Maurice Geuskens

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12 – 24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.

A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

N-linked glycans containing linear poly-N-acetyllactosamine as sorting signals in endocytosis in Trypanosoma brucei

African trypanosomes, such as Trypanosoma brucei, are protozoan parasites that are transmitted by the tsetse fly and cause sleeping sickness in humans and Nagana in cattle. Trypanosomes evade the immune responses of their hosts by varying their surface coat protein (VSG) and restricting exocytosis and endocytosis to an invagination of the plasma membrane called the flagellar pocket (FP). The FP represents only 0.5% of the cellular surface but membrane turnover here occurs at high rates [1] [2] [3]. No model has yet been proposed to account for the sequestration of membrane proteins and the rate of membrane turnover that occur in the FP. Recent data have suggested that glycans are involved in the sorting of membrane proteins in polarized cells [4] [5] [6] [7]. Here, we show that N-linked glycans containing linear poly-N-acetyllactosamine (pNAL) are only associated with proteins of the FP/endocytic pathway in T. brucei and are present only in bloodstream forms of the parasite. These glycoproteins bind to tomato lectin (TL), a property that allowed their single-step isolation. Chito-oligosaccharides that compete specifically for pNAL binding to TL also inhibited receptor-mediated uptake of several ligands. These results suggest a model in which N-linked linear pNAL acts as a sorting signal for endocytosis in trypanosomes.

Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity.

We report the characterization of aTrypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of theTBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem.227, 780–786).

Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos

The development of the nucleoli and the sites of rDNA transcription have been studied by high-resolution autoradiography during the cleavage stages of mouse embryos. The appearance of fibrillar centres at the periphery of the fibrillar primary nucleoli has been observed at the 4-cell stage. Several fibrillar centres, interconnected by electron-dense fibrillar strands, form a reticulated region around the fibrillar mass at the 6-to 8-cell stage. After a 10 min pulse with [3H]uridine, only this peripheral network is labelled. At the late morula and at the blastocyst stage, the fibrillar component (nucleolonema) of the reticulated nucleoli is labelled after 10 min [3H]uridine incorporation. When the embryos are reincubated for 2 h in cold medium, the label is localized mainly in the granular component. Fibrillar centres are not labelled. Autoradiograms of in vitro developed embryos pulsed for 2 h with [3H]uridine confirm that the central fibrillar core of the nucleoli of 6-to 8-cell embryos is never labelled. Thus, the fibrillar constituent of this core is not homologous to the fibrillar component of the nucleoli of later stage embryos, which is the site of active rDNA transcription. An interpretation of nucleologenesis during early mouse embryogenesis is proposed.

Apoptosis and karyogamy in syncytia induced by the HIV-1-envelope glycoprotein complex.

One of the several mechanisms accounting for HIV-1induced lymphodepletion resides in the capacity of the envelope glycoprotein complex (Env) expressed on HIV-1 infected cells to interact with CD4 and a suitable coreceptor (CXCR4 or CCR5) expressed on non-infected cells, thereby triggering cell fusion. The vast majority of syncytium-inducing HIV-1 variants employs CXCR4 as a coreceptor, and a strong correlation between CD4 T cell decline and infection by syncytium-induced HIV-1 variants has been established. Formation of syncytia in vitro is generally followed by cell death, either by apoptosis or necrosis, depending on the cell types engaged in the process. In vivo, in lymphoid tissues from AIDS patients, syncytium formation is accompanied by overexpression of tissue transglutaminase, a marker of apoptosis. In a model culture system of syncytium-dependent cell death, HeLa cells stably transfected with a lymphotropic HIV-1 Env gene (HeLa Env) were fused by co-culture with CD4/CXCR4-expressing HeLa cells (Hela CD4). As an internal control, HeLa cell expressing a monotropic HIV-1 envelope (ADA, that does not interact with the CXCR4 co-receptor expressed on HeLa CD4) failed to form syncytia with HeLa CD4 cells and did not undergo apoptosis (Figure 1A). Fusion events were monitored by means of two stable, non-toxic CellTracker fluorescent dies with which HeLa Env (CellTracker Green) or HeLa CD4 cells (CellTracker Orange) were pre-incubated. After 24 h of co-culture, juxtaposed nuclei from both cell types could be clearly distinguished within a common cytoplasm, the center of syncytia (Figure 1A) whose overall organization roughly recapitulates that of normal cells. After 48 h, however, nucleoplasm fusion (karyogamy) occurred in *50% of syncytia, as detectable by the blending of the two CellTracker dies within the nucleus. Concomitantly, an increasing percentage of syncytia spontaneously exhibited apoptotic chromatin condensation, as detected with the Hoechst 33342 dye (Figure 1A,B). Karyogamy induced by the ENV-CD4/CXCR4 interaction involved circumscript fusions of the nuclear envelopes and occurred in a fraction of cells which are lacking signs of nuclear chromatin condensation (Figure 1C). It thus may be uncoupled from apoptotic chromatin condensation, as also suggested by kinetic analyses (Figure 1B). What is then the functional relationship between syncytium formation, karyogamy, and apoptosis? Freshly formed syncytia (24 h) driven into apoptosis by a shortterm incubation (2 h) with staurosporin (STS) exhibited a similar degree of chromatin condensation as syncytia undergoing apoptosis spontaneously (48 h). However, neither STS (Figure 1) nor other pro-apoptotic agents such as etoposide (not shown) did increase the frequency of syncytia exhibiting karyogamy, indicating that apoptosis induced by exogenous stimuli obeys other principles than spontaneous syncytial apoptosis. The two pan-caspase inhibitors Boc-D.fmk and Z-VAD.fmk (but not the chemically related cathepsin inhibitor Z-FA.fmk) suppressed nuclear chromatin condensation (Figure 1A,B) as well as the detachment of HeLa Env/HeLa CD4 syncytia from the culture substrate (not shown). Moreover, inhibition of apoptosis by the caspase inhibitor Z-VAD.fmk allowed for the formation of much larger syncytia as compared to control HeLa Env/HeLa CD4 cocultures (Figure 1D), suggesting that caspase activation with the consequent irreversible loss of cellular functions limits the recruitment of cells into growing syncytia. In strict contrast to their effects on chromatin condensation (Figure 1A,B) and syncytial size (Figure 1D), Boc-D.fmk or Z-VAD.fmk failed to prevent nuclear fusion (Figure 1A,B), suggesting that, karyogamy precedes in a caspase-independent fashion. To further investigate the impact of ongoing fusion events between individual cells/nuclei and existing syncytia (or fusion between syncytia), the monoclonal antibody Leu 3a (which blocks the CDR2 region of the CD4 D1 domain) was added to cocultures. Blockade of CD4 by adding Leu3a from the beginning of cocultures completely abolished cell fusion (Figure 1A). When added after 24 h of culture, Leu 3a led to a net reduction of the median number of nuclei per syncytium, strongly inhibited karyogamy (Figure 1A), and, concomitantly, reduced the frequency of apoptotic nuclear events observed 72 h (Figure 1D) after initiation of coculture. Altogether, these data suggest that the size of syncytia determines the probability of karyogamy and spontaneous apoptosis and that, inversely, apoptotic caspase activation limits ongoing syncytium formation. More importantly, these data suggest also that both karyogamy and apoptosis may be mechanistically coupled to nonphysiologial syncytium formation. To our knowledge, this is the first description of nuclear fusion in somatic mammalian cells. The molecular mechanisms of nuclear fusion or karyogamy has previously only been explored in the context of sexual reproduction of unicellular fungi such as Saccharomyces cerevisiae. It will be interesting to learn whether similar mechanisms also participate in cell fusion-induced karyogamy of somatic mammalian cells. Cell Death and Differentiation (2000) 7, 1137 ± 1139 ã 2000 Macmillan Publishers Ltd All rights reserved 1350-9047/00

Alphafoetoprotein uptake by cloned cell lines derived from a nickel-induced rat rhabdomyosarcoma.

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Comparative Analysis of Antibody Responses against HSP60, Invariant Surface Glycoprotein 70, and Variant Surface Glycoprotein Reveals a Complex Antigen-Specific Pattern of Immunoglobulin Isotype Switching during Infection by Trypanosoma brucei

Rat, mouse, pig and chicken alphafoetoproteins (AFP), rat serum albumin and egg albumin, or their fluoresceinated conjugates were added to cultures of several cloned cell lines isolated from a nickel-induced rat rhabdomyosarcoma. The intracellular uptake of assayed proteins was revealed by the indirect immunoperoxidase technique and/or by direct fluorescence microscopy. All the clones examined bound AFP, and all but one internalized the protein. The protein localized in the membrane and the cytoplasm, as well as along straight processes interconnecting cells. Nuclei were always AFP negative. The protein uptake of fluoresceinated conjugates of AFP and serumalbumin was already visible 15 min after incubation and progressed with time to reach a plateau 4-5 h later. Ultrastructural radioautographs of cells incubated with [3H]-AFP (rat) showed protein accumulation in several organelles and particularly in lipid droplets. Parallel to these observations, the intracellular presence of AFP within myofibrillar structures was demonstrated in tongue sections of rat foetuses and neonates. The results presented here provide experimental evidence of the reappearance in cloned cell lines derived from a primary rhabdomyosarcoma of a property pertaining to foetal striated muscle.

Alpha-Fetoprotein Binding and Uptake by Primary Cultures of Human Skeletal Muscle

ABSTRACT During Trypanosoma brucei infections, the response against the variant surface glycoprotein (VSG) of the parasite represents a major interaction between the mammalian host immune system and the parasite surface. Since immune recognition of other parasite derived factors also occurs, we examined the humoral host response against trypanosome heat shock protein 60 (HSP60), a conserved antigen with an autoimmune character. During experimental T. bruceiinfection in BALB/c mice, the anti-HSP60 response was induced when parasites differentiated into stumpy forms. This response was characterized by a stage-specific immunoglobulin isotype switching as well as by the induction of an autoimmune response. Specific recognition of trypanosome HSP60 was found to occur during the entire course of infection. Immunoglobulin G2a (IgG2a) and IgG2b antibodies, induced mainly in a T-cell-independent manner, were observed during the first peak of parasitemia, whereas IgG1 and IgG3 antibodies were found at the end of the infection, due to a specific T-cell-mediated response. Comparative analysis of the kinetics of anti-HSP60, anti-invariant surface glycoprotein 70 (ISG70), and anti-VSG antibody responses indicated that the three trypanosome antigens give rise to specific and independent patterns of immunoglobulin isotype switching.

In vitro binding of 3H-estradiol to eosinophil and neutrophil granulocytes in various tissues (normal and neoplastic) of newborn and adult rats

alpha-Fetoprotein (AFP), a serum alpha-globulin mainly synthesized by the fetal liver and the yolk sac, is the major carrier of polyunsaturated fatty acids during embryo-fetal development. One property characteristic of fetal cells undergoing growth and differentiation is their ability to bind and internalize AFP. In the present work, we have studied the binding and endocytosis of AFP by human muscular cells developing in vitro. Primary cultures of human skeletal muscle, obtained from biopsies and examined at two stages of differentiation (myoblasts and myotubes), were incubated for different times, at 0 and 37 degrees C, with a colloidal-gold-conjugated human AFP probe and studied by light and electron microscopy, as well as by laser scanning confocal microscopy in the reflection mode. The results obtained show that (a) human myoblasts in primary culture bind and internalize the protein, probably through specific AFP receptors, (b) this property is strongly reduced or lost in well-differentiated myotubes, and (c) AFP is also bound, throughout culture development, to the extracellular matrix of fusing myoblasts and differentiated myotubes. The physiological significance of AFP uptake by human myoblasts undergoing growth and differentiation may be based on the ability of AFP to carry and deliver fatty acids to fetal cells.