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Arrest of cell membrane movements by in vitro incubation with polycation reversed by polyanion.

A total arrest of cell membrane movement occurred after incubation for 2 h of cultured fibroblasts with the polycation DEAE-dextran. The immobilized cells resumed normal membrane undulations after exposure for 5 min to the polyanion dextran-sulphate. The influence of DEAE-dextran on the electrophoretic mobility, which was seen after 10 min of incubation was also erased by a subsequent incubation with dextran-sulphate. No influence on growth of the culture was seen.

Animals

Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells.

Cytochalasin B was used as a tool to study the inter-relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 mug/ml inhibits serum-stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis. While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 mug/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine. Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (less than 1 mug/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10-fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated. Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S-phase associated increase in thymidine transport.

Animals

Three-dimensional scroll waves organize Dictyostelium slugs.

To test the hypothesis that periodic signals and chemotaxis direct later morphogenesis in Dictyostelium discoideum, we investigated cell behavior and cell movement in slugs. Trails of neutral red-stained prestalk and anterior-like cells were recorded by high-resolution digital image processing. Neutral red-stained anterior-like cells in the prespore zone of slugs move straight forward in the direction of slug migration and, furthermore, show coherent periodic cell movement. In contrast, cells in the prestalk zone move along completely different trajectories. Prestalk cells move perpendicular to the direction of slug migration; that is, they rotate around the tip. The cell movement data show that the chemotactic signal in the slug propagates as a three-dimensional scroll wave in the prestalk zone and as a planar wave in the prespore zone. The different behavior of prestalk and prespore cells is most likely caused by a difference in the oscillatory properties of the two cell types. We provide evidence that the slug stage of Dictyostelium behaves like an excitable system and that a (twisted) scroll wave organizes slug formation and migration.

Animals

A cinematographical study of the development of vitally stained Dictyostelium discoideum.

We report observations from time-lapse films of the development of Dictyostelium discoideum (Dd) stained with the vital dye neutral red. We have used this dye to enable us to track individual cells, as they move through multicellular tissues in later Dd stages. Our observations lead us to new conclusions about the control of cell movement and cell contact during Dd development, including the tentative conclusion that the aggregation control competences regulate cell behaviour throughout the whole of Dd. development. We are led to specific hypotheses for the mechanisms of later morphogenetic movements and pattern formation.

Dictyostelium

Active cell edge and movements of concanavalin A receptors of the surface of epithelial and fibroblastic cells.

Movements of the receptors of concanavalin A on various parts of the surfaces of substrate-attached cells were compared. Cultured mouse embryo cells of several types were used: epithelial kidney cells and normal and transformed fibroblasts. Initial distribution of receptors was random on the cells of all types. Binding of concanavalin A induced patching of its receptors on all the cell parts. In contrast, directional centripetal movement of receptors was observed only on the surface of certain cell parts, namely, only the surface of peripheral lamellar cytoplasm was cleared of the receptors. Clearing was always initiated in the zone of lamellar cytoplasm located near active cell edges. In epithelial sheets, clearing was not observed on the surface of central cells that had no lamellar cytoplasm. Concanavalin A receptors on the cleared areas of cell surface were gradually restored after the end of incubation. It is suggested that anchoring of the patches of membrane receptors by cortical microfilaments is possible only on the surface of pseudopods and of lamellar cytoplasm but not on the surface of other cell parts. Besides receptor movements, this hypothesis may be explain differences in the adhesive properties of various parts of the cell surface.

Animals

Involvement of a protein kinase C and protein phosphatases in adhesion of CD4+ T cells to and detachment from extracellular matrix proteins.

For immune surveillance and function to be effective, T lymphocytes constantly recirculate via lymph and blood between lymphoid organs and body tissues. To enable efficient cell movement and migration, cell adhesion to components of the basement membrane and the extracellular matrix (ECM) must be a rapid and transitory process. Whether phosphorylation and dephosphorylation of cellular proteins are involved in this phenomena was explored by monitoring the adhesion of T cells to immobilized ECM proteins. A short exposure of 51Cr-labeled human CD4+ T cells to phorbol esters in vitro induced a rapid beta 1-integrin-mediated adhesion to both fibronectin and laminin, as determined by inhibition with anti-integrin antibodies. Adhesion was reversible; detachment from the immobilized ECM ligands occurred between 20 and 120 min without further intervention. This T cell adhesion was regulated by the activation of protein kinase C because (a) staurosporine and H-7 inhibitors of protein kinase C suppressed T cell adhesion, and (b) PMA-induced down-regulation of intracellular levels of protein kinase C was associated with the abrogation of the T cell adhesiveness to fibronectin and laminin. Furthermore, inhibition of protein phosphatases activity by okadaic acid delayed the detachment of the T cells from fibronectin or laminin. Thus, we suggest that T cell-ECM interactions such as adhesion and detachment are regulated, respectively, by protein kinase C and protein phosphatases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine

Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy.

A recently introduced extension of video-enhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-microns colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK2 cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 micron2/s. A diffusion coefficient in the 0.1 micron2/s range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2 microns in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lamelloplasm and perinuclear cytoplasm at a velocity of 0.5-1 micron/min by a directed ATP-dependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.

Adenosine Triphosphate

[Reversible suppression of cellular aggregation by cytochalasin B and kolkhamin].

Aggregation of normal mouse fibroblast-like cells plated on the surface of millipore filter was studied quantitatively. It was shown that addition of 10 mug/ml of cytochalasin B (a drug which blocked active cell locomotion) or 0.1 mug/ml of colcemid (a drug which destroyed polarization processes in the cells and therefore interfered with the oriented cell movement) completely inhibited such aggregation. This inhibition was reversible: after the drugs were washed off the cells did aggregate. It was concluded that cell aggregation in this experimental system required active cell movement and cell polarization.

Animals

Action of cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface.

The projection of knobby protuberances at the cell surface (zeiosis) is a general cellular response to cytochalasin D (CD), resulting from herniation of endoplasm through undefended places of the cortex during cell contractions and displacement of microfilaments induced by CD. Zeiosis is prevented by agents that interfere with the contractile response to CD, such as inhibitors of energy metabolism or cyclic AMP. The developed protrusions, which remain relatively stable in the presence of CD, contain chiefly mono- or subribosomes, and occasionally other organelles normally resident in endoplasm; compact microfilament felt occupies their bases and extends into their proximal stalks. Protein synthesis in the knobs is less than half of that in the polyribosome-containing endoplasm residual in the main body of the cell. Knobs first protrude singly near the margin of the contracting cells and rapidly cluster into small groups in the periphery even at lower temperature. The clusters then migrate centripetally and coalesce into a large aggregate near the apex of the immobilized and retracted cell: this movement is energy- and temperature-dependent. Aggregation is more prominent and stable in cell lines of epithelial derivation than in fibroblastic or other lines in which nuclear extrusion occurs more readily. The latter is regarded as a special manifestation of zeiosis. Macromarkers, such as latex spherules, migrate like the zeiotic knobs on the cell surfaces in the presence of CD. The aggregated knobs, although persistent for days in the presence of CD, are rapidly recessed after withdrawal of the agent as ruffling is resumed and the cells spread. These movements are discussed in terms of current concepts of mobility of the cell membrane.

Amino Acids

Histogenesis of mouse cerebellum in microwell cultures. Cell reaggregation and migration, fiber and synapse formation.

A microwell culture system was developed for analysis of cell movements and interactions during nervous system histogenesis. Cells from trypsinized 7-day-old mouse cerebellum reaggregated within hours into clusters which later developed interconnections consisting of either sheets of migrating cells and cell processes or cables of fiber bundles with cells migrating along their surfaces. Granule cells in several stages of differentiation, basket and/or stellate neurons, some larger neurons, and two types of neuroglial cells were identified in reproducible, nonrandom patterns by scanning and transmission electron microscopy. Axonal and dendritic processes, both with growth cones, and numerous synapses were generated in vitro.

Animals

Nucleotide sequence of carnation ringspot dianthovirus RNA-2.

RNA-2 of carnation ringspot virus (CRSV), the type member of the dianthovirus group, has been cDNA cloned and sequenced. CRSV RNA-2 is 1394 nucleotides in length and contains a single open reading frame encoding a 304 amino acid polypeptide of 33.8K. Amino acid sequence alignment of this polypeptide with the cell-to-cell movement proteins encoded by RNA-2 of red clover necrotic mosaic virus (RCNMV) Australian (Aus) and Czechoslovakian (TpM-34) isolates indicates 59.6% and 55.7% sequence identity, respectively. The N-terminal 230 amino acids are more highly conserved, with 64.3% and 62.6% sequence identity, respectively. The cell-to-cell movement proteins of the two RCNMV isolates are themselves 82.5% and 91.7% identical when the amino-terminal 230 amino acids are compared. Structural prediction comparison of the RCNMV-Aus, RCNMV-TpM-34 and tobacco mosaic virus cell-to-cell movement proteins to the putative CRSV RNA-2-encoded movement protein suggests that even though no primary amino acid sequence similarity exists, the movement protein polypeptides are possibly similar in structure and function.

Amino Acid Sequence

Cyclic nucleotides, thioldisulfide status of proteins, and cellular control processes.

It is shown that cyclic nucleotides can have a variety of effects on cell division, cell shape, cell adhesion, and cell movement, depending on the cells selected and the conditions under which they are used. For example, while CHO cells elongate under the influence of exogenous dibutyryl CAMP, Y-1 adrenal tumor cells round up and polyoma-transformed 3T3 cells show no change in shape. The totality of experience with cyclic nucleotides suggests that where they have been used by cells as control elements involving the four processes listed above, they are superimposed on basic cellular processes that progress in their absence--that is, they must be acting indirectly. In attempting to understand the inhibitory action of methyl xanthines on egg development, we were forced to abandon the idea that they acted through cyclic nucleotides. We found that methyl xanthines inhibited the activation of glutathione reductase and that glutathione oxidizing agents act as mitotic inhibitors. Further, we found that tubulin polymerizability, NAD-kinase activity, and a mitotic apparatus associated Ca+2-ATP-ase were all inhibited by oxidation of some of their sulfhydryls and were activated by reduction of the resulting disulfides. These results are discussed in terms of reported cycles and activations of glutathione reductase (GR) in cells and reports that mixed disulfides of glutathione and proteins can act as substrates for GR. Using the fact that a CAMP-dependent protein kinase has been reported to be activated by glutathione, we have suggested potential sites where sulfhydryl control processes and cyclic nucleotide control processes and cyclic nucleotide control processes may interact in certain restricted cases.

Adenosine Triphosphatases

Sequential effects of thyroxine on the developing cerebellum of rats made hypothyroid by propylthiouracil.

Young rats made hypothyroid by propylthiouracil (PTU) received a daily physiological dose of thyroxine (T4) from day 0, 4, 6, 8, 10, 11, 12 or 13 and their cerebella were studied on day 14. With the very low doses of T4 used and when the treatment was started at birth, cerebellar development was nearly normal in terms of the parameters studied (cell formation, migration, maturation and death). The effect of T4 on cell formation appeared after two days. With the same latency, T4 induced migration of the newly-formed granule cells. The effects on those processes requiring cell movements over long distances, e.g. the number of cells in the internal granular layer or the thickness of the molecular layer, were longer to appear. The most rapidly affected parameter was the pyknotic index in the internal granular layer. This index was half. The increased cell death in the cerebellum of hypothyroid rats is probably related to the decreased synaptogenetic competence of Purkinje cells. The rapidity of the effect of T4 on the pyknotic index may be related to an important effect of this hormone on the formation of synapses and, more generally, on the mechanisms of neuronal maturation.

Animals

Vertebrate gastrulation.

Our understanding of the mechanisms that control gastrulation is still in its infancy. One problem is that gastrulation is a complex set of coordinated behaviours involving directional cell movements, several types of cell interactions, changes in cell fate and gene expression. Therefore, the successful analysis of its control mechanisms requires simultaneous analysis of more than one of these, or at least some way of separating them. Although progress has been slow, some recent studies have made significant advances in the field and we can probably look forward to some major breakthroughs in the near future.

Animals