Limits...
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.

Show MeSH

Related in: MedlinePlus

Capping and internalization but not SPOTS depend on caspase activity. (A) SKW 6.4 cells were stimulated as shown and then labeled for surface and internalized Fas as described in the Flow cytometric quantitation of Fas surface levels section of Materials and methods. The top panels show Alexa-488 staining of surface Fas in green, the middle panels show surface and intracellular staining by Alexa-594 in red, and the bottom panels show combined fluorescence with colocalized proteins staining in yellow. Separate experiments showed that the two anti-IgG secondary antisera did not cross-block (not depicted). Each panel shows medium and high power magnifications from left to right. Bars: (left) 5 μm; (right) 1 μm. (B) Flow cytometric quantitation of surface Fas expression after similar treatment of SKW 6.4 cells. Numbers are derived from the geometric mean fluorescence normalized a value of 100 for untreated cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172594&req=5

fig2: Capping and internalization but not SPOTS depend on caspase activity. (A) SKW 6.4 cells were stimulated as shown and then labeled for surface and internalized Fas as described in the Flow cytometric quantitation of Fas surface levels section of Materials and methods. The top panels show Alexa-488 staining of surface Fas in green, the middle panels show surface and intracellular staining by Alexa-594 in red, and the bottom panels show combined fluorescence with colocalized proteins staining in yellow. Separate experiments showed that the two anti-IgG secondary antisera did not cross-block (not depicted). Each panel shows medium and high power magnifications from left to right. Bars: (left) 5 μm; (right) 1 μm. (B) Flow cytometric quantitation of surface Fas expression after similar treatment of SKW 6.4 cells. Numbers are derived from the geometric mean fluorescence normalized a value of 100 for untreated cells.

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)

Capping and internalization but not SPOTS depend on caspase activity. (A) SKW 6.4 cells were stimulated as shown and then labeled for surface and internalized Fas as described in the Flow cytometric quantitation of Fas surface levels section of Materials and methods. The top panels show Alexa-488 staining of surface Fas in green, the middle panels show surface and intracellular staining by Alexa-594 in red, and the bottom panels show combined fluorescence with colocalized proteins staining in yellow. Separate experiments showed that the two anti-IgG secondary antisera did not cross-block (not depicted). Each panel shows medium and high power magnifications from left to right. Bars: (left) 5 μm; (right) 1 μm. (B) Flow cytometric quantitation of surface Fas expression after similar treatment of SKW 6.4 cells. Numbers are derived from the geometric mean fluorescence normalized a value of 100 for untreated cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Capping and internalization but not SPOTS depend on caspase activity. (A) SKW 6.4 cells were stimulated as shown and then labeled for surface and internalized Fas as described in the Flow cytometric quantitation of Fas surface levels section of Materials and methods. The top panels show Alexa-488 staining of surface Fas in green, the middle panels show surface and intracellular staining by Alexa-594 in red, and the bottom panels show combined fluorescence with colocalized proteins staining in yellow. Separate experiments showed that the two anti-IgG secondary antisera did not cross-block (not depicted). Each panel shows medium and high power magnifications from left to right. Bars: (left) 5 μm; (right) 1 μm. (B) Flow cytometric quantitation of surface Fas expression after similar treatment of SKW 6.4 cells. Numbers are derived from the geometric mean fluorescence normalized a value of 100 for untreated cells.
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