Apoptosis and eryptosis: Striking differences on biomembrane level.

The cell plasma membrane plays an essential role in programmed cell death of nucleated cells (apoptosis) and erythrocytes (eryptosis), and its changes due to loss of transmembrane asymmetry are quite similar. However, nucleated cells possess the network of intracellular membranes, which are missing in erythrocytes. Providing comparative studies with series of molecular probes, we observe dramatic differences in membrane lipid order in the course of apoptosis and eryptosis. In contrast to nucleated cells, in which a significant drop of the lipid order in the plasma membrane is observed, the erythrocyte membrane retains the relatively high level of the lipid order. Observation in nucleated cells of significant differences between inner and plasma membranes and detection of apoptotic bodies with different organization suggest that the decrease in the lipid order of their plasma membrane could be at least partially explained by the phospholipid and/or cholesterol exchange between membranes. Such features are absent in erythrocytes.

Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns--thereby forced into a bipolar morphology--displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.

Interaction of poly(L-lysine)-g-poly(ethylene glycol) with supported phospholipid bilayers.

Interactions between the graft copolymer poly(L-lysine)-g-poly(ethylene glycol), PLL-g-PEG, and two kinds of surface-supported lipidic systems (supported phospholipid bilayers and supported vesicular layers) were investigated by a combination of microscopic and spectroscopic techniques. It was found that the application of the copolymer to zwitterionic or negatively charged supported bilayers in a buffer of low ionic strength led to their decomposition, with the resulting formation of free copolymer-lipid complexes. The same copolymer had no destructive effect on a supported vesicular layer made up of vesicles of identical composition. A comparison between poly(L-lysine), which did not induce decomposition of supported bilayers, and PLL-g-PEG copolymers with various amounts of PEG side chains per backbone lysine unit, suggested that steric repulsion between the PEG chains that developed upon adsorption of the polymer to the nearly planar surface of a supported phospholipid bilayer (SPB) was one of the factors responsible for the destruction of the SPBs by the copolymer. Other factors included the ionic strength of the buffer used and the quality of the bilayers, pointing toward the important role defects present in the SPBs play in the decomposition process.

Exon skipping of cathepsin B: mitochondrial targeting of a lysosomal peptidase provokes cell death.

The alternatively spliced messenger RNA of the human cysteine peptidase cathepsin B missing exons 2 and 3 encodes a truncated form of the enzyme lacking the signal peptide and part of the inhibitory propeptide. This deletion results in a new N-terminal leader sequence characteristic of proteins predestined for transport into mitochondria. We determined enzyme targeting to intracellular organelles by transfecting HeLa cells with constructs containing segments of variable length of the N terminus of truncated cathepsin B fused to green fluorescent protein. Co-localization of the constructs with mitochondria and the endoplasmic reticulum was probed with specific markers. None of the chimeric products were found in the endoplasmic reticulum, showing that truncated cathepsin B is misrouted from its regular biosynthetic pathway and forced to enter the mitochondria instead of lysosomes as its final destination. The first 20 amino acids of the new N terminus were necessary and sufficient for mitochondrial targeting, but only cells expressing the complete truncated cathepsin B sequence died by nuclear fragmentation. This new and unexpected behavior draws attention to an additional extralysosomal role for a cysteine peptidase with several recognized important pathophysiological functions. Mitochondrial targeting of cathepsin B may have significant consequences on cell life in pathological or physiological situations characterized by excessive transcription of the cathepsin B message lacking exons 2 and 3, as observed for instance in osteoarthritic cartilage.

Exploring the role of 5' alternative splicing and of the 3'-untranslated region of cathepsin B mRNA.

The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase. Transfection of fusion constructs with GFP and luciferase containing the full-length 5'-UTR, the variant lacking exon 2, and that lacking exons 2 and 3 into mammalian cells, resulted in modulation of the biosynthetic rate of cathepsin B in a cell-specific manner. Constructs missing these exons were biosynthetically more efficient than the full-length counterpart. Luciferase was cloned upstream of the 3'-UTR, downstream of the 5'-UTR, or sandwiched between the 5'- and the 3'-UTR. The UTRs of cathepsin B downregulated luciferase biosynthesis moderately when present individually, with the 3'-UTR being more efficient than the 5'-UTR, and downregulated it even more when present simultaneously. A truncated cathepsin B-GFP chimeric product derived from the 5'-UTR missing exons 2 and 3 induced cell death. The increased biosynthetic rate and abnormal trafficking of cathepsin B observed in pathologies such as cancer and osteoarthritis may depend on alternative splicing of pre-mRNA.

The alternative use of exons 2 and 3 in cathepsin B mRNA controls enzyme trafficking and triggers nuclear fragmentation in human cells.

Pathological overexpression and trafficking of the cysteine peptidase cathepsin B depend in part on the composition of its mRNA. To investigate the roles of the alternatively spliced exons 2 and 3 in the 5'-untranslated region of cathepsin B mRNA we produced constructs of cathepsin B fused to green fluorescent protein. Expression and trafficking of the fluorescent chimeric products was followed in living human immortalized chondrocytes and HeLa cells. Although synthesized at different rates, proteins encoded by the full transcript and by that missing exon 2 followed a classic route, with the endosomal-lysosomal compartment as the final target. The point-mutated variant missing the glycosylation site for lysosomal targeting followed the secretory pathway. A truncated form of cathepsin B lacking the signal peptide and part of the propeptide, and encoded by the construct missing exons 2 and 3, was neither found in the Golgi apparatus nor in vesicles, but rather in the cytoplasm as patches associated with membranous and short fibrillar elements. This particular form of truncated cathepsin B produced nuclear damage and shrinking of the trans Golgi network and of the acidic compartment. The C-terminal, six-amino acid-long propeptide of cathepsin B did not exhibit a discernible role in protein trafficking.

Ligand-specific targeting of microspheres to phagocytes by surface modification with poly(L-lysine)-grafted poly(ethylene glycol) conjugate.

PURPOSE: The purpose of this study was to demonstrate specific receptor-mediated targeting of phagocytes by functional surface coatings of microparticles, shielding from nonspecific phagocytosis and allowing ligand-specific interactions via molecular recognition. METHODS: Coatings of the comb polymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were investigated for potential to inhibit 1) nonspecific spreading of human blood-derived macrophages (MOs) and dendritic cells (DCs) on glass and 2) nonspecific phagocytosis of PLL-g-PEG-coated, carboxylated polystyrene (PS) or biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Coating was performed by adsorption of positively charged PLL-g-PEG on negatively charged microparticles or plasma-cleaned glass through electrostatic interaction. The feasibility of ligand-specific interactions was tested with a model ligand, RGD, conjugated to PEG chains of PLL-g-PEG to form PLL-g-PEG-RGD and compared with inactive ligand conjugate, PLL-g-PEG-RDG. RESULTS: Coatings with PLL-g-PEG largely impaired the adherence and spreading of MOs and DCs on glass. The repellent character of PLL-g-PEG coatings drastically reduced phagocytosis of coated PS and PLGA microparticles to 10% in presence of serum. With both MOs and DCs, we observed ligand-specific interactions with PLL-g-PEG-RGD coatings on glass and PS and PLGA microspheres. Ligand specificity was abolished when using inactive ligand conjugate PLL-g-PEG-RDG, whereas repellency of coating was maintained. CONCLUSIONS: Coatings of PLL-g-PEG-ligand conjugates provide a novel technology for ligand specific targeting of microspheres to MOs and DCs while reducing nonspecific phagocytosis.