Effects of the myosin inhibitor 2,3-butanedione monoxime (BDM) on cell shape, locomotion and fluid pinocytosis in human polymorphonuclear leucocytes

We investigated the role of myosin in polymorphonuclear leucocyte (PMN) shape changes, locomotion, and fluid pinocytosis using the myosin inhibitor 2,3 butanedione monoxime (BDM). Treatment of resting spherical PMNs with BDM produced spheroid cells showing small continuous shape changes (IC(50)=15.5 m m BDM) and occasionally small blebs. Cell polarity, as induced by the chemotactic peptide fNLPNTL or by colchicine, and locomotion were completely suppressed (IC(50)=8.4 to 10 m m). Suppression of fNLPNTL- or colchicine-induced cell polarity produced spheroid cells, suppression of PMA-induced shape changes and fluid pinocytosis produced non-motile spherical cells (IC(50)=25 to 30 m m BDM). BDM suppressed formation of lamellipodia but not formation of blebs. Suppression of microvilli by BDM as observed in resting spherical cells was partially antagonized by PMA. The results suggest that myosin is involved in stabilizing the shape of resting spherical cells, including microvilli, and that myosin is required for cell polarity, locomotion, fluid pinocytosis and for formation of lamellipodia, but not for formation of blebs.     

Chemotactically-induced redistribution of CD43 as related to polarity and locomotion of human polymorphonuclear leucocytes

Leukocyte motility involves pseudopods extension at the leading edge and uropod contraction at the cell rear. Previous studies have shown that the glycoprotein CD43 redistributes to the uropod, when the cells develop polarity and locomotion. The present study addresses the question whether the accumulation of specific membrane molecules, such as CD43 at the contracted uropod precedes or follows development of polarity and locomotion. PMNs were labeled with fluorescent anti-CD43 antibodies and guided to polarize in the direction of a chemoattractant-containing micropipette or, once polarized, they were forced to reverse polarity and movement direction by placing the micropipette behind the uropod. This chemotactically-induced reversal of polarity was used as an efficient tool to analyse the sequence of events. CD43, but not another abundant surface glycoprotein CD45, was concentrated at the uropod. This documents that CD43 redistribution is a selective phenomenon. During reversal of polarity and of locomotion direction, the geometric center of the cell clearly changed direction earlier than the center of anti-CD43 fluorescence intensity. Thus, CD43 redistribution to the new uropod follows rather than precedes reversal of polarity, suggesting that CD43 redistribution is a consequence rather than a prerequisite for polarity and locomotion. PMNs making a U-turn maintained the pre-existing polarity and CD43 remained concentrated at the uropod, even when the front was moving in the opposite direction. Our data show that anterior pseudopod formation, rather than capping of CD43 at the uropod or the position of the uropod determines the direction of locomotion.     

Head regeneration and polarity reversal inHydra attenuata can occur in the absence of DNA synthesis

Summary Regeneration in hydra is considered to be morphallactic because it can occur in the absence of cell division. Whether DNA synthesis is required for regeneration or other repatterning events is not known. The question was investigated by blocking DNA synthesis with hydroxyurea and examining several developmental processes. Head regeneration, reversal of regeneration polarity and battery cell differentiation all took place in the absence of DNA synthesis. Hence, morphallactic regulation in hydra is independent of both DNA synthesis and mitosis.     

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on cytotoxicity of PSK-induced peritoneal polymorphonuclear leukocytes (PMNs)

We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) en hanced the cytotoxicity of PSK-induced polymorphonuclear leukocytes (PMNs) in the peritoneal cavity. Male C3H/He mice, 8- to 10-week-old, received single subcutaneous (s.c.) or intraperitoneal (i.p.) injection of 2.5 µg/animal of rhG-CSF at different time points before or after an i.p. administration of PSK. In other experiments, mice were s.c. or i.p. treated with the same dosage of rhG-CSF every day for 7 or 14 consecutive days and i.p. injected with 2.5 mg/animal of PSK on the last day. Peritoneal PMNs were harvested 6 hrs after the administration of PSK and purified to more than 95% by Ficoll-Paque for in vitro cytotoxic assay.In vitro cytotoxic assays with⁵¹Cr labeled MM46 mammary carcinoma cells were added with 5–20 µg/ml of Nocardia rubra cell wall skeleton (N-CWS) at the beginning of the assay to augment the cytotoxic activity of PMNs.In vitro addition of rhG-CSF to the assay did not enhance the cytotoxicity of PSK-induced PMNs. However, the cytotoxicity was signifi cantly increased when rhG-CSF was s.c. administered 12 hrs before a PSK injection or 2 or 5 hrs after that. On the other hand, the cytotoxicity was rather weak when mice s.c. or i.p. received consecutive injections of rhG-CSF. This cytotoxicity may be mediated by H₂O₂, since H₂O₂ production of PMNs during the cytotoxic assay appears to correlate with the levels of cytotoxicity under suppressed H₂O₂ generation by catalase or enhanced generation by rhG-CSF. These results suggest that rhG-CSF augments the cytotoxicity of PSK-induced PMNs when administeredin vivo timely.     

Subcellular localization of NAD(P)H oxidase(s) in human neutrophilic polymorphonuclear leucocytes.

NADH and NADPH oxidase activities in a homogenate of human neutrophils co-sediment in a linear sucrose density gradient under either velocity or isopycnic conditions of centrifugation. The position of these activities in the gradient does not correspond to any known subcellular granule or to the cell-membrane fraction. These data suggest that the oxidase activities may reside in a unique granule that has previously not been recognized.     

Biphasic response of human polymorphonuclear leucocytes and keratinocytes (epitheliocytes) fromXenopus laevis to mechanical stimulation

Summary Human polymorphonuclear leucocytes and epitheliocytes isolated from tadpole tails ofXenopus laevis were used to observe the responses of cells to mechanical stimulation with a microneedle. Biphasic responses were observed: a retraction phase lasting 1–3 s was followed by an extension phase lasting 10–40s. Weak stimulation evoked alocal response whilst on strong stimulation the whole cells rounded up. Spreading after induced rounding was at least one order of magnitude faster (it lasted less than 1–2min) than cell spreading after chemical dissociation of cell cultures. Local or extended loss of cell attachment to the substratum (observed with reflection interference contrast microscopy) preceded changes in cell morphology, visible with phase contrast microscopy. Repeated weak stimulation of one cell side induced extension and locomotion of the cell in this direction. The reported biphasic responses of cells to mechanical stimulation highlight the significance of exact timing when following any cell response to external stimuli.     

Regulation of epithelial cell shape and polarity by cell - cell adhesion (Review)

Among all cell types that exhibit a polarized phenotype, epithelial cells are unique in that their polarity depends on the integration of the cell into a tissue, the epithelium. In recent years, the analysis of epithelial cell polarity in different epithelia and organisms has contributed to an understanding of the components involved and has further demonstrated that cell polarity and cell adhesion are intimately related to each other. Therefore, processes that mediate and modulate cell adhesion and coordinate adhesion and cell shape are fundamental for the function of epithelia. Recent results obtained in Drosophila melanogaster and Caenorhabditis elegans have provided further insight into the complex circuits regulating these processes, and have laid the direction for future analysis.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant for human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B4. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant fo human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy - 5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B₄. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

Factors affecting the measurement of chemiluminescence in stimulated human polymorphonuclear leucocytes

Optimum conditions were established for the generation and measurement of luminoldependent chemiluminescence (CL) in human polymorphonuclear leucocytes (PMNL) stimulated with a variety of particulate and soluble agents. Several factors had a particular influence on the kinetics of CL stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Two peaks, both azide-sensitive, were observed at 21°C and 25°C. but these increased in magnitude and merged t o give a single, early peak when the temperature was increased t o 37°C. Pre-exposure of PMNL to a buffer containing calcium was essential for the expression of both phases of fMLP-stimulated CL, while the second peak decreased dramatically if the cells were stored at 4°C for 4 hours before assay. In contrast, storage of PMNL at 4°C for up t o 8 hours in a buffer without divalent cations did not alter the kinetics or magnitude of CL induced by other stimuli, and had the benefit of minimizing the rate of cell aggregation. This study confirms that measurement of luminol-dependent CL in stimulated PMNL is a useful analytical tool, but shows that careful attention t o experimental design is required t o ensure that the observed CL provides a true measure of the parameter under investigation.     

The cytoskeleton and polarization during pollen development in Carex blanda (Cyperaceae)

Patterns of cytoskeletal organization during distinct polarizations that characterize pollen development in the sedge Carex blanda (Cyperaceae) were studied by correlated methods of immunohistochemistry and confocal and transmission electron microscopy. As is typical of the family Cyperaceae, Carex produces a unique pollen type known as a pseudomonad in which all four microspores of the tetrad are enclosed within the wall of a single pollen grain. Only one member of the tetrad is functional and the other three abort. The pseudomonads are precisely oriented in the locule with the functional microspore in the wide abaxial portion of the wedge-shaped cytoplasm adjacent to the tapetum, and the degenerative microspores are packed tightly in the pointed adaxial portion. A unique sequence of post-meiotic developmental events reflects both intracellular and intercellular polarity. Development includes: (1) random placement of tetrad nuclei in the coenocytic sporocyte after meiosis, (2) interrupted cytokinesis resulting in a tetrad of nuclei that migrates as a unit into the narrow adaxial tip, (3) completion of unequal cytokinesis and centering of the functional nucleus in the wide abaxial portion of the microsporocyte via a radial array of microtubules and microfilaments, (4) unequal mitosis resulting in a small generative cell at the proximal surface of the functional microspore (adjacent to the abortive microspores), and (5) recentering of the vegetative nucleus in the abaxial cytoplasm via a radial cytoskeletal array.     

Division polarity and plasticity of the meiosis I spindle inCypripedium californicum (Orchidaceae)

Summary Establishment of division polarity and meiotic spindle organization in the lady's slipper orchidCypripedium californicum A. Gray was studied by immunocytochemistry, confocal and transmission electron microscopy. Prior to organization of the spindle for meiosis I, the cytoplasmic domains of the future dyad and spindle polarity are marked by: (1) constriction of the prophase nucleus into an hourglass shape; (2) reorganization of nuclear-based radial microtubules into two arrays that intersect at the constriction; and (3) redistribution of organelles into a ring at the boundary of the newly defined dyad domains. It is not certain whether the opposing microtubule arrays contribute directly to the anastral spindle which is organized in the perinuclear areas of the two hemispheres. By late prophase each half-spindle consists of a spline-like structure from which depart the kinetochore fibers. This peculiar spindle closely resembles the spline-like spindle of generative-cell mitosis in certain plants where the spindle is distorted by physical constraints of the slender pollen tube. In the microsporocyte, the elongate spindle of late prophase/metaphase is curved within the cell so that the poles are not actually opposite each other and chromosomes do not form a plate at the equator. By late telophase the poles of the shortened halfspindles lie opposite each other. Plasticity of the physically constrained plant spindle appears to be due to its construction from multiple units terminating in minipoles. Cytokinesis does not follow the first meiosis. However, the dyad domains are clearly defined by radial microtubules emanating from the two daughter nuclei and the domains themselves are separated by a disc-like band of organelles.     

Morphological study of cytotoxicity produced by PSK-induced polymorphonuclear leukocytes (PMNs) andNocardia rubra cell wall skeleton

The morphologic changes in PMNs induced by an i.p. injection of PSK, a polysaccharide from the mycelia ofCoriolus versicolor, and tumor cells undergoing cell death, were evaluated by immunohistochemical staining and electron microscopy. Male C3H/He mice, 8–10-weeks old, received an i.p. injection of 125 mg/kg of PSK. Their PMNs were obtained 6 h after the PSK injection by peritoneal lavage. N-CWS (Nocardia rubra cell wall skeleton) was added at the start of the chromium release assay using the MM46 mammary carcinoma cell line, which is syngeneic to C3H/He mice, as target cells. During the cytotoxic assay, the cells were fixed at various time points. The MM46 cells expressed ICAM-1 while the PMNs expressed both ICAM-1 and LFA-1 as determined by immunohistochemical staining and immunoelectron microscopy using anti-ICAM-1 and anti-LFA-1 antibodies. PMNs with ruffle-like microvilli adhered to the MM46 tumor cells 30 min after the addition of N-CWS. Immunoelectron microscopic findings suggested that the adhesion molecules were LFA-1 on the PMNs and ICAM-1 on the MM46 tumor cells, but cell fusion between the PMNs and tumor cells was not observed. The MM46 tumor cells gradually lost their microvilli, which showed cell damage, and died 6–7 h after the addition of the N-CWS. This time course of tumor cell death is compatible with the results of the cytotoxic assay. Pretreatment of PMNs by anti-LFA-1 antibody suppressed % lysis of MM46 tumor cells from 90 % to 10 %(p<0.01). These data suggest that adhesion molecule on the surface of PMNs such as LFA-1 might play an important role on signal transduction of these PMNs cytotoxic function in this experimental system.     

Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. The role of lyso-PAF disposal and free arachidonic acid.

Tumour necrosis factor (TNF) is a potent mitogen for some fibroblast cell lines. Here we have examined the TNF-mediated changes in protein phosphorylation in Swiss 3T3 and human FS-4 fibroblasts, and compared them with changes observed after the treatment of cells with other mitogens, such as platelet-derived growth factor (PDGF) and bombesin. TNF stimulated the rapid phosphorylation of two 41,000-Mr and two 43,000-Mr cytosol proteins on tyrosine, threonine and/or serine, as did PDGF, epidermal growth factor and fibroblast growth factor; the increased levels of this mitogen-induced protein-tyrosine phosphorylation correlated well with the extent of mitogen-induced DNA synthesis as determined by the percentage of labelled nuclei. In contrast, bombesin, which is an even better mitogen for Swiss 3T3 cells than TNF, stimulated the tyrosine phosphorylation of 41,000-Mr and 43,000-Mr proteins only to a limited extent. On the other hand, bombesin and PDGF stimulated the rapid serine phosphorylation of an 80,000-Mr acidic protein, a major substrate for protein kinase C; increased phosphorylation of the 80,000-Mr protein was not observed at all when cells were stimulated with TNF. These results suggest significant differences among the mitogenic signalling pathways of TNF, PDGF and bombesin as regards the involvement of protein kinases; the mitogenic signalling pathway of TNF involves the activation of tyrosine kinase, but not of protein kinase C, whereas bombesin seems to transduce its mitogenic signal mainly through the activation of protein kinase C, and the activation of both kinases seems to be involved in the mitogenic signalling pathway of PDGF.     

Platelet derived growth factor (PDGF) contained in Platelet Rich Plasma (PRP) stimulates migration of osteoblasts by reorganizing actin cytoskeleton

Platelet-rich plasma (PRP) is a platelet concentrate in a small volume of plasma. It is highly enriched in growth factors able to stimulate the migration and growth of bone-forming cells. PRP is often used in clinical applications, as dental surgery and fracture healing. Platelet derived growth factor (PDGF), is highly concentrated in PRP and it was shown in our previous studies to provide the chemotactic stimulus to SaOS-2 osteoblasts to move in a microchemotaxis assay. Aim of the present studies is to analyze the effects of a PRP pretreatment (short time course: 30–150 min) of SaOS-2 cells with PRP on the organization of actin cytoskeleton, the main effector of cell mobility. The results indicate that a pretreatment with PRP increases chemokinesis and chemotaxis and concomitantly induces the organization of actin microfilaments, visualized by immunocytochemistry, in a directionally elongated phenotype, which is characteristic of the cells able to move. PRP also produces a transient increase in the expression of PGDF α receptor. This reorganization is blocked by the immunoneutralization of PDGF demonstrating the responsibility of this growth factor in triggering the mechanisms responsible for cellular movements.     

Platelet derived growth factor (PDGF) contained in Platelet Rich Plasma (PRP) stimulates migration of osteoblasts by reorganizing actin cytoskeleton

Platelet-rich plasma (PRP) is a platelet concentrate in a small volume of plasma. It is highly enriched in growth factors able to stimulate the migration and growth of bone-forming cells. PRP is often used in clinical applications, as dental surgery and fracture healing. Platelet derived growth factor (PDGF), is highly concentrated in PRP and it was shown in our previous studies to provide the chemotactic stimulus to SaOS-2 osteoblasts to move in a microchemotaxis assay. Aim of the present studies is to analyze the effects of a PRP pretreatment (short time course: 30–150 min) of SaOS-2 cells with PRP on the organization of actin cytoskeleton, the main effector of cell mobility. The results indicate that a pretreatment with PRP increases chemokinesis and chemotaxis and concomitantly induces the organization of actin microfilaments, visualized by immunocytochemistry, in a directionally elongated phenotype, which is characteristic of the cells able to move. PRP also produces a transient increase in the expression of PGDF α receptor. This reorganization is blocked by the immunoneutralization of PDGF demonstrating the responsibility of this growth factor in triggering the mechanisms responsible for cellular movements.     

Evidence for division and polarity in apical cells of bryophytes and pteridophytes

Apical cells on the verge of dividing, or having recently formed a new segment, or actually dividing, are not uncommonly encountered in bryophytes and pteridophytes. This is interpreted as evidence for the classical concept of active apical segmentation in these plants (versus the concept of a quiescent apical cell). In certain species a polarized organization of the cytoplasm of the dividing apical cells is identifiable.     

An MEK-cofilin signalling module controls migration of human T cells in 3D but not 2D environments

T cells infiltrate peripheral tissues to execute immunosurveillance and effector functions. For this purpose, T cells first migrate on the two‐dimensional (2D) surface of endothelial cells to undergo transendothelial migration. Then they change their mode of movement to undergo migration within the three‐dimensional (3D)‐extracellular matrix of the infiltrated tissue. As yet, no molecular mechanisms are known, which control migration exclusively in either 2D or 3D environments. Here, we describe a signalling module that controls T‐cell chemotaxis specifically in 3D environments. In chemotaxing T cells, Ras activity is spatially restricted to the lamellipodium. There, Ras initiates activation of MEK, which in turn inhibits LIM‐kinase 1 activity, thereby allowing dephosphorylation of the F‐actin‐remodelling protein cofilin. Interference with this MEK‐cofilin module by either inhibition of MEK or by knockdown of cofilin reduces speed and directionality of chemotactic migration in 3D‐extracellular matrices, but not on 2D substrates. This MEK‐cofilin module may have an important function in the tissue positioning of T cells during an immune response.     

Chloroplast division polarity,a marker of cell division planes during morphogenesis inBangia vermicularis Harvey (Rhodophyceae)

Summary Ultrastructural observations of dividing cells inBangia vermicularis revealed a type of chloroplast division (plastokinesis) not previously reported in the red algae. The polarity of this prekaryokinetic process serves as a reliable marker of the plane of cytokinesis, a key factor in establishing thallus morphology. At the onset of division one or more invaginations develop in the envelope of the large, lobed chloroplast and proceed centripetally through the stroma in the plane of the thylakoids, forming narrow cytoplasmic channels (CCs). The thylakoids are realigned somewhat, but are not constricted as the chloroplast is divided into two or more units. The number of resulting chloroplasts and the orientation of the CCs are dependent on cell type. Distinctive cylindrical cells at the base of the filamentous region, immediately distal to the holdfast, are shorter than broad and contain a central nucleus surrounded by a doughnutshaped chloroplast. The cylindrical morphology of the thallus is established early in the first periclinal division as multiple plastokinesis commences, generating several radially-arranged daughter chloroplasts. Cleavage of the original chloroplast is completed during subsequent cell divisions in the initial developmental stage, finally resulting in eight chloroplasts that are distributed to an equal number of wedge-shaped radial cells. Cells distal to the actively dividing basal cells are cuboidal and have a peripheral nucleus. Division of the single chloroplast prior to karyokinesis in these cells results in two or four daughter chloroplasts according to cell type. During or following plastokinesis, multilamellar bodies derived from the CE appear to serve as the source of membranes for the developing septum in the channels. Septa link to proliferations of the plasmalemma in areas of slight cell wall (CW) indentations, and are completed between daughter nuclei after karyokinesis, producing a cleavage channel. Subsequently, primary CW material is deposited between the two septal membranes. The shape and arrangement of daughter cells in each of the developmental stages in the thallus are defined by the planes of cell division. These are indicated by both the orientation of CCs and the polar orientation of nuclear division which is always at right angles to the CC.Abbreviations CC cytoplasmic channel - CE chloroplast envelope - CW cell wall - ER endoplasmic reticulum - MLB multilamellar body     

Changes in fused cells induced by hvj (sendai virus): redistribution of cytoplasmic organelles and cytoskeletal reorganization

To understand the relationship between the location of organelles and cellular function, we examined the dynamic state of cytoplasmic organelles and cytoskeleton in polynuclear Ehrlich ascites tumor (EAT) cells fused with hemagglutinating virus of Japan (HVJ; Sendai virus) by confocal laser scanning microscopy. Irregular fused cells gradually became spherical during culture, and nuclei and mitochondria were redistributed in the fused cell; nuclei formed a cluster surrounded by mitochondria. F-actin, vimentin, and microtubules were also reorganized with the redistribution of cell organelles. Further, when the morphological change was inhibited by L4-1, a chlorophyll-like substance derived from silkworm faeces, or pyropheophorbide-a, the arrangement of organelles and cytoskeleton remained disturbed, suggesting that the movement of the cytoskeleton is closely associated with cell shape and the distribution of cytoplasmic organelles.