Monika Vöth

Short- and long-term alterations of mitochondrial morphology, dynamics and mtDNA after transient oxidative stress

Cells are exposed during their life span to fluctuating levels of reactive oxygen species (ROS). To investigate the effects of a single ROS boost in vitro, human endothelial cells (HUVEC) were treated with one short-term dose of hydrogen peroxide. This treatment resulted in a short, dose-dependent ROS peak that caused transient changes in the mitochondrial morphology and fine structure, in the frequency of mitochondrial fission and fusion and in the mRNA levels of distinct fission and fusion factors. Treatment with a higher dose induced prolonged mtDNA damage; these cells exhibited a significantly shortened replicative lifespan, indicating dose-dependent effects of oxidative stress on mitochondria.

Morpho-dynamic changes of mitochondria during ageing of human endothelial cells

Mitochondrial morphology is regulated in many cultured eukaryotic cells by fusion and fission of mitochondria. A tightly controlled balance between fission and fusion events is required to ensure normal mitochondrial and cellular functions. During ageing, mitochondria are undergoing significant changes on the functional and morphological level. The effect of ageing on fusion and fission of mitochondria and consequences of altered fission and fusion activity are still unknown although theoretical models on ageing consider the significance of these processes. Human umbilical vein endothelial cells (HUVECs) have been established as a cell culture model to follow mitochondrial activity and dysfunction during the ageing process. Mitochondria of old and postmitotic HUVECs showed distinct alterations in overall morphology and fine structure, and furthermore, loss of mitochondrial membrane potential. In parallel, a decrease of intact mitochondrial DNA (mtDNA) was observed. Fission and fusion activity of mitochondria were quantified in living cells. Mitochondria of old HUVECs showed a significant and equal decrease of both fusion and fission activity indicating that these processes are sensitive to ageing and could contribute to the accumulation of damaged mitochondria during ageing.

Structural implications of mitochondrial dynamics

Mitochondrial components are continuously distributed throughout the whole chondriome of a cell by fusion and fission. Thus, a single mitochondrion represents a transient fraction of the chondriome. Mitochondrial dynamics are responsible for intracellular distribution and reaction of mitochondria to functional requirements. Dynamics occur on different levels: overall morphology, inner membrane–matrix compartment, turnover and rearrangements of mitochondrial proteins and DNA. Electron micrographs of serial sections of human umbilical vein endothelial cells reveal perinuclear mitochondria of extreme length and with branches in those cells that also have short peripheral mitochondria. Interactions of mitochondria with cytoskeletal elements are revealed in cells treated with cytochalasin D to destroy actin fibrillar structures or after disassembling microtubule by nocodazole. In the latter case mitochondria not only become immobilized, they also acquire a multiple ring structure. In F‐actin‐disturbed cells, motility (shape changes in particular) is increased and the mitochondria become elongated. Mechanisms of how F‐actin might render mitochondria immobile may involve dynamin‐related protein 1 (DRP1) or interaction with anion channels. This may be responsible for the lack of mitochondrial motility in senescent cells. Fusion between mitochondria revealed local fluctuations of mitochondrial red fluorescent protein (mtRFP), indicating novel fast inner membrane reorganizations. Mitochondrial dynamics result from a complex interplay between the molecular organization of the inner membrane–matrix complex and cytoskeletal elements outside.

Filament organization and formation of microridges at the surface of fish epidermis.

The surface of fish epidermis cells is characterized by a varying pattern of microridges. Their width is about 0.3 μm and the height may reach values up to 1 μm. Under certain conditions these microridges can be maintained in cultures of epidermis derived from guppy embryos after outgrowth on glass coverslips. In such preparations indirect immunofluorescence microscopy allows the identification of actin and α-actinin as characteristic components of the microridges. The distribution of prekeratin-related tonofilaments (7- to 10-nm filaments) was visualized by the same method. A good correlation between the display of the microridges and accumulations of tonofilaments was observed, while the organization of microtubules seemed to be without any direct relation to the surface structures. The highly dynamic activity of microridges and their changing length is demonstrated by time-lapse cinemicrography. Ridges and their precursors, which are small zones of increased optical density, display in histochemical procedures a strong ATPase activity. The course of filaments in the ridges and in the subjacent terminal web has been studied also by electron microscopy. Epidermis cells of the midbody region of female guppies (Poecilia reticulata, Peters), the sea horse (Hippocampus kuda, Bleeker), and embryonic guppy epidermis in culture have been used. Treatment of adult guppy epidermis with Triton X-100 prior to fixation facilitated the interpretation of the organization of filaments in the microridges and the terminal web. From these observations a similarity between microridges and microvilli is discussed. The major similarities involve a core of actin filaments attached to the plasma membrane and a thickened glycocalyx. Filaments are arranged as bundles along the length of the ridges, a behavior which is most pronounced in the concentric ridges of the flame cone cells of sea horse epidermis. A model for the formation of the microridges is proposed which explains the strong interdependence of the development of surface protrusions and the organization of a terminal web. Folding up of the plasmalemma is thought to be brought about by the force generated by filaments inserting at the prospective flank region of the developing microridge. These filaments are supposed to stick in the subjacent terminal web and intermingle with tonofilaments. The course of filaments from opposite flanks is marked by a crossover under the center of the ridge. The relation of these findings to observations on the reformation of microvilli is briefly discussed.

Identification of cultivation-independent markers of human endothelial cell senescence in vitro.

Human aging processes are regulated by many divergent pathways and on many levels. Thus, to understand such a complex system and define conserved mechanisms of aging, the use of cell culture-based models is a widespread practice. An often stated advantage of in vitro aging of primary cells is the high reproducibility compared to the much more intricate aging of organisms. However, the aging process of cultured cells is, like aging of organisms, not only defined by genetic but also by environmental factors, making it difficult to distinguish between cell culture condition-induced artefacts and true aspects of aging. Therefore we investigated aging of HUVEC (human umbilical vascular endothelial cells), a well-known and widely used model system for in vitro aging, with different, already well-established cell culture protocols. Culturing conditions had indeed a strong impact on cell proliferation, the replicative lifespan and apoptosis rates. However, despite these significant differences, we found also various robust markers that define senescent HUVEC: morphological changes, increased senescence-associated β-galactosidase staining, cell cycle arrest in the G1 phase, lowered mitochondrial membrane potential and increased oxidatively modified proteins were displayed independent of cell culture protocols and could therefore be considered also as markers for in vivo aging.

Interaction of metabolic inhibitors with actin fibrils

SummaryThe dependence of the arrangement of fibrillar actin in cultured endothelial cells on metabolic conditions was investigated with cellular elements derived from the heart of Xenopus laevis tadpoles. Either primary culture or an established cell line (XTH-2) were used in these studies The metabolic stage of the cells was influenced by inhibiting respiration and lactate production. The actin pattern was revealed either by indirect immunofluorescence or by tetramethylrhodaminyl (TRITC)-phalloidin fluorescence. Total block of energy supply causes in all cases a distinct loss of actin fibrils, while inhibition of respiration alone increases the variability of actin organization. In primary XTH cells but not in XTH-2 cells cyanide disintegrates most of the actin fibres during 3 h of treatment. This effect is independent of the inhibition of respiration, since actin gels prepared from skeletal muscle also undergo destruction in the presence of cyanide. It is concluded that the actin fibrils of the primary cells and the established line behave differently to changing metabolic conditions and to application of KCN.

Do single mitochondria contain zones with different membrane potential

Observations of Lan Bo Chen’s group using a mitochondria-selective fluorochrome 5,5’,6,6’- tetrachloro- 1,1’,3,3’— tetraethylbenzimidazolocarbocyanine iodide (JC-1) indicate that mitochondriain situ may have zones of different electrochemical potential along their length. This was indicated by the formation of J-aggregates of this dye at distinct sites along a single mitochondrion. Also, intensity variations along single mitochondria were found with diamino-styryl-pyridinium methiodide (DASPMI), another fluorochrome that selectively stains mitochondria depending on their electrochemical potential. DASPMI exchanges easily with the cytoplasm and changes its quantum yield when bound to mitochondria) membranes. Therefore, fluorescence intensity is primarily controlled by the membrane environment rather than by mass accumulation. Two possible explanations of intramitochondrial fluorescence intensity variations have to be discussed: variations in the amount of mitochondria) inner membrane per unit of projection area (or voxel), and differences in the electrochemical gradient. This problem has been approached by comparing fluoro-micrographs of mitochondria in endothelial cells stained with either JC-1 or DASPMI with electron micrographs of the same mitochondria after fixation with glutardialdehyde and osmium tetroxide and ultrathin sectioning. JC-1 red fluorescence (revealing J-aggregate formation) as well as high-intensity staining with DASPMI correlate roughly with the local thickness of mitochondria; no differences in the crista organization are revealed for those areas or mitochondria exhibiting red JC-i fluorescence and those with green fluorescence. The distance between red fluorescing areas in a single mitochondrion seem to be caused by competition for dye molecules placed in between centres of JC-1 aggregation. Isolated mitochondria are of uniform small size and spherical shape; therefore, no differences in shape interfere with JC-1 staining. Thus JC-1 may be an appropriate indicator of membrane potential in isolated mitochondria. In living cells mitochondria often are large and elongated, and thus the situation is not straightforward to interpret. However, evidence is provided that there are sub-mitochondrial zones, which differ in membrane potential from one adjacent area to another, because DASPMI staining of intramitochondrial zones reveals differences in fluorescence along the total length of a mitochondrion. However, this type of bleaching develops over tens of seconds, not in the very short time range (e.g. ms) expected from the discharge of all the membranes if they were electrically coupled.

Biphasic response of human polymorphonuclear leucocytes and keratinocytes (epitheliocytes) fromXenopus laevis to mechanical stimulation

SummaryHuman polymorphonuclear leucocytes and epitheliocytes isolated from tadpole tails ofXenopus laevis were used to observe the responses of cells to mechanical stimulation with a microneedle. Biphasic responses were observed: a retraction phase lasting 1–3 s was followed by an extension phase lasting 10–40s. Weak stimulation evoked alocal response whilst on strong stimulation the whole cells rounded up. Spreading after induced rounding was at least one order of magnitude faster (it lasted less than 1–2min) than cell spreading after chemical dissociation of cell cultures. Local or extended loss of cell attachment to the substratum (observed with reflection interference contrast microscopy) preceded changes in cell morphology, visible with phase contrast microscopy. Repeated weak stimulation of one cell side induced extension and locomotion of the cell in this direction. The reported biphasic responses of cells to mechanical stimulation highlight the significance of exact timing when following any cell response to external stimuli.

Continuity of Movement and Preservation of Architecture during Cell Locomotion

Continuous fluxes of cytoplasm and membrane material are required to sustain cell locomotion. In some examples, e. g. epidermal cells of various vertebrates very small changes in the overall morphology of the cells take place during locomotion, in others e. g. fibroblasts or lymphocytes, at least the general appearance remains unaltered. Thus any model on cell locomotion has to include the continuous organization and disorganization of cell architecture under steady state conditions. Such a model is presented, based on intracellular pressure differences providing the source for the motive force and on the distribution of cytoskeletal elements providing the structural basis for force generation and cell shape. A detailed description of very small changes in cell surface topography presents the basis on which the models of cell locomotion and the control of this event can be discussed appropriately. Cytosolic calcium controls force generation and the direction of locomotion. Ca2+ concentration is highest in the lamella/cell body transition region and at the leading front, as has been revealed by scanning fluorometry.