Atomic Force Microscopy of height fluctuations of fibroblast cells

We investigated the nanometer scale height fluctuations of 3T3 fibroblast cells with the atomic force microscope (AFM) under physiological conditions. Correlation between these fluctuations and lateral cellular motility can be observed. Fluctuations measured on leading edges appear to be predominantly related to actin polymerization-depolymerization processes. We found fast (5 Hz) pulsatory behavior with 1--2 nm amplitude on a cell with low motility showing emphasized structure of stress fibres. Myosin driven contractions of stress fibres are thought to induce this pulsation.Comment: 6 pages, 5 figures, 1 tabl

Mitochondria-Targeted Antioxidant SkQ1 Improves Dermal Wound Healing in Genetically Diabetic Mice

Oxidative stress is widely recognized as an important factor in the delayed wound healing in diabetes. However, the role of mitochondrial reactive oxygen species in this process is unknown. It was assumed that mitochondrial reactive oxygen species are involved in many wound-healing processes in both diabetic humans and animals. We have applied the mitochondria-targeted antioxidant 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) to explore the role of mitochondrial reactive oxygen species in the wound healing of genetically diabetic mice. Healing of full-thickness excisional dermal wounds in diabetic C57BL/KsJ-db−/db− mice was significantly enhanced after long-term (12 weeks) administration of SkQ1. SkQ1 accelerated wound closure and stimulated epithelization, granulation tissue formation, and vascularization. On the 7th day after wounding, SkQ1 treatment increased the number of α-smooth muscle actin-positive cells (myofibroblasts), reduced the number of neutrophils, and increased macrophage infiltration. SkQ1 lowered lipid peroxidation level but did not change the level of the circulatory IL-6 and TNF. SkQ1 pretreatment also stimulated cell migration in a scratch-wound assay in vitro under hyperglycemic condition. Thus, a mitochondria-targeted antioxidant normalized both inflammatory and regenerative phases of wound healing in diabetic mice. Our results pointed to nearly all the major steps of wound healing as the target of excessive mitochondrial reactive oxygen species production in type II diabetes

Separation of Propulsive and Adhesive Traction Stresses in Locomoting Keratocytes

Strong, actomyosin-dependent, pinching tractions in steadily locomoting (gliding) fish keratocytes revealed by traction imaging present a paradox, since only forces perpendicular to the direction of locomotion are apparent, leaving the actual propulsive forces unresolved. When keratocytes become transiently “stuck” by their trailing edge and adopt a fibroblast-like morphology, the tractions opposing locomotion are concentrated into the tail, leaving the active pinching and propulsive tractions clearly visible under the cell body. Stuck keratocytes can develop ∼1 mdyn (10,000 pN) total propulsive thrust, originating in the wings of the cell. The leading lamella develops no detectable propulsive traction, even when the cell pulls on its transient tail anchorage. The separation of propulsive and adhesive tractions in the stuck phenotype leads to a mechanically consistent hypothesis that resolves the traction paradox for gliding keratocytes: the propulsive tractions driving locomotion are normally canceled by adhesive tractions resisting locomotion, leaving only the pinching tractions as a resultant. The resolution of the traction pattern into its components specifies conditions to be met for models of cytoskeletal force production, such as the dynamic network contraction model (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G.G. Borisy. 1997. J. Cell Biol. 139:397–415). The traction pattern associated with cells undergoing sharp turns differs markedly from the normal pinching traction pattern, and can be accounted for by postulating an asymmetry in contractile activity of the opposed lateral wings of the cell

Prospects for Drugs Based on the Mitochondria-Targeting Antioxidant SkQ1 in Treatment of Wounds with Impaired Healing

Chronic wounds with impared wound healing that require prolong time for healing remain unsolved problem of modern medicine. Excessive oxidative stress plays an important role in the pathogenesis of chronic wounds caused by aging, diabetes and other pathologies. This review is aimed at the role of mitochondria in oxidative stress and to the future prospects for using the innovative mitochondria targeted antioxidants for treatment of impaired wounds. Recent studies in old mice and mice with type 2 diabetes showed that the mitochondrial antioxidant SkQ1 [10- (6'- plastoquinonyl) decyltriphenylphosphonium] stimulates healing of full-thickness dermal wounds. SkQ1 accelerates inflammatory stage of wound healing, maturation of granulation tissue, angiogenesis and epithelization of wounds. The anti-inflammatory effect of SkQ1 is possibly connected to decreased inflammatory activation of the vascular endothelium, which is typical for aging, diabetes and other pathologies. Local administration of SkQ1 also accelerates wound healing and provides strong anti-inflammatory effect in the model of acute aseptic inflammation. In addition, SkQ1 to stimulate apoptosis of neutrophils and suppresses their activation, as well as suppresses inflammatory activation of mast cells. In the wound model in vitro, SkQ1 accelerates movement of epithelial cells and fibroblasts into the «wound» and stimulates differentiation of human subcutaneous fibroblasts to myofibroblasts. Reviewed data suggest that SkQ1-based topical drugs have a great potential to treat wounds that exhibit impaired healing also in patients suffering from chronic critical illness

Long astral microtubules uncouple mitotic spindles from the cytokinetic furrow

Depletion of MCAK, which lengthens astral microtubules, induces oscillations of the mitotic spindle, and displaces the plane of cell division

Bistability in the actin cortex

Multi-color fluorescence imaging experiments of wave forming Dictyostelium cells have revealed that actin waves separate two domains of the cell cortex that differ in their actin structure and phosphoinositide composition. We propose a bistable model of actin dynamics to account for these experimental observation. The model is based on the simplifying assumption that the actin cytoskeleton is composed of two distinct network types, a dendritic and a bundled network. The two structurally different states that were observed in experiments correspond to the stable fixed points in the bistable regime of this model. Each fixed point is dominated by one of the two network types. The experimentally observed actin waves can be considered as trigger waves that propagate transitions between the two stable fixed points

Electric Field Exposure Triggers and Guides Formation of Pseudopod-Like Blebs in U937 Monocytes

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to regular, round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10-20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb formation. In contrast to round-shaped blebs, PLBs retracted within several minutes after exposure. Cells treated with 1 nM of the actin polymerization blocker cytochalasin D were unable to form PLBs and instead produced stationary, spherical blebs with no elongation or retraction capacity. Live cell fluorescent actin tagging showed that during elongation actin promptly entered the PLB interior, forming bleb cortex and scaffold, which was not seen in stationary blebs. Overall, PLB formation was governed by both passive (physicochemical) effects of membrane permeabilization and active cytoskeleton assembly in the living cell. To a certain extent, PLB mimics the membrane extension in the process of cell migration and can be employed as a nonchemical model for studies of cytomechanics, membrane-cytoskeleton interaction and cell motility

Mitochondrial fragmentation and superoxide anion production in coronary endothelial cells from a mouse model of type 1 diabetes

Mitochondria frequently change their shapes by fusion and fission and these morphological dynamics play important roles in mitochondrial function and development as well as programmed cell death. The goal of this study is to investigate whether: (1) mitochondria in mouse coronary endothelial cells (MCECs) isolated from diabetic mice exhibit increased fragmentation; and (2) chronic treatment with a superoxide anion (O2 −) scavenger has a beneficial effect on mitochondrial fragmentation in MCECs. MCECs were freshly isolated and lysed for protein measurement, or cultured to determine mitochondrial morphology and O2 − production. For the ex vivo hyperglycaemia experiments, human coronary endothelial cells were used. Elongated mitochondrial tubules were observed in MCECs isolated from control mice, whereas mitochondria in MCECs from diabetic mice exhibited augmented fragmentation. The level of optic atrophy 1 (OPA1) protein, which leads to mitochondrial fusion, was significantly decreased, while dynamin-related protein 1 (DRP1), which leads to mitochondrial fission, was significantly increased in MCECs from diabetic mice. Diabetic MCECs exhibited significantly higher O2 − concentrations in cytosol and mitochondria than control MCECs. Administration of the O2 − scavenger TEMPOL to diabetic mice for 4 weeks led to a significant decrease in mitochondrial fragmentation without altering the levels of OPA1 and DRP1 proteins in MCECs. High-glucose treatment for 24 h significantly induced mitochondrial fragmentation, which was restored by TEMPOL treatment. In addition, excess O2 − production, either in cytosol or in mitochondria, significantly increased mitochondrial fragmentation. These data suggest that lowering the O2 − concentration can restore the morphological change in mitochondria and may help improve mitochondrial function in diabetic MCECs