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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

Distinct caspase-dependent and -independent morphological stages in Fas signaling. Immunostaining of Fas performed after the following treatments at 37°C: (A) isotype control; (B) 15-min anti-Fas; (C) 30-min anti-Fas; (D) 60-min anti-Fas with 60-min pretreatment with 50 μM zVAD-fmk; (E) 60-min Anti-FLAG control; (F) 60-min FLAG-FasL and anti-FLAG. For each panel, a low-power mid-cell confocal section and a high power reconstruction of a z-stack of a representative cell is shown. Results are representative of three independent experiments.
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fig1: Distinct caspase-dependent and -independent morphological stages in Fas signaling. Immunostaining of Fas performed after the following treatments at 37°C: (A) isotype control; (B) 15-min anti-Fas; (C) 30-min anti-Fas; (D) 60-min anti-Fas with 60-min pretreatment with 50 μM zVAD-fmk; (E) 60-min Anti-FLAG control; (F) 60-min FLAG-FasL and anti-FLAG. For each panel, a low-power mid-cell confocal section and a high power reconstruction of a z-stack of a representative cell is shown. Results are representative of three independent experiments.

Mentions: To follow the subcellular localization of endogenous Fas during signaling, we treated the lymphoblastoid B cell line SKW6.4, which expresses relatively high levels of Fas, with anti-Fas mAb or FasL at 37°C, and then fixed and stained the cells with a secondary antibody to reveal the pattern of Fas subcellular localization. In these cells, we observed small spotlike foci of staining beginning 15 min after receptor ligation, followed by caplike structures visible after 30 min (Fig. 1). The small foci measured 0.3–0.5 μm in size. As we will show that these foci represent oligomerized receptors and require signaling protein recruitment for their formation, we will refer to them as SPOTS. Interestingly, in the presence of the caspase inhibitor zVAD-fmk, cells with SPOTS were readily seen, but cells with capped Fas were rarely seen. To determine whether or not SPOTS form before internalization of the receptor, we used a double-labeling strategy to specifically mark surface Fas complexes. As can be seen in Fig. 2 A, in SKW6.4 cells treated for 60 min with anti-Fas, Fas complexes were largely internalized. FACS staining experiments confirmed that surface receptor levels were significantly reduced in these cells (Fig. 2 B). However, in the presence of zVAD-fmk, surface-labeled SPOTS predominated, and FACS staining of zVAD-treated cells revealed normal or even increased levels of surface Fas. Thus, formation of SPOTS occurs at the plasma membrane during the same time frame that the Fas signaling complex assembles at the cytoplasmic tail of the receptor.


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)

Distinct caspase-dependent and -independent morphological stages in Fas signaling. Immunostaining of Fas performed after the following treatments at 37°C: (A) isotype control; (B) 15-min anti-Fas; (C) 30-min anti-Fas; (D) 60-min anti-Fas with 60-min pretreatment with 50 μM zVAD-fmk; (E) 60-min Anti-FLAG control; (F) 60-min FLAG-FasL and anti-FLAG. For each panel, a low-power mid-cell confocal section and a high power reconstruction of a z-stack of a representative cell is shown. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Distinct caspase-dependent and -independent morphological stages in Fas signaling. Immunostaining of Fas performed after the following treatments at 37°C: (A) isotype control; (B) 15-min anti-Fas; (C) 30-min anti-Fas; (D) 60-min anti-Fas with 60-min pretreatment with 50 μM zVAD-fmk; (E) 60-min Anti-FLAG control; (F) 60-min FLAG-FasL and anti-FLAG. For each panel, a low-power mid-cell confocal section and a high power reconstruction of a z-stack of a representative cell is shown. Results are representative of three independent experiments.
Mentions: To follow the subcellular localization of endogenous Fas during signaling, we treated the lymphoblastoid B cell line SKW6.4, which expresses relatively high levels of Fas, with anti-Fas mAb or FasL at 37°C, and then fixed and stained the cells with a secondary antibody to reveal the pattern of Fas subcellular localization. In these cells, we observed small spotlike foci of staining beginning 15 min after receptor ligation, followed by caplike structures visible after 30 min (Fig. 1). The small foci measured 0.3–0.5 μm in size. As we will show that these foci represent oligomerized receptors and require signaling protein recruitment for their formation, we will refer to them as SPOTS. Interestingly, in the presence of the caspase inhibitor zVAD-fmk, cells with SPOTS were readily seen, but cells with capped Fas were rarely seen. To determine whether or not SPOTS form before internalization of the receptor, we used a double-labeling strategy to specifically mark surface Fas complexes. As can be seen in Fig. 2 A, in SKW6.4 cells treated for 60 min with anti-Fas, Fas complexes were largely internalized. FACS staining experiments confirmed that surface receptor levels were significantly reduced in these cells (Fig. 2 B). However, in the presence of zVAD-fmk, surface-labeled SPOTS predominated, and FACS staining of zVAD-treated cells revealed normal or even increased levels of surface Fas. Thus, formation of SPOTS occurs at the plasma membrane during the same time frame that the Fas signaling complex assembles at the cytoplasmic tail of the receptor.

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