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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

Kinetics of SPOTS formation and internalization of Fas-YFP fusion proteins. (A) Selected frames from a confocal time series of Jurkat cells transfected with wild-type Fas-YFP fusion protein and treated with anti-Fas at the indicated time point (minutes:seconds). Midsection confocal images of the whole cell are shown in the top panels, and an enlarged view of a portion of the plasma membrane is shown at each time point with the intracellular (i) and extracellular (e) compartments in the first frame of each row.
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fig5: Kinetics of SPOTS formation and internalization of Fas-YFP fusion proteins. (A) Selected frames from a confocal time series of Jurkat cells transfected with wild-type Fas-YFP fusion protein and treated with anti-Fas at the indicated time point (minutes:seconds). Midsection confocal images of the whole cell are shown in the top panels, and an enlarged view of a portion of the plasma membrane is shown at each time point with the intracellular (i) and extracellular (e) compartments in the first frame of each row.

Mentions: When transfected into Jurkat cells, Fas-YFP also formed SPOTS after receptor ligation, followed by receptor internalization, although with less prominent cap formation. An example of time-lapse images from a typical transfectant is shown in Fig. 5 and Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200406101/DC1). To determine whether or not FADD and caspase-8 are required for formation of SPOTS, we transfected wild-type Fas-YFP into mutant Jurkat cells that have been shown to lack FADD or caspase-8 and are completely resistant to Fas-induced apoptosis (Juo et al., 1998). As shown in Table I, FADD-deficient Jurkat cells as well as wild-type Jurkat cells transfected with a DD-deficient truncated Fas-YFP were almost completely deficient in SPOTS formation. Caspase-8–deficient Jurkat cells were partially deficient in SPOTS formation. As in other cell types, zVAD-fmk treatment of wild-type cells did not affect SPOTS formation although apoptosis was completely inhibited.


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)

Kinetics of SPOTS formation and internalization of Fas-YFP fusion proteins. (A) Selected frames from a confocal time series of Jurkat cells transfected with wild-type Fas-YFP fusion protein and treated with anti-Fas at the indicated time point (minutes:seconds). Midsection confocal images of the whole cell are shown in the top panels, and an enlarged view of a portion of the plasma membrane is shown at each time point with the intracellular (i) and extracellular (e) compartments in the first frame of each row.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Kinetics of SPOTS formation and internalization of Fas-YFP fusion proteins. (A) Selected frames from a confocal time series of Jurkat cells transfected with wild-type Fas-YFP fusion protein and treated with anti-Fas at the indicated time point (minutes:seconds). Midsection confocal images of the whole cell are shown in the top panels, and an enlarged view of a portion of the plasma membrane is shown at each time point with the intracellular (i) and extracellular (e) compartments in the first frame of each row.
Mentions: When transfected into Jurkat cells, Fas-YFP also formed SPOTS after receptor ligation, followed by receptor internalization, although with less prominent cap formation. An example of time-lapse images from a typical transfectant is shown in Fig. 5 and Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200406101/DC1). To determine whether or not FADD and caspase-8 are required for formation of SPOTS, we transfected wild-type Fas-YFP into mutant Jurkat cells that have been shown to lack FADD or caspase-8 and are completely resistant to Fas-induced apoptosis (Juo et al., 1998). As shown in Table I, FADD-deficient Jurkat cells as well as wild-type Jurkat cells transfected with a DD-deficient truncated Fas-YFP were almost completely deficient in SPOTS formation. Caspase-8–deficient Jurkat cells were partially deficient in SPOTS formation. As in other cell types, zVAD-fmk treatment of wild-type cells did not affect SPOTS formation although apoptosis was completely inhibited.

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