusion proteins in Jurkat clones stably overexpressing Bcl-x and v-FLIP and a control clone transfected with the drug-resistance plasmid alone. Results are shown as in A.
Mentions: Cells were treated for 60 min with crosslinked anti-Fas APO-1-3 mAb and microscopically scored for receptor clustering with fluorescence microscopy by a blinded observer. Numbers represent the average and SD of at least two independent experiments. Specific Fas downmodulation was calculated from the median channel fluorescence data shown in Fig. 6. Fas-induced apoptosis was calculated from annexin and PI staining and normalized to the percent specific death in the appropriate control cells.

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