Autophagic and apoptotic response to stress signals in mammalian cells

Autophagy is a highly conserved catabolic programme for degrading proteins and organelles. This process has been shown to act as a pro-survival or pro-death mechanism in different physiological and pathological conditions. Several stress stimuli can induce autophagy, such as nutrient deprivation or critical steps in development of lower and higher eukaryotes. Apoptosis is an orchestrated form of cell death in which cells are actively involved in their own demise. Again, stress is a positive regulator of apoptosis and, in particular, of its apoptosome-mediated mitochondrial pathway. Besides discussing the individual roles played by the key molecules involved in autophagy in mammals in response to stress signals, we discuss here the interrelations between autophagy and apoptosis under these conditions.     

Structure of the nuclear pore complex in mammalian cells. Two annular components

The ultrastructure of the nuclear pore complex has been investigated in isolated nuclei of an in vitro cultured bovine liver cell line. In shadow-cast replicas of the surface of nuclei isolated in Tris buffer containing low K⁺ and Mg²⁺ concentrations (RSB) the rims of the pores appeared as annular projections with an outer diameter of 100 to 120 nm. When the nuclei were isolated in Tris buffer containing 0.1% Triton the projections were essentially lost, together with the outer membrane of the nuclear envelope. In electron micrographs of whole-mount preparations the Triton-Tris nuclei—but not the RSB nuclei—were surrounded by numerous circular structures, which obviously had been detached from the nuclear surface during the preparation. They consisted of eight granules of about 20 nm diameter which were connected in a circular fashion by fibrous material. The circular structures had an inside diameter close to 65 nm. In broken nuclei many of these circular structures contained a second, smaller circular component and a central granule. From these observations it is concluded that the annulus of the nuclear pore consists of two components and that the outer component is located in the perinuclear space in intimate association with the membrane limiting the pore. A modified model of the nuclear pore complex which accounts for this location is proposed.     

Autophagic degradation of leaf starch in plants

Autophagy is an evolutionarily conserved process in eukaryotic cells that functions to degrade cytoplasmic components in the vacuole or lysosome. Previous research indicates that the core molecular machinery of autophagosome formation works well in plants, and plant autophagy plays roles in diverse biological processes such as nutrient recycling, development, immunity and responses to a variety of abiotic stresses. Recently, we reported that autophagy contributed to leaf starch degradation, which had been thought to be a process confined to chloroplasts. This finding demonstrated a previously unidentified pathway of leaf starch depletion and a new role of basal autophagy in plants.     

Autophagic degradation of active caspase-8

Apoptotic defects endow tumor cells with survival advantages. Such defects allow the cellular stress response to take the path of cytoprotective autophagy, which either precedes or effectively blocks an apoptotic cascade. Inhibition of the cytoprotective autophagic response shifts the cells toward apoptosis, by interfering with an underlying molecular mechanism of cytoprotection. The current study has identified such a mechanism that is centered on the regulation of caspase-8 activity. The study took advantage of Bax-/- Hct116 cells that are TRAIL-resistant despite significant DISC processing of caspase-8, and of the availability of a caspase-8-specific antibody that exclusively detects the caspase-8 large subunit or its processed precursor. Utilizing these biological tools, we investigated the expression pattern and subcellular localization of active caspase-8 in TRAIL-mediated autophagy and in the autophagy-to-apoptosis shift upon autophagy inhibition. Our results suggest that the TRAIL-mediated autophagic response counter-balances the TRAIL-mediated apoptotic response by the continuous sequestration of the large caspase-8 subunit in autophagosomes and its subsequent elimination in lysosomes. The current findings are the first to provide evidence for regulation of caspase activity by autophagy and thus broaden the molecular basis for the observed polarization between autophagy and apoptosis.     

Stability of nuclear RNA in mammalian cells

The kinetics of entry of [³H]adenosine into ATP, cellular RNA, and nuclear RNA of mouse L cells were determined and analyzed. A molar accumulation curve for RNA was estimated from the specific radioactivities of RNA and ATP; this curve was resolved graphically into stable and unstable components. The stability of the unstable component (mostly heterogeneous nuclear RNA) was estimated by applying first-order decay analysis. Heterogeneous, nuclear RNA decays with an apparently uniform half-life of 23 minutes, considerably greater than some previous estimates. It is synthesized at an instantaneous rate of 5.4 × 10^?2 pg/cell per minute and reaches a steady-state level of 1.8 pg/cell in the nucleus, or 7% of the total cellular RNA. Only about 2% of the heterogeneous RNA synthesized in L cells enters polysomes as messenger RNA. The implications of these values are discussed with reference to similarly determined values for sea urchin embryos.     

Autophagic degradation in rat liver after vinblastine treatment

Mechanisms of autophagic proteolysis in the liver have been studied using vinblastine. Vinblastine stimulated degradation by induced autophagy in a dose-related fashion [1, 2]. Insulin partially inhibits the increased rate of degradation and the formation of autophagosomes as well as the lysosomal fragility induced by vinblastine. Insulin has little effect, however, on basal, non-induced degradation rates. Vinblastine-induced autophagy enhances the degradation of both ‘old’ and ‘newly’ synthesized proteins and is therefore in that sense a random process. The administration of high doses of colchicine also augments proteolysis. This effect is attributed to increase in autophagy. The very nascent vinblastine-induced autophagosomes appear to lack hydrolytic enzymes and acquire them only after fusion with lysosomes. The autophagolysosomal population induced by vinblastine is heterogeneous with respect to shape, size, content and density. Isolated secondary lysosomes (‘residual bodies’) lacking morphologically recognizable sequestered membranes (degradable substrate) show no release of degradation products. Autophagosomes fuse with secretory vesicles originating from the Golgi apparatus.     

Autophagic degradation of GZMB/granzyme B

The crucial issue for defining successful natural killer (NK)-based anticancer therapy is the ability of tumor cells to activate resistance mechanisms leading to escape from NK-mediated killing. It is now well established that such mechanisms are likely evolved under hypoxia in the tumor microenvironment. Here, we show that hypoxia-induced autophagy impairs breast cancer cell susceptibility to NK-mediated lysis and that this impairment is reverted by targeting autophagy. We provide evidence that activation of autophagy in hypoxic cells is involved in selective degradation of the pro-apoptotic NK-derived serine protease GZMB/granzyme B, thereby blocking NK-mediated target cell apoptosis. Our in vivo data validate the concept that targeting autophagy in cancer cells promotes tumor regression by facilitating their elimination by NK cells. This study provides a cutting-edge advance in our understanding of how hypoxia-induced autophagy impairs NK-mediated lysis and might pave the way for formulating more effective NK-based antitumor therapy by combining autophagy inhibitors.     

Autophagic degradation of peroxisomes in isolated rat hepatocytes.

Degradation of the peroxisomal enzymes fatty acyl-CoA oxidase and catalase was studied in hepatocytes isolated from rats treated with clofibrate and from control rats. Hepatocytes were incubated in the absence of amino acids in order to ensure maximal flux through the autophagic pathway and in the presence of cycloheximide to inhibit protein synthesis. (1) Degradation of the two peroxisomal enzymes in hepatocytes from clofibrate-fed rats, but not in hepatocytes from control rats, was much faster than that of other intracellular enzymes. This increased degradation of the peroxisomal enzymes was almost completely prevented by 3-methyladenine, an inhibitor of macroautophagic sequestration. (2) The increased degradation of the peroxisomal enzymes was also inhibited by a long-chain (C16:0) and a very-long-chain (C26:0) fatty acid, but not by C12:0, a medium-chain fatty acid, or by C8:0, a short-chain fatty acid. These results provide direct evidence for the proposal that autophagic sequestration can be highly selective [(1987) Exp. Mol. Pathol. 46, 114-122]. It is concluded that preferential autophagy of peroxisomes is prevented when these organelles are supplied with their fatty acid substrates.     

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.     

Autophagic degradation of HAS2 in endothelial cells: A novel mechanism to regulate angiogenesis

Hyaluronan plays a key role in regulating inflammation and tumor angiogenesis. Of the three transmembrane hyaluronan synthases, HAS2 is the main pro-angiogenic enzyme responsible for excessive hyaluronan production. We discovered that HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin. Using live-cell and super-resolution confocal microscopy, we found that protracted autophagy evoked a dynamic interaction between HAS2 and ATG9A, a key transmembrane autophagic protein. This regulatory axis of HAS2 degradation occurred in various cell types and species and in vivo upon nutrient deprivation. Inhibiting in vivo autophagic flux via chloroquine showed increased levels of HAS2 in the heart and aorta. Functionally, autophagic induction via endorepellin or mTOR inhibition markedly suppressed extracellular hyaluronan production in vascular endothelial cells and inhibited ex vivo angiogenic sprouting. Thus, we propose autophagy as a novel catabolic mechanism regulating hyaluronan production in endothelial cells and demonstrate a new link between autophagy and angiogenesis that could lead to potential therapeutic modalities for angiogenesis.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

Fluorine nuclear magnetic resonance-based assay in living mammalian cells

Nuclear magnetic resonance (NMR)-based screening has been recognized as a powerful approach for the identification and characterization of molecules interacting with pharmaceutical targets. Indeed, several NMR methods have been developed and successfully applied to many drug discovery projects. Whereas most of these approaches have targeted isolated biomolecular receptors, very few cases are reported with the screening performed in intact cells and cell extracts. Here we report the first successful application of the fluorine NMR-based assay n-FABS (n-fluorine atoms for biochemical screening) in living mammalian cells expressing the membrane protein fatty acid amide hydrolase (FAAH). This method allows the identification of both weak and potent inhibitors and the measurement of their potency in a physiological environment.     

Auto-reverse nuclear migration in bipolar mammalian cells on micropatterned surfaces

A novel assay based on micropatterning and time-lapse microscopy has been developed for the study of nuclear migration dynamics in cultured mammalian cells. When cultured on 10-20-microm wide adhesive stripes, the motility of C6 glioma and primary mouse fibroblast cells is diminished. Nevertheless, nuclei perform an unexpected auto-reverse motion: when a migrating nucleus approaches the leading edge, it decelerates, changes the direction of motion, and accelerates to move toward the other end of the elongated cell. During this process, cells show signs of polarization closely following the direction of nuclear movement. The observed nuclear movement requires a functioning microtubular system, as revealed by experiments disrupting the main cytoskeletal components with specific drugs. On the basis of our results, we argue that auto-reverse nuclear migration is due to forces determined by the interplay of microtubule dynamics and the changing position of the microtubule organizing center as the nucleus reaches the leading edge. Our assay recapitulates specific features of nuclear migration (cell polarization, oscillatory nuclear movement), while it allows the systematic study of a large number of individual cells. In particular, our experiments yielded the first direct evidence of reversive nuclear motion in mammalian cells, induced by attachment constraints.     

Accumulation of mature mRNA in the nuclear fraction of mammalian cells

Little is known about the nuclear mRNA content of mammalian cells. In this study, we analyzed by Northern blotting with a panel of probes the nuclear and cytoplasmic fractions derived from several rodent cell lines. For most of the genes under study, mature mRNAs could easily be detected in the nuclear fraction and accumulated to higher levels than the corresponding precursors. In addition, significant differences in the nucleo-cytoplasmic partition of mature mRNAs were observed between genes as well as between cell types (NIH 3T3, CTLL-2, D3-ES, PC-12), indicating that this nuclear accumulation of mRNA is regulated. Thus, while it is usually considered that splicing is the limiting step of pre-mRNA processing, these results point towards transport or nuclear retention of mRNA as a key determinant of nuclear mRNA metabolism.     

Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells

The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.