We have previously reported that the exposure of human peripheral blood lymphocytes (PBL) to a variety of stimulants caused rapid changes in intracellular fluorescein fluorescence polarization (IFFP) in the activated cells. In the present study we further analyzed possible mechanisms responsible for the changes in IFFP in PBL exposed to phytohaemagglutinin (PHA) and anti-CD3 antibody. By employing several agents which are known to affect the polymerization of the cytoskeleton we showed that both cytochalasin B, which regulates the microfilaments structure, and vinblastine and colchicine, which affect the microtubules, completely abolished the changes induced in IFFP of human PBL by both PHA and anti-CD3. This effect was dose dependent and was noted at concentrations ranging from 10 to 100 μM of cytochalasin B and 10 /M of vinblastine and colchicine. The effect of these cytoskeleton modulators occurred within 20 minutes after the initiation of activation with PHA. Our results indicate that activation with PHA and anti-CD3 causes early changes in the microtubules and microfilaments components of the cytoskeleton. The possible application of IFFP measurement in analyzing early changes in the cytoskeleton following cell activation is discussed.

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The effects of hydrostatic pressure on ultrastructure, microtubules and microfllaments of Schizosaccharomyces pombe were investigated by fluorescence microscopy, conventional electron microscopy and immunoelectron microscopy. Cells were treated with hydrostatic pressure from 0.1 to 400 MPa for 10 min at room temperature. The nuclear membrane was disrupted at above 100 MPa. At 150 MPa the matrixes of mitochondria had an electron dense area. At 250 MPa the cytoplasmic substances changed dramatically, the cellular organelles could hardly be detected and the fragmented nuclear membrane was barely visible. The fluorescence in α-tubulin was lost in most of the cells at 100 MPa. The gold particles for anti α-tubulin were not visible in the cells at the same level. Cell cycle specific actin distribution was lost even at 50 MPa, although actin dots localized at the central region remained unchanged. Thick actin cables appeared at 100 MPa. Complete depolymerization of F-actin was observed at 150 MPa. These results suggest that S. pombe cells were more sensitive than Saccharomyces cerevisiae cells. The damage to microtubules and nuclear membrane caused by hydrostatic pressure was thought to be followed by breakdown of nuclear division apparatus and the inhibition of nuclear division. This damage might contribute to the frequent formation of polyploidy in S. pombe.

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Expression of fusion proteins between prepro-α-factor and somatostatin (SRIF) in yeast, resulted in the correct processing and secretion of the heterologous 14-amino acid SRIF peptide (1). When the chimeric genes were placed under the control of yeast acid phosphatase (PHO5) promoter, significant amount of an unglycosylated form of the fusion precursor molecule accumulated intracellularly, suggesting disruption of an endoplasmic reticulum-mediated function. We report here that the appearance of the precursor is due to an alteration in the three amino terminal residues of the chimera, i.e., Met-Arg-Phe in native prepro-α-factor is changed to Met-Phe-Lys in the hybrids. The unglycosylated precursor represents a population of molecules that are disrupted at an early stage of targeting to or translocation across the endoplasmic reticulum membrane. Our data demonstrate that the N-terminus plays an important role in topogenesis. Furthermore, these results show that translocation and glycosylation can be uncoupled from protein synthesis ]itin vivo, and therefore can be post-translational events in yeast.

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Myosin light chain kinase (MLCK) is a calmodulin-dependent protein kinase which phosphorylates the 20, 000 dalton regulatory light chain of myosin II. Here we show that activation of chicken gizzard MLCK by Ca²⁺/calmodulin is inhibited by autophosphorylation at 2 sites in the absence of Ca²⁺/calmodulin. Two phosphorylation sites are located in the functional domain of the kinase, the threonine site toward the actin binding domain near the N-terminus of MLCK and the serine site in immediate proximity to the calmodulin binding site. The autophosphorylation was significantly inhibited by the binding of calmodulin to MLCK in the presence of Ca²⁺.

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Actin from Dictyostelium was labeled with iodoacetamide tetramethylrhodamine (IAR). The labeled actin retained the ability to polymerize into filaments. The labeled actin was introduced into Dictyostelium cells by electroporation. The introduced IAR-labeled actin was diffusely distributed in the cytoplasm but some of it was concentrated in small and large projections at the cell periphery. IAR-labeled actin was concentrated in pseudopods and in the tail cortical region in actively migrating cells. Intense fluorescence due to labeled actin appeared rapidly and disappeared during the extension and retention of pseudopods. During cytokinesis, IAR-labeled actin was concentrated in both polar regions and slightly concentrated in the furrow region. However, the ratio of intensities due to IAR-labeled actin and fluorescently labeled bovine serum albumin that had been introduced simultaneously into cells showed the absence of any concentration of IAR-labeled actin in the furrow region. Staining of fixed cells with fluorescently labeled phalloidin revealed that filamentous actin was rich in the furrow region. These observations indicate that actin is concentrated at this region in higher ratio of filamentous actin to monomeric actin than in other regions of the cytoplasm. Image analysis revealed that the concentration of IAR-labeled actin was high in a pseudopod of an actively migrating cell. Comparisons with staining by fluorescently labeled phalloidin of fixed cells revealed that there was a decreasing gradient in the concentration of filamentous actin from the tip to the base of a pseudopod. These results reveal the high rate at which actin is coordinately organized during mitosis and locomotion in highly motile Dictyostelium cells.

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Bistheonellide A, an inhibitor of actin polymerization from the marine sponge Theonella sp., was introduced at a concentration of 100 nM into rat 3Y1 fibroblast of 2.4 × 10⁴ cells/ml. Within 1 h, it disrupted stress fibers, accompanied by a marked change of the cell morphology, resulting in the formation of processes from the cell surface. Further incubation for 24 h in the presence of 100 nM bistheonellide A led to binucleation in most cells and subsequent inhibition of cell cycle progression. When bistheonellide A was withdrawn from the culture medium, binuclear cells began to grow again within 20 h and reverted to mononuclear morphology. Flow cytometric analysis by fluorescence-activated cell sorting showed that 2C diploid DNA content in G1 phase was changed into 4C content of tetraploid for the bistheonellide A treated-cells in G1 phase and into SC content during G2 and M phase. Therefore, we suggested that the bistheonellide A treatment inhibited cytokinesis, but not mitosis in M phase, and that treated cells were arrested at the early G1 phase. These effects of bistheonellide A on the cell cycle progression of 3Y1 fibroblast were also observed more prominently in cells synchronized in S phase with hydroxyurea. Cells in GO phase were then activated by the addition of fetal calf serum in the presence of 100 nM bistheonellide A. Cell cycle progression of the bistheonellide A-treated cells was obviously slowed down or completely inhibited during G1 phase. These results reveal that actin filaments are not only essential to cytokinesis but also for promoting the progression of cell cycle from G1 to S phase.

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An ultrastructural and functional study was performed during methotrexate (MTX) induced apoptosis on HL-60 leukemia cells. The fine preservation of plasma membrane architecture and organellar components was present untill latest apoptotic stages, despite strong nuclear changes. This observation suggests the presence of a residual cell metabolic activity, which is here investigated. DNA agarose gel electrophoresis demonstrated its fragmentation, which was also confirmed in situ by nick translation confocal analysis. Nuclear purification was subsequently performed to investigate DNA polymerase activities. DNA polymerase α activity appeared strongly reduced from the early phases of methotrexate treatment, while the rate of DNA polymerase β synthesis was found to increase progressively along with the time of drug treatment. Low levels of DNA polymerase γ activity were revealed both in control and in treated cells, suggesting the irrelevant involvement of this enzyme in the DNA metabolism of this model. DNA polymerase β appears thus to be the enzyme prominently correlated to cell attempts to repair the methotrexate-induced DNA damage.

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