Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A).

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.

Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro.

Although many types of membrane-bound organelles rely upon microtubule-based transport for their proper placement within the cytoplasm, the molecular mechanisms that regulate intracellular motility remain largely unknown. To address this problem, we have studied the microtubule-dependent dispersion and aggregation of pigment granules from an immortalized Xenopus melanophore cell line. We have reconstituted pigment granule motility along bovine brain microtubules in vitro using a microscope-based motility assay. Pigment granules, or melanosomes, move along single microtubules bidirectionally; however, analysis of the polarities of this movement shows that melanosomes that have been purified from dispersed cells exhibit mostly plus end-directed motility, while movement of organelles from aggregating cells is biased toward the minus end. Removal of all soluble proteins from the melanosome fractions by density gradient centrifugation does not diminish organelle motility, demonstrating that all the components required for transport have a stable association with the melanosome membranes. Western blotting shows the presence of the plus end-directed motor, kinesin-II, and the minus end-directed motor, cytoplasmic dynein in highly purified melanosomes. Therefore, purified melanosomes retain their ability to move along microtubules as well as their regulated state. Direct biochemical comparison of melanosomes from aggregated and dispersed cells may elucidate the molecular mechanisms that regulate organelle transport in melanophores.

Role of the microtubular system in morphological organization of normal and oncogene-transfected epithelial cells.

To understand better the role of the microtubular system in the development and maintenance of morphological organization of nonpolarized and polarized cells of the same origin we examined the effects of two microtubule-specific drugs, colcemid and taxol, on discoid cultured epithelial rat cells of the IAR-2 line and on polarized cells obtained from this line by transfection of mutated N-ras oncogene; morphometric, immunomorphologic, and videomicroscopic methods were used. Depolymerization of microtubules by colcemid did not cause major changes in the discoid shape of IAR cells but altered organization of actin cortex; in particular, it led to disappearance of circumferential bundle of actin microfilaments. Taxol reorganized the normal network of microtubules radiating from the perinuclear centers into numerous arrays of short microtubules not associated with any centers. Taxol-treated cells had wider circumferential bundles of microfilaments than control cells and morphometric analysis showed that their contours were closer to geometric circle than those of control or of colcemid-treated cells. These data show that function of the microtubular system is essential for maintenance of the characteristic morphological organization of discoid cells; we propose to name this function "contra-polarization." Contra-polarization is not prevented and is even promoted by taxol; this result suggests that a decentralized system of microtubules is sufficient for this function. In contrast, maintenance of polarized morphology of IAR-2 cells transfected by the N-ras oncogene is inhibited not only by colcemid but also by taxol and thus requires the presence of a normal centralized microtubular system.

Reversible structural alterations of undifferentiated and differentiated human neuroblastoma cells induced by phorbol ester.

Morphological alterations in the structure of undifferentiated and morphologically differentiated human neuroblastoma cells induced by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were examined by video microscopy and immunomorphology. In undifferentiated cells, PMA induced the formation of motile actin-rich lamellas and of stable cylindrical processes rich in microtubules. Formation of stable processes resulted either from the collapse of lamellas or the movement of the cell body away from the base of a process. In differentiated cells, PMA induced the rapid extension of small lamellas and subsequent formation of short-lived elongated processes from the lateral edges of neurites. Additionally, growth cones exhibited enhanced modulation in shape after PMA treatment. These reversible reorganizations were similar to the actinoplast-tubuloplast transformations exhibited by PMA-treated fibroblasts. We suggest that actinoplast-tubuloplast reorganizations play essential roles in morphogenesis where stable cytoplasmic extensions are induced by external stimuli. In particular, PMA-induced reorganizations of neural cells in culture may be a model for morphological modulations that occur in nerve tissue.

[Changes in the organization of the intermediate filament system of human fibroblasts in lysosomal storage diseases and their modeling]. / Izmenenie organizatsii sistemy promezhutochnykh filamentov v fibroblastakh cheloveka pri lizosomnykh bolezniakh nakopleniia i ikh modelirovanii.

The organization of the system of the vimentin intermediate filaments (IFs) in human fibroblasts in lysosomal storage diseases (Fabry's disease, mannosidosis) and their modelling has been studied in vitro. It was shown that during accumulation of nonhydrolyzable compounds, hypertrophy of the lysosomal compartment is accompanied by formation of ring-shaped bundles IFs, surrounding apparently these increased organelles. The changed organization of IFs is characteristic of polarised pathological cells in monolayer, and after repassage it is retained only at the spreading state; on transition from the discoid to extended cellular form there occurred the centrifugal shift of ring-shaped structures of IFs to active cell border and gradual restoration of radial fibrillar state of IFs. It is suggested that on intralysosomal storage of unsplit compounds reorganization of the vimentin-type IFs in ring-shaped structure is necessary for optimal distribution and stabilization into the cytoplasm of large amounts of increased lysosomes with exo- and endogenous contents. In condition of free spreading (i. e. with diminished cell density) the restoration of normal fibrillar IF organization may be due to the loss of considerable number of hypertrophied lysosomes; the involvement of lysosomal membrane in formation of active cellular border is not to be ruled out.

Fluorenone-azomethines, a novel class of microtubule inhibitors that specifically affect cell proliferation.

The effects of various azomethine derivatives on microtubule (MT) assembly in vitro as well as on cell proliferation, cell shape, and the cytoskeleton of some cultured murine cell lines were studied. 3 of them, the alpha-diphenylene-N-(p-[bis-(beta-hydroxyethyl)-amino]-phenyl)-nit rone (DHPN), alpha-diphenylene-N-(p-[N-(hydroxyethyl)-N-(gamma-hydroxypropyl)- amino]-phenyl)-nitrone, and alpha-diphenylene-N-(p-diethylaminophenyl)-nitrone, strongly inhibit the assembly of microtubules (MTs) in vitro (50% inhibition at 4 to 7 mumol/l). The same compounds are also able to disrupt preformed microtubules. Moreover, they were found to inhibit proliferation of leukaemia L 1210, melanoma B16 K, fibroblast L 929, and embryo fibroblast cells down to 1 to 10 mumol/l, completely. Immunofluorescence microscopy revealed that DHPN, used as a representative of the active azomethines, causes a reversible destruction of the microtubule part of the cytoskeleton. Apparently resulting from microtubule disruption, the intermediate filament system collapsed whereas the microfilament system remained unaffected. The results indicate that the antiproliferative action of the azomethines is based, at least partially, on their ability to attack microtubules.

Post-translational modification of microtubules is a component of synergic alterations of cytoskeleton leading to formation of cytoplasmic processes in fibroblasts.

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) induces rapid and reversible shape changes in cultured fibroblasts: extension of motile lamellas is followed by transformation of these lamellas into nonmotile stalklike processes. This "lamella-to-stalk" transformation was found to be associated with the formation of microtubules enriched in detyrosinated alpha-tubulin. This change was local: microtubules in motile lamellas at the distal ends of the processes and in the cell bodies were not enriched in detyrosinated alpha-tubulin. Detyrosinated microtubules in the processes were more resistant to Colcemid treatment than other microtubules of PMA-treated and control cells. The effects of PMA were reversible and could be abolished by sphingosine, a specific inhibitor of protein kinase C. Besides modification of microtubules, lamella-to-stalk transformation is associated with the ingrowth of intermediate filaments into the extensions. Earlier it was found that this transformation is also associated with the profound reorganization of the system of actin microfilaments. Thus, all three cytoskeletal systems are altered simultaneously during PMA-induced formation of processes. Similar "cytoskeletal synergies" may play essential roles in many morphogenetic processes--e.g., in the growth of neurites.

Evidence that intermediate filament reorganization is induced by ATP-dependent contraction of the actomyosin cortex in permeabilized fibroblasts.

Intermediate filaments (IFs) undergo specific rearrangements in cells, some aspects of which can be induced experimentally. Centripetal aggregation of the IF network, for example, can be produced by a variety of perturbations. However, the source of motive force is clear for neither in vivo nor experimentally generated IF movements, since, unlike microtubules and actin filaments, IFs have no known force-generating system directly associated with them. We recently obtained evidence that the drug-induced aggregation of vimentin IFs in fibroblasts is an active event, which requires ATP and involves the actin cytoskeleton. In the present study, we sought to test the hypothesis that IF aggregation is driven by a centripetally directed contraction of the actomyosin cortex. To that end, we have permeabilized fibroblasts with Triton X-100 in a stabilizing buffer and reactivated cytoskeletal movements in vitro, under defined solution conditions. Upon nucleotide treatment, these permeabilized cells undergo a nucleotide-dependent centripetal aggregation of vimentin IFs similar in appearance and time course to that induced in intact cells by drug treatment. During in vitro IF aggregation, the permeabilized cells remain fully spread and adherent to the substratum, and the distal ends of the microtubules and actin microfilaments retain their positions in the cell periphery, IF aggregation is accompanied by a contraction of F-actin and myosin into focal aggregates in the same perinuclear region in which the IFs accumulate. If permeabilized cells are treated with the actin-severing protein gelsolin prior to the reactivation of IF movement, the actin cytoskeleton is eliminated and IF aggregation fails to occur when ATP is added. These results strongly support a model in which the motive force for IF movement is supplied indirectly by association with a contracting actomyosin network.

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.

[Formation of an actin cytoskeleton and adhesive structures during the spreading of cultured cells]. / Formirovanie aktinovogo tsitoskeleta i adgezionnykh struktur v khode rasplastyvaniia kul'tiviruemykh kletok.

Fibroblast spreading was studied using immunofluorescent method that provided visualization of actin structures and adhesion contacts in the same cell. Four stages of actin system formation were observed. 1. Actin concentration in ruffles at the cell periphery. Formation of numerous dot-like contacts along the whole perimeter of the cell. 2. Formation of a circumferential actin bundle. Focal contacts are located at the outer edge of the bundle. 3. Gradual transformation of the circumferential bundle into actin network with triangular meshes. Peripheral (rather than internal) filaments of the network are associated with the focal contacts. 4. Appearance of the system of long straight actin bundles (stress fibers) associated with dash-like focal contacts. The stress fibers are supposed to arise from the triangular actin network which in its turn arises from the circumferential bundle. It is suggested that the formation of actin cytoskeleton is a process driven by the development of tensions in actin structures attached to the focal contacts at the cell periphery.

Organization of stress fibers in cultured fibroblasts after extraction of actin with bovine brain gelsolin-like protein.

Mouse and quail embryo fibroblasts were extracted with Triton X-100 and the resulting cytoskeletons were treated with gelsolin-like actin-capping protein (the 90-kDa protein-actin complex isolated from bovine brain). Staining of cells with rhodamine-conjugated phalloin or an antibody to actin did not reveal any actin-containing structures after treatment with the 90-kDa protein-actin complex. Extraction of actin was confirmed by SDS-gel electrophoresis. Immunofluorescence microscopy showed that vinculin and alpha-actinin were released from the cytoskeletons together with actin. However, myosin remained associated with the cytoskeleton after treatment with the 90-kDa protein-actin complex. The distribution of myosin in treated cells showed no significant difference from that in control cells: in both cases myosin was localized mainly in the stress fibers. Double-fluorescence staining showed the absence of actin in myosin-containing stress fibers of treated cells. The ultrastructural organization of actin-depleted stress fibers was studied by transmission electron microscopy of platinum replicas. On electron micrographs these fibers appeared as bundles of filaments containing clusters of globular material. It is concluded that myosin localization in stress fibers does not depend on actin.

[Focal contacts and the cytoskeleton]. / Fokal'nye kontakty i tsitoskelet.

Cultured cells attach to the substratum by means of specialized domains of cell surface, called focal contacts. The inner side of the cell membrane is associated in these structures with cytoskeletal elements, while the outer side is connected with extracellular matrix. The present review describes both light and electron microscopic methods of studying the focal contacts and ultrastructure of adhesion plaque, that is the cytoskeletal domain of focal contact. The proteins of adhesion plaque and focal contact membranes are also characterized. The processes of the formation of focal contacts and their association with the bundles of actin microfilaments in normal cultured fibroblasts are described in detail. Association of focal contacts with other cytoskeletal elements microtubules and intermediate filaments is discussed. The neoplastic transformation induced changes of focal contact system and cytoskeletal structures associated with contact sites are described.

[Organization of stress fibrils in cultured fibroblasts studied by using an actin filament-fragmenting protein]. / Issledovanie organizatsii stress-fibrill kul'tiviruemykh fibroblastov s pomoshch'iu belka, fragmentiruiushchego aktinovye filamenty.

Earlier we isolated a 1:1 complex of 90 kD-protein and actin from bovine brain. This complex was able to fragment actin filaments. Effects of this complex on the cytoskeleton of mouse and quail embryo fibroblasts are described. The cells were extracted with Triton X-100, and the resulting cytoskeletons were treated with the complex. Tetramethylrhodaminylphalloin and actin antibody staining failed to detect actin in preparations treated with the 90 kD protein-actin complex. Electrophoretic data confirmed actin solubilization upon this treatment. Immunofluorescent microscopy showed that actin solubilization was accompanied by extraction of vinculin and alpha-actinin from focal contacts and stress-fibers. In contrast, myosin distribution in treated cytoskeletons did not differ significantly from that in control extracted cells: in both the cases myosin was found mainly in the stress-fibers. Thus, myosin localization in stress-fibers does not depend on actin and is probably controlled by some other cytoskeletal component(s).

Association of intermediate filaments with vinculin-containing adhesion plaques of fibroblasts.

Double immunofluorescence staining of quail embryo fibroblasts with rabbit antibody to vinculin and mouse monoclonal antibody to vimentin revealed a coincidence between fluorescence patterns for cell-substrate focal contacts and intermediate filaments. Most of the vinculin-containing adhesion plaques coincided with the ends of vimentin-positive fibrils. This association was further corroborated by immunoelection microscopic observations of the cytoskeletons of quail and mouse fibroblasts using a platinum replica technique. The intermediate filaments were identified either by direct treatment with antivimentin IgM or by an indirect immunogold staining method. Colcemid treatment of the cells caused a collapse of intermediate filaments and destroyed their association with focal contacts. During the early stages of the colcemid-induced collapse of the intermediate filaments, single vimentin fibrils appeared to retain their association with focal contacts. The possible role of the intermediate filaments in the formation and maintenance of focal contacts is discussed.

Focal contacts of normal and RSV-transformed quail cells. Hypothesis of the transformation-induced deficient maturation of focal contacts.

Morphology and distribution of cell-substrate contacts and their association with microfilament bundles in normal and RSV-transformed quail fibroblasts (16Q line) were studied by indirect immunofluorescence. The focal contacts were visualized by antibody exclusion method using monoclonal antibody to 80 kD serum protein adsorbed on the substratum. Embryo quail cells formed focal contacts of two morphological types: (1) small punctate; and (2) elongated large contacts. These two variants of contacts were designated respectively as dot and dash contacts. Both of focal contacts contained vinculin and alpha-actinin. Double immunofluorescence staining with polyclonal antibody to actin and monoclonal antibody to vinculin revealed that the dot contacts, in contrast to the dash ones, were not associated with microfilament bundles. The dot contacts were localized mostly near the active cell edges, while the dash contacts were found near the retracted cell edges and also under the central parts of the cell. We suppose that dot contacts represent initial structures which then can undergo maturation transforming them into dash contacts. RSV-transformed 16Q cells had predominantly the dot contacts which were not only located at the edges but also in the more central parts of the lamella. The dash contacts were present only in the minority of 16Q cells. RSV transformation is assumed to affect not the ability of cells to form initial dot contacts, but the maturation of dot contacts into dash contacts.

Visualization of cellular focal contacts using a monoclonal antibody to 80 kD serum protein adsorbed on the substratum.

We have obtained a monoclonal antibody to 80 kD protein of calf serum; this protein easily and uniformly adsorbs on glass from serum-containing media. Indirect immunofluorescence staining of chick and mouse embryo fibroblasts cultured in the presence of calf serum, fixed with formaldehyde and permeabilized with Triton X-100, revealed black non-fluorescent strips and dots under the ventral cell surface, whereas all other parts of the substratum under and between cells were highly fluorescent. The distribution of non-fluorescent regions coincided with the distributed of focal contacts of cells with the substratum, revealed by interference reflection microscopy, as well as with the distribution of vinculin-containing plaques. The dark regions were also associated with the ends of microfilament bundles revealed by immunofluorescence with an anti-actin antibody. Thus, non-fluorescent regions seen after anti-80 kD staining are parts of the substratum under the focal contacts. Visualization of focal contacts with anti-80 kD provides very contrasting and high resolution pictures. Evidence is presented that 80 kD protein is adsorbed to glass in the areas of focal contacts, but the antibodies used for staining cannot penetrate these contacts.

Cell shape and organization of cytoskeleton and surface fibronectin in non-tumorigenic and tumorigenic rat liver cultures.

Morphological changes associated with neoplastic transformation of epithelial cells were studied in a series of IAR cell lines derived from rat liver. The series included three independently obtained, non-tumorigenic lines and five derived, tumorigenic lines. The morphology of cell surfaces was observed by scanning electron microscopy; the distribution of actin, tubulin and fibronectin was determined by indirect immunofluorescence. All the non-tumorigenic lines had a typical epithelioid morphology: isolated cells of these lines spread on the substratum had a discoid shape and contained circular, marginal bundles of microfilaments and microtubules. In denser areas, the cells formed monolayered sheets with characteristic marginal bundles of microfilaments near the free edges. Decreased spreading of isolated cells on the substratum was the characteristic feature that distinguished tumorigenic lines from their non-tumorigenic parent lines. In particular a decrease in the size of the ring-like, peripheral lamella and its disintegration into several discrete lamellar zones were often observed; as a result, the cell shape was altered from discoid to polygonal or elongated. The altered distribution of microfilament bundles and microtubules was characteristic in elongated cells; the pattern of the cytoskeletal elements of these cells resembled that of polarized fibroblasts. Complete disappearance of microfilament bundles was observed in cells of only one tumorigenic line. Various degrees of disorganization of monolayered cell sheets were observed in tumorigenic cultures, accompanied by an altered distribution of microfilament bundles. The alterations in the fibronectin-containing structures were more complex: there were often fewer fibronectin "spots' and fibrils at the lower surfaces of cells of tumorigenic cultures as compared with those of non-tumorigenic ones; there were more fibrils in dense cultures of certain lines but fewer in others. It is concluded that alterations in the ability to spread on the substratum and to form cell-cell contacts are common features of morphologically transformed fibroblastic and epithelial cultures. However, the actual changes in the cytoskeletal structures that accompany these alterations are different in transformed cultures of various tissue types.

[Morphology of the microtubule system during the sphering, layering and polarization of normal and transformed fibroblasts]. / Morfologiia sistemy mikrotrubochek pri okruglenii, rasplastyvanii i poliarizatsii normal'nykh i transformirovannykh fibrolbastov.

The ultrastructure of the genital system (ductus seminalis, vestibulum, ductus tortuosus and canaliculus fecundans) has been studied during mating and oviposition. All these organs have a common organization and contain three parts: muscular and epithelial layer and chitin intima lining the interior surface of the female genital system. Ultrastructures of the muscular tissue and of the basal part of epidermis are similar. The luminal surface and microvilli are different in various organs of the female genital system. The ultrastructural pattern and the thickness of chitin intima may also vary, and can be used as a character for identificating different organs of the female system in ultrathin sections.