Association of nuclear matrix proteins with granular and threaded nuclear bodies in cell lines undergoing apoptosis.

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyer et al., Exp. Cell Res. 221,27-40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-alpha, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequences in vitro, thus mediating the formation of looped DNA structures in vivo. Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

Theileria parva-transformed T cells show enhanced resistance to Fas/Fas ligand-induced apoptosis.

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L) are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.

Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles.

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2) and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.

Proliferation is a prerequisite for bacterial superantigen-induced T cell apoptosis in vivo.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex class II molecules and selectively interacts with T cells that bear certain T cell receptor (TCR) V beta domains. Administration of SEB in adult mice results in initial proliferation of V beta 8+ T cells followed by a state of unresponsiveness resulting from a combination of clonal deletion and clonal anergy in the SEB-reactive population. At this time, it is unclear what relationship exists between the T cells that have proliferated and those that have been deleted or have become anergic. Here we show that only a fraction of the potentially reactive V beta 8+ T cells proliferate in response to SEB in vivo, and that all the cells that have proliferated eventually undergo apoptosis. Virtually no apoptosis can be detected in the nonproliferating V beta 8+ T cells. These data demonstrate a causal relationship between proliferation and apoptosis in response to SEB in vivo, and they further indicate that T cells bearing the same TCR V beta segment can respond differently to the same superantigen. The implications of this differential responsiveness in terms of activation and tolerance are discussed.

Crystal structure of yeast peroxisomal multifunctional enzyme: structural basis for substrate specificity of (3R)-hydroxyacyl-CoA dehydrogenase units.

(3R)-hydroxyacyl-CoA dehydrogenase is part of multifunctional enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric multifunctional enzyme is very different.

Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments.

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.

Hepatitis C Virus Nonstructural 5A Protein Inhibits Lipopolysaccharide-Mediated Apoptosis of Hepatocytes by Decreasing Expression of Toll-Like Receptor 4.

Background. Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to modulate multiple cellular processes, including apoptosis. The aim of this study was to assess the effects of HCV NS5A on apoptosis induced by Toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Methods. Apoptotic responses to TLR4 ligands and the expression of molecules involved in TLR signaling pathways in human hepatocytes were examined with or without expression of HCV NS5A. Results. HCV NS5A protected HepG2 hepatocytes against LPS-induced apoptosis, an effect linked to reduced TLR4 expression. A similar downregulation of TLR4 expression was observed in Huh-7-expressing genotype 1b and 2a. In agreement with these findings, NS5A inhibited the expression of numerous genes encoding for molecules involved in TLR4 signaling, such as CD14, MD-2, myeloid differentiation primary response gene 88, interferon regulatory factor 3, and nuclear factor-κB2. Consistent with a conferred prosurvival advantage, NS5A diminished the poly(adenosine diphosphate-ribose) polymerase cleavage and the activation of caspases 3, 7, 8, and 9 and increased the expression of anti-apoptotic molecules Bcl-2 and c-FLIP. Conclusions. HCV NS5A downregulates TLR4 signaling and LPS-induced apoptotic pathways in human hepatocytes, suggesting that disruption of TLR4-mediated apoptosis may play a role in the pathogenesis of HCV infection.

FLIP prevents apoptosis induced by death receptors but not by perforin/granzyme B, chemotherapeutic drugs, and gamma irradiation.

FLICE-inhibitory protein, FLIP (Casper/I-FLICE/FLAME-1/CASH/CLARP/MRIT), which contains two death effector domains and an inactive caspase domain, binds to FADD and caspase-8, and thereby inhibits death receptor-mediated apoptosis. Here, we characterize the inhibitory effect of FLIP on a variety of apoptotic pathways. Human Jurkat T cells undergoing Fas ligand-mediated apoptosis in response to CD3 activation were completely resistant when transfected with FLIP. In contrast, the presence of FLIP did not affect apoptosis induced by granzyme B in combination with adenovirus or perforin. Moreover, the Fas ligand, but not the perforin/granzyme B-dependent lytic pathway of CTL, was inhibited by FLIP. Apoptosis mediated by chemotherapeutic drugs (i.e., doxorubicin, etoposide, and vincristine) and gamma irradiation was not affected by FLIP or the absence of Fas, indicating that these treatments can induce cell death in a Fas-independent and FLIP-insensitive manner.

Cornea graft endothelial cells undergo apoptosis by way of an alternate (caspase-independent) pathway.

PURPOSE: To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process. MATERIALS AND METHODS: Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry. RESULTS: Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells. CONCLUSION: During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.

The Transcription Factor NFATc2 Controls Apoptosis and Activation of T Cells by IL-6 in Colitis

The nuclear factor of activated T cells (NFAT) family of transcription factors controls calcium signaling in T lymphocytes. In this study, we have identified a crucial regulatory role of the transcription factor NFATc2 in T cell-dependent experimental colitis. Similar to ulcerative colitis in humans, the expression of NFATc2 was up-regulated in oxazolone-induced chronic intestinal inflammation. Furthermore, NFATc2 deficiency suppressed colitis induced by oxazolone administration. This finding was associated with enhanced T cell apoptosis in the lamina propria and strikingly reduced production of IL-6, -13, and -17 by mucosal T lymphocytes. Further studies using knockout mice showed that IL-6, rather than IL-23 and -17, are essential for oxazolone colitis induction. Administration of hyper-IL-6 blocked the protective effects of NFATc2 deficiency in experimental colitis, suggesting that IL-6 signal transduction plays a major pathogenic role in vivo. Finally, adoptive transfer of IL-6 and wild-type T cells demonstrated that oxazolone colitis is critically dependent on IL-6 production by T cells. Collectively, these results define a unique regulatory role for NFATc2 in colitis by controlling mucosal T cell activation in an IL-6-dependent manner. NFATc2 in T cells thus emerges as a potentially new therapeutic target for inflammatory bowel diseases.

ß-Catenin Regulation during the Cell Cycle: Implications in G2/M and Apoptosis

ß-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. ß-Catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. However, in some developmental contexts and cell systems ß-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of ß-Catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous ß-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase¿synchronized epithelial cells. ß-Catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of ß-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not ß-catenin, is detected in M phase. Interestingly, overexpression of a stable form of ß-catenin, or inhibition of endogenous ß-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for ß-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.