Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants--the "Skulachev-ion" derivatives.

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1-2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of "bad" mitochondria in cell physiology is discussed.

Novel mitochondria-targeted antioxidants, "Skulachev-ion" derivatives, accelerate dermal wound healing in animals.

It is shown that the novel mitochondria-targeted antioxidant SkQ1, (10-(6'-plastoquinonyl) decyltriphenylphosphonium) stimulates healing of full-thickness dermal wounds in mice and rats. Treatment with nanomolar doses of SkQ1 in various formulations accelerated wound cleaning and suppressed neutrophil infiltration at the early (7 h) steps of inflammatory phase. SkQ1 stimulated formation of granulation tissue and increased the content of myofibroblasts in the beginning of regenerative phase of wound healing. Later this effect caused accumulation of collagen fibers. Local treatment with SkQ1 stimulated re-epithelization of the wound. Lifelong treatment of mice with SkQ1 supplemented with drinking water strongly stimulated skin wounds healing in old (28 months) animals. In an in vitro model of wound in human cell cultures, SkQ1 stimulated movement of epitheliocytes and fibroblasts into the "wound". Myofibroblast differentiation of subcutaneous fibroblasts was stimulated by SkQ1. It is suggested that SkQ1 stimulates wound healing by suppression of the negative effects of oxidative stress in the wound and also by induction of differentiation. Restoration of regenerative processes in old animals is consistent with the "rejuvenation" effects of SkQ1, which prevents some gerontological diseases.

Selective elimination of mitochondria from living cells induced by inhibitors of bioenergetic functions.

The inhibitors of oxidative phosphorylation induced fragmentation of mitochondria without any signs of apoptosis in CV-1 and HeLa cells. Prolonged treatment with the uncouplers (alone or in combination with the inhibitors of respiration) caused perinuclear clusterization of mitochondria, followed by their selective elimination. The fraction of mitochondria-depleted cells remained viable.

Marginal blebbing during the early stages of TNF-induced apoptosis indicates alteration in actomyosin contractility.

Dynamics of alterations of cell surface topography during TNF-induced apoptosis of HeLa cells was examined by phase-contrast videomicroscopy and immunomorphological analysis. The final stage of apoptosis accompanied by cell rounding and general blebbing of the cell surface became after 4-6 h of incubation but much earlier, after 1.5-3 h, essentially flattened lamellipodia at the active edges transformed into the small blebs that were continuously extended and retracted during the next 1-2 h. This phenomenon was called "marginal blebbing". It took place before the cytochrome c release from mitochondria to cytosol. Marginal blebbing was inhibited by drugs that depolymerized actin microfilaments (cytochalasin, latrunculin) or decreased Rho-kinase-dependent contractility of actin-myosin cortex (H7, HA-1077, Y27632). A pancaspase inhibitor, zVAD-fmk, completely prevented marginal and general blebbing, and TNF-induced apoptosis. DEVD-fmk, a specific inhibitor of caspase-3, inhibited both marginal and general blebbing but not cell rounding and death. Thus, marginal blebbing is an early microfilament-dependent apoptotic event. It is suggested that it is initiated by minimal activation of caspase-3 and the following local Rho-kinase-dependent stimulation of actin-myosin cortex contractility. Localization of marginal blebs at the active edge may be associated with special organization of cortex in that zone.

Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing.

Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton- matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction.

Intermediate filament collapse is an ATP-dependent and actin-dependent process.

In this study, we have investigated the properties of intermediate filament rearrangements using experimentally induced collapse of vimentin intermediate filaments in mouse fibroblasts. In these cells, depolymerizing microtubules by colchicine or vinblastine treatment at 37 degrees C results in a two-stage collapse of intermediate filaments. First, the vimentin filaments aggregate into large cables; then, the cables coil into a dense mass surrounding the nucleus. By using inhibitors of oxidative phosphorylation along with glucose deprivation to lower intracellular ATP levels by 95%, we have found that both stages of intermediate filament collapse require ATP. However, once collapse has occurred, only the second stage can be reversed in the absence of microtubules by lowering ATP levels. An additional difference between the two stages of collapse was revealed by treating cells with cytochalasin D: the formation of intermediate filament cables still occurs after disruption of the actin filament system by cytochalasin, but the subsequent coiling of cables to form a perinuclear mass is strongly inhibited by these conditions, and can be reversed by applying cytochalasin to cells in which intermediate filaments have already undergone complete collapse. We propose that the formation of vimentin cables involves a phosphorylation event, while the coiling of cables into a perinuclear mass relies on interaction of intermediate filaments with a component of the actin cortex.

Effects of small doses of cytochalasins on fibroblasts: preferential changes of active edges and focal contacts.

The effects of low doses of cytochalasin B (2 micrograms/ml) and cytochalasin D (0.2 microgram/ml) on the spreading of normal mouse fibroblasts in culture were investigated to find out which components of cell-substrate interactions are most sensitive to alterations of the state of actin cytoskeleton. Cytochalasin B disorganized the cortical layer of actin microfilaments and caused partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Diffuse close contacts were apparently insensitive to cytochalasin B. Low doses of cytochalasin B did not inhibit the outgrowth and maintenance of lamellas at the cell periphery. However, in contrast to controls, these lamellas had no distal zones with convex outer edges and ruffles at the upper surfaces. The disappearance of these ruffling active edges was accompanied by loss of the ability to clear the surface of the lamellas from the concanavalin A receptors crosslinked by the corresponding ligand. The effects of cytochalasin D were similar to those of cytochalasin B. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of crosslinked surface receptors.

Spreading of fibroblasts in medium containing cytochalasin B: formation of lamellar cytoplasm as a combination of several functional different processes.

Normal cultured mouse fibroblasts spreading on solid substrate extend and attach numerous pseudopods; lamellar cytoplasm is eventually formed from the attached pseudopods. Fibroblasts spreading in the presence of cytochasin B (CB) from de novo a system of arbor-like branched processes rather than lamellar cytoplasm. The growing and fully formed arbor-like processes, in contrast to normal lamellar cytoplasm, have low contractility and are unable to clear patched concanavalin A receptors from their surfaces; their attachement sites are not associated with microfilament bundles. The cells spreading in medium containing CB and Colcemid do not form well-organized branched structures but extend and attach numerous unstable pseudopods. It is suggested that normal formation of lamellar cytoplasm can be regarded as a combination of several functionally different processes: (a) of rudimentary pseudopodial reactions resistant to CB and Colcemid; (b) of CB-sensitive lamellization; and (c) of Colcemid-sensitive stabilization.