Myosin is involved in postmitotic cell spreading

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time- lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.     

Monoclonal antibodies to cell surface antigens involved in sex pheromone induced mating in Streptococcus faecalis

Hybridoma cell lines producing monoclonal antibodies to Streptococcus faecalis cell surface antigens were constructed. Some of the antibodies isolated were directed against surface components involved in pheromone-induced mating. This paper describes the use of the monoclonal antibodies to identify antigenically related surface components detected by immunoprecipitation and Western blotting, their use in pheromone response assays, and their use as functional inhibitors in mating experiments.     

Investigation of signal transduction pathways involved in melanoma cell spreading

Integrins are a major family of heterodimeric adhesion receptors that are responsible for anchoring cells to extracellular matrix and they also can initiate intracellular signal pathways. Here parental and alpha 4-expressing human malignant melanoma cell lines were used to study the effect of protein kinase C (PKC), protein tyrosine kinases (PTKs) and intracellular Ca2+ on alpha 4 beta 1-mediated cell spreading on VCAM-1. Incubation of melanoma cells with PKC inhibitor inhibited alpha 4 beta 1-mediated melanoma cell spreading completely. Effect of intracellular Ca2+ on melanoma cell spreading was also investigated by non-phorbol ester tumor promotor, thapsigargin, which blocks the ability of the endoplasmic reticulum to replenish stocks of calcium which naturally leak out into the cytosol leading to a transient increase in concentration of intracellular calcium. The results showed that alpha 4 beta 1-mediated spreading was also required intracellular calcium involvement. However, in the presence of PTKs inhibitor melanoma cells showed long, thin dendiritic projections compared to control cells. Previously, data was obtained from immunofluorescense experiments showed that after genistein treatment, alpha 4-expressing cells exhibited considerable amounts of alpha 4 integrin and PTKs in both the focal contact points as well as over the whole cell. PTKs inhibitor did not have any effect on alpha 4-expressing cells spreading. This could be related to the amount of the PTKs present in these cells.     

The cell surface during mammalian spermiogenesis

In this paper the origin of the membrane investing the newly formed elongating organelles during mammalian spermiogenesis is studied. According to previous authors, the beginning axoneme is hollowed in a deep membrane invagination. We demonstrate that in man, rat, and bull this new surface is formed by several clusters of Golgi-originated vesicles which form a periaxonemal double cylinder which finally fuses at its end with the old plasma membrane. So the new periaxonemal plasma membrane is preformed in the spermatid body. The membrane surrounding the elongating head is, on the contrary, simply an extension of the old one, because migration of Golgi vesicles and preformed new membranes have not been observed in this region. Con A properties of new and old membranes are the same and will change only after the transit through the epididymis.     

Investigation of the surface heteroorganic antigens of rat hepatoma cells involved in process of cell proliferation

The investigation of antigenic diversion of hepatoma cells resulting from the expression of heteroorganic kidney antigens has been continued. Tumor-associated heteroorganic antigens 110-115 and 125-130 kDa were detected by immunoserum of narrow specificity in fractions of plasmatic membranes of cells of rat ascitic hepatoma Zajdela and cultured hepatoma HTC; the antigen 75-80 kDa was revealed only for hepatoma Zajdela cells. It has been shown by methods of radioisotope analysis and flow DNA-cytometry that heteroorganic antigens 110-130 kDa can be involved in process of cell proliferation.     

Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity- purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.     

Cell type-specific neural cell surface antigens.

Immunological methods have served to define several cell surface antigens that are differentially expressed among neural cell types and are developmentally regulated. These antigens have served as useful markers for cell identification and isolation of several neural cell types. The molecular nature and functional properties of almost all of these antigens are presently unknown.     

Characterization of a 36,000-dalton protein from the surface of Madin- Darby canine kidney cells involved in cell attachment and spreading

We have identified and immunochemically characterized a 36,000-dalton membrane glycoprotein from Madin-Darby canine kidney cells. This protein is surface-labeled by lactoperoxidase-mediated iodination and metabolically labeled by [35S]methionine. It binds to Concanavalin A and incorporates 2-D-3H-mannose residues, thus indicating it is a glycoprotein. Rabbit polyclonal antibodies against this protein evenly decorate the external surface of trypsinized, unpolarized cells. The external apical surface of confluent monolayers, grown under culture conditions in which the tight junctions are closed and the cells have acquired polarity, is also evenly stained. The basolateral aspects of the external surface are stained only when the tight junctions are opened by removal of Ca++ or when the antibody has access to the monolayer from the basal side, which indicates an even distribution of this antigen on the surface of polarized cells. The antibody has no inhibitory effect on the opening and resealing of tight junctions in dense cultures, but does inhibit the attachment and spreading of cells on a substrate, which then blocks the establishment of a confluent functional monolayer.     

Cross-reactivity between parasitic antigens and T lymphocyte surface antigens: a possible host-escape mechanism for Echinococcus multilocularis

We report an immunocytochemical analysis of E. m. protoscolices obtained in 2 strains of mice (AKR, Balb c) which were experimentally infected. Sections of hepatic and peritoneal lesions and spreading of protoscolices from peritoneal vesicles were analyzed. Five monoclonal antibodies, specific of murine T lymphocyte populations, produced an intense and regular staining of the anterior area of the protoscolices. This immunostaining has not yet been explained; it could bear witness to particular mode of parasite protection against host immunological responses.     

Induction of cell spreading by substratum-adsorbed ligands directed against the cell surface

Studies were carried out to compare the spreading of baby hamster kidney (BHK) cells, which occurs by an interaction between the cells and a specific serum glycoprotein (ASF) adsorbed onto the substratum surface, with the spreading of BHK cells that occurs by an interaction between the cells and substrata coated with ligands directed at various cell surface determinants. The ligands tested were polycationic ferritin, concanavalin A (ConA) and antibody directed against BHK plasma membranes. Cell spreading onto ASF and ligand-coated substrata were similar even though different cell surface components were apparently involved. The similarities were:

1. 1. The shape of the spread cells.

2. 2. The inhibition of cell spreading by conditions that interfere with metabolic activity, block free sulfhydryl groups, or interfere with microtubules and microfilaments.

3. 3. The similar reorganization of certain cell surface antigenic determinants during cell spreading onto any of the substrata.

The results indicate that cell spreading is a general cellular response to specific cell-substratum interactions but does not depend upon binding between a unique cell surface receptor and the substratum.     

Immunolatex visualisation of cell surface Forssman and polyamine antigens

Specific antibodies were chemically linked to latex spheres. These immunolatex spheres, which are stable on storage for up to 4 weeks at 4°C have been used to visualise the corresponding antigens on chick embryo cells under the scanning electron microscope. The Forssman antigen has been successfully visualised using the indirect method and the polyamine antigen by the direct method. Using the immunolatex technique, visualisation of Forssman antigenic sites was achieved with a dilution of antiserum 80-fold greater than that necessary for a positive reaction using the immunofluorescence technique.     

Heparanase facilitates cell adhesion and spreading by clustering of cell surface heparan sulfate proteoglycans

Heparanase is a heparan sulfate (HS) degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys(158)-Asp(171), termed KKDC) was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity.     

Rotenone inhibits mammalian cell proliferation by inhibiting microtubule assembly through tubulin binding

Rotenone, a widely used insecticide, has been shown to inhibit mammalian cell proliferation and to depolymerize cellular microtubules. In the present study, the effects of rotenone on the assembly of microtubules in relation to its ability to inhibit cell proliferation and mitosis were analyzed. We found that rotenone inhibited the proliferation of HeLa and MCF-7 cells with half maximal inhibitory concentrations of 0.2 +/- 0.1 microm and 0.4 +/- 0.1 microm, respectively. At its effective inhibitory concentration range, rotenone depolymerized spindle microtubules of both cell types. However, it had a much stronger effect on the interphase microtubules of MCF-7 cells compared to that of the HeLa cells. Rotenone suppressed the reassembly of microtubules in living HeLa cells, suggesting that it can suppress microtubule growth rates. Furthermore, it reduced the intercentrosomal distance in HeLa cells at its lower effective concentration range and induced multipolar-spindle formation at a relatively higher concentration range. It also increased the level of checkpoint protein BubR1 at the kinetochore region. Rotenone inhibited both the assembly and the GTP hydrolysis rate of microtubules in vitro. It also inhibited the binding of colchicine to tubulin, perturbed the secondary structure of tubulin, and reduced the intrinsic tryptophan fluorescence of tubulin and the extrinsic fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid complex, suggesting that it binds to tubulin. A dissociation constant of 3 +/- 0.6 microm was estimated for tubulin-rotenone complex. The data presented suggest that rotenone blocks mitosis and inhibits cell proliferation by perturbing microtubule assembly dynamics.     

Positional cell surface antigens in an insect appendage

Summary Mice were immunized with membrane preparations of epidermal cells taken from different parts (internal and external face of femur and apex and base of tibia) of the metathoracic legs of cockroach larvae. Using indirect immunofluorescence, anti-internal face of femur antibodies were observed to bind preferentially to membranes from the internal face of the femur; similarly, anti-external face of femur antibodies bound preferentially to membranes from the external face of the femur. We also found a preferential binding of anti-apex of tibia antibodies to membranes from the apex of the tibia and anti-base of the tibia antibodies to membranes from the base of the tibia. When anti-tibia sera were tested on membranes from the femur, anti-apex of tibia antibodies bound preferentially to membranes from the apex of the femur, and anti-base of tibia antibodies bound preferentially to membranes from the base of the femur.This demonstrates that epidermal cell membranes from the different parts of the leg differ in their antigenic properties, and that these differences are related to their position around the appendage and along the proximodistal axis of segments.These results are in agreement with those of previous graft experiments and with the concept of ordered sequences in insect appendages.