A toolbox for the immunomagnetic purification of signaling organelles.

Homeostasis and the complex functions of organisms and cells rely on the sophisticated spatial and temporal regulation of signaling in different intra- and extracellular compartments and via different mediators. We here present a set of fast and easy to use protocols for the target-specific immunomagnetic enrichment of receptor containing endosomes (receptosomes), plasma membranes, lysosomes and exosomes. Isolation of subcellular organelles and exosomes is prerequisite for and will advance their detailed subsequent biochemical and functional analysis.

Cell fate decisions regulated by K63 ubiquitination of tumor necrosis factor receptor 1.

Signaling by tumor necrosis factor (TNF) receptor 1 (TNF-R1), a prototypic member of the death receptor family, mediates pleiotropic biological outcomes ranging from inflammation and cell proliferation to cell death. Although many elements of specific signaling pathways have been identified, the main question of how these selective cell fate decisions are regulated is still unresolved. Here we identified TNF-induced K63 ubiquitination of TNF-R1 mediated by the ubiquitin ligase RNF8 as an early molecular checkpoint in the regulation of the decision between cell death and survival.

Immunomagnetic isolation of pathogen-containing phagosomes and apoptotic blebs from primary phagocytes.

Macrophages and polymorphonuclear neutrophils are professional phagocytes essential in the initial host response against intracellular pathogens such as Mycobacterium tuberculosis. Phagocytosis is the first step in phagocyte-pathogen interaction, where the pathogen is engulfed into a membrane-enclosed compartment termed a phagosome. Subsequent effector functions of phagocytes result in killing and degradation of the pathogen by promoting phagosome maturation, and, terminally, phago-lysosome fusion.

Separation of magnetically isolated TNF receptosomes from mitochondria.

We previously demonstrated that tumor necrosis factor receptor-1 (TNF-R1) initiates distinct TNF signaling pathways depending on the localization of the receptor. While TNF-R1 at the plasma membrane transmits proinflammatory and antiapoptotic signals, internalized TNF-R1 forms signaling endosomes (TNF receptosomes) that transmit proapoptotic signals. These findings were obtained by a novel technique for the isolation of morphologically intact endocytic vesicles containing magnetically labeled TNF-R1 complexes using a high-gradient, free-flow magnetic chamber.

Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes.

Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system.

Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes.

We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression.

Subcellular compartmentalization of TNF receptor-1 and CD95 signaling pathways.

Receptors that belong to the family of death-receptors including TNF receptor-1 (TNF-R1), CD95 (Fas, APO-1) and TRAIL receptors (TRAIL-R1, TRAIL R2/DR4/DR5) transduce signals resulting in entirely different biological outcomes: They promote cell death via apoptosis but are also capable of inducing anti-apoptotic signals through the transcription factor nuclear factor NF-κB or activation of the proliferative MAPK/ERK protein kinase cascade resulting in cell protection and tissue regeneration. Recent findings revealed a regulatory role of receptor internalization and its intracellular trafficking in selectively transmitting signals that lead either to apoptosis or to the survival of the cell, providing a clue to the understanding of these contradictory biological phenomena. In this chapter we review our data obtained during the Collaborative Research Center 415 (CRC 415) focusing on the compartmentalization of TNF-R1 and CD95 pro and anti-apoptotic signaling.

TRAIL signaling is mediated by DR4 in pancreatic tumor cells despite the expression of functional DR5.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and agonistic anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antibodies are currently under clinical investigation for treatment of different malignancies. TRAIL activates DR4 and DR5 and thereby triggers apoptotic and non-apoptotic signaling pathways, but possible different roles of DR4 or DR5 in these responses has poorly been addressed so far. In the present work, we analyzed cell viability, DISC formation as well as IL-8 and NF-kappaB activation side by side in responses to TRAIL and agonistic antibodies against DR4 (mapatumumab) and against DR5 (lexatumumab) in pancreatic ductal adenocarcinoma cells.

The Polycomb group protein EED couples TNF receptor 1 to neutral sphingomyelinase.

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer's, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2.

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood.

Immunomagnetic isolation of tumor necrosis factor receptosomes.

Internalized tumor necrosis factor (TNF) receptor-1 (TNF-R1) recruits the adaptor proteins TRADD and FADD, as well as caspase-8, to establish the "death-inducing signaling complex" (DISC). DISC formation and apoptosis depend strictly on TNF-R1 internalization, whereas recruitment of TRAF-2 and RIP-1 to signal for NF-kappaB activation occurs from TNF-R1 at the cell surface. Findings revealed that TNF-R1 establishes divergent TNF signaling pathways depending on compartmentalization of TNF-R1 to the plasma membrane or to plasma membrane-derived endocytic vesicles harboring the TNF-R1-associated DISC.

Regulation of TNFR1 and CD95 signalling by receptor compartmentalization.

The death receptors tumour-necrosis factor receptor-1 (TNFR1) and CD95 (also known as FAS and APO-1) transduce signals that promote cell death by apoptosis. However, these receptors are also capable of inducing anti-apoptotic signals through the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) or through activation of the proliferative mitogen-activated protein kinase (MAPK) cascade. Recent findings reveal a role for receptor internalization and endosomal trafficking in selectively transmitting the signals that lead either to apoptosis or to the survival of the cell.

CARP-2 is an endosome-associated ubiquitin ligase for RIP and regulates TNF-induced NF-kappaB activation.

Background: The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) elicits cellular responses by signaling through a receptor complex that includes the essential adaptor molecule RIP. One important consequence of signaling is activation of the transcription factor NF-kappaB, and failure to downregulate TNF-induced NF-kappaB transcriptional activity results in chronic inflammation and death. Internalization of the receptor complex plays an important regulatory role in TNF signaling.

Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling.

Apoptosis signaling through CD95 (Fas/APO-1) involves aggregation and clustering of the receptor followed by its actin-dependent internalization. Internalization is required for efficient formation of the death-inducing signaling complex (DISC) with maximal recruitment of FADD, caspase-8/10 and c-FLIP occurring when the receptor has reached an endosomal compartment. The first detectable event during CD95 signaling is the formation of SDS-stable aggregates likely reflecting intense oligomerization of the receptor.

Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism.

The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively.

The role of receptor internalization in CD95 signaling.

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis.

Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles.

The molecular regulation of the recruitment of initial signaling complexes at the TNF-R1 is poorly defined. We demonstrate here that within minutes internalized TNF-R1 (TNF receptosomes) recruits TRADD, FADD, and caspase-8 to establish the "death-inducing signaling complex" (DISC). In addition, we identified the TNF-R1 internalization domain (TRID) required for receptor endocytosis and provide evidence that TNF-R1 internalization, DISC formation, and apoptosis are inseparable events.