Regulatory functions of microtubules.

This mini-review summarizes literature and original data about the role of microtubules in interphase animal cells. Recent data have shown that functioning of microtubules is essential for such diverse phenomena as directional cell movements, distribution of organelles in the cytoplasm, and neuronal memory in the central nervous system. It is suggested that microtubules can act as an important regulatory system in eukaryotic cells.

Functions of nonmuscle myosin II in assembly of the cellular contractile system.

The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system's maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process.

Rearrangements of the actin cytoskeleton and E-cadherin-based adherens junctions caused by neoplasic transformation change cell-cell interactions.

E-cadherin-mediated cell-cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell-cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells.

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53(-/-) mice.

Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow.

Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells.

Controlling cell length.

Control of cell dimensions is an important but poorly understood aspect of morphogenesis. In this review, our primary focus is on control of cell length in different types of cells and cytoskeletal regulation of this parameter. Cell length is not a constant characteristic of certain cell type but of cells of fibroblastic morphology.

Regulation of polarity in cells devoid of actin bundle system after treatment with inhibitors of myosin II activity.

Interplay of two cytoskeletal systems--microfilaments and microtubules is essential for directional cell movement. To better understand the role of those cytoskeletal systems in polarization of cells, rat fibroblasts were incubated with drugs inhibiting activity of myosin II: blebbistatin and Y-27632. Both drugs led to disappearance of actin-myosin bundles and mature focal cell-matrix adhesions but did not affect polarization and directional motility.

Reorganization of molecular morphology of epitheliocytes and connective-tissue cells in morphogenesis and carcinogenesis.

Complete and incomplete transitions of epitheliocytes into cells of mesenchymal type, so-called epithelial-mesenchymal transitions (EMT), take place in many types of normal morphogenesis and in epithelial carcinogenesis. Connective tissue cells (fibroblasts) also undergo considerable morphological changes during normal morphogenesis and carcinogenesis, but their dynamics are less known. It is suggested that EMT and fibroblast dynamics may have some common step that is some united precursor cell type.

Transformation by RAS oncogene decreases the width of substrate-spread fibroblasts but not their length.

We compared morphometric parameters of the contours of cells in four pairs of non-transformed mouse and rat lines and of the same lines transformed by oncogenes of the RAS family. As expected, the mean areas of all RAS-transformed lines were much smaller than those of their non-transformed counterparts. At the same time the average length of cell projection did not regularly decrease after transformation.

Cellular search migrations in normal development and carcinogenesis.

This review describes the large group of morphogenetic processes designated as search migrations. Search migrations typically include two stages: i) search, when a group of cells or of the cytoplasmic processes migrate over the cell-free spaces, and ii) choice, the stage when migrating cells reach specific loci where they stop and undergo specific differentiations induced by local factors such as cell-cell contacts and humoral agents. Migrating cells that do not meet their targets usually undergo apoptosis.

ROS up-regulation mediates Ras-induced changes of cell morphology and motility.

Expression of activated Ras causes an increase in intracellular content of reactive oxygen species (ROS). To determine the role of ROS up-regulation in mediation of Ras-induced morphological transformation and increased cell motility, we studied the effects of hydrogen peroxide and antioxidant NAC on morphology of REF52 rat fibroblasts and their ability to migrate into the wound in vitro. Treatment with low dosages of hydrogen peroxide leading to 1.

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal.

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient).

Cytoskeletal mechanisms responsible for invasive migration of neoplastic cells.

Cytoskeletal reorganizations, especially alterations of contractile tension generated by the actin-myosin cortex, are of central importance in the development of the phenotype of morphologically transformed neoplastic cells with invasive behavior. These reorganizations can be regarded as genetically determined aberrations of the physiological reactions of normal cells which are responsible for their ability to undergo exploratory migrations, including epithelio-mesenchymal transformations, invasion of matrix by epithelial tubules etc. It is suggested that these physiological and neoplastic transformations are based on Rho-dependent alterations in contractility.

Rho overexpression leads to mitosis-associated detachment of cells from epithelial sheets: a link to the mechanism of cancer dissemination.

Dissemination of neoplastic cells from the primary tumor (invasion and metastasis) is a fundamentally dangerous step in multistage carcinogenesis. Recent evidence suggests that Rho GTPase-mediated signaling is linked to dissemination of cells from several different types of human tumors. The Rho family of proteins is typically associated with the regulation of cytoskeletal activity, including actin assembly, microtubule dynamics, and myosin II-dependent contractility of the actin-rich cortex.

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.

Length control is determined by the pattern of cytoskeleton.

In our previous experiments with linear strips of adhesive substrate, we found that elongated cultured fibroblasts preserve their length regardless of cell width and the number of cytoplasmic processes. This constancy of length was called 'length control'. In contrast to fibroblasts, single cultured epitheliocytes have nearly discoid shape on the plane substrata and have no length-controlling mechanism: their length on the narrow strips of adhesive substrate increased significantly in comparison with the diameter on the plane substrate.

Thread-grain transition of mitochondrial reticulum as a step of mitoptosis and apoptosis.

Association of mitochondrial population to a mitochondrial reticulum is typical of many types of the healthy cells. This allows the cell to organize a united intracellular power-transmitting system. However, such an association can create some difficulties for the cell when a part of the reticulum is damaged or when mitochondria should migrate from one cell region to another.

Rho-dependent formation of epithelial "leader" cells during wound healing.

The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special "leader" cells at the tops of outgrowths and "follower" cells along the sides.

Mechanism of filopodia initiation by reorganization of a dendritic network.

Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells.

Mechanisms of polarization of the shape of fibroblasts and epitheliocytes: Separation of the roles of microtubules and Rho-dependent actin-myosin contractility.

Cultured fibroblasts possess a characteristic polarized phenotype manifested by an elongate cell body with an anterior lamella whose cell edge is divided into protrusion-forming and inactive zones. Disruption of the fibroblast microtubule cytoskeleton leads to an increase in Rho-dependent acto-myosin contractile activity and concomitant loss of structural polarity. The functional relationship of myosin-driven contractile activity to loss of fibroblast anterior-posterior polarity is unknown.

Effects of the inhibitors of dynamics of cytoskeletal structures on the development of apoptosis induced by the tumor necrosis factor.

Changes in cytoskeletal structures have been investigated during apoptosis of epithelial HeLa cells induced by tumor necrosis factor-alpha (TNF-alpha). Shape and surface cell activity were investigated by time-lapse video microscopy, and changes of the cytoskeletal structure were studied by immune fluorescent microscopy. Addition of TNF-alpha to HeLa cell culture caused early disruption of the actin cytoskeleton and vinculin-containing focal contacts, keratin filaments, and microtubules.

Cytoskeletal control of fibroblast length: experiments with linear strips of substrate.

In order to understand the factors determining the length of fibroblasts, three cell lines (mouse embryonic fibroblasts plus human fibroblast lines AGO 1523 and M19) were cultivated on the usual planar substrate (glass) and on specially prepared narrow linear strips of the same substrate, where the cells could spread only linearly. Morphometric measurements showed that the average length of cells of each type on the 'unidimensional' strips was no different from that on the usual 'bidimensional' substrate. The addition of colcemid significantly decreased cell length on both substrates, whereas cytochalasin D increased the length.

Responses of epithelial and fibroblast-like cells to discontinuous configuration of the culture substrate.

The behaviour of epitheliocytes, their transformed analogues, and fibroblasts was studied on special culture substrates--lattices with large square openings (the area of an opening was 2000 microm2). It was shown that normal epithelocytes and fibroblasts initially attached to and spread on the lattice bars, were soon displaced into the lattice openings and appeared to be "sagged" in the substrate-free spaces. The cells remained attached to the bars only by their edges (epitheliocytes) or lateral processes (fibroblasts), whereas basal surfaces of the cells had no contacts with the substrate.

Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors.

Transforming growth factor beta (TGFbeta), the most established promoter of myofibroblast differentiation, induces ED-A cellular fibronectin and alpha-smooth muscle actin expression in fibroblastic cells in vivo and in vitro. ED-A fibronectin exerts a permissive action for alpha-smooth muscle actin expression. A morphological continuity (called fibronexus), a specialized form of focal adhesion, has been described between actin stress fibers that contain alpha-smooth muscle actin, and extracellular fibronectin, which contains the ED-A portion, in both cultured fibroblasts and granulation tissue myofibroblasts.