Ruffled Edges Research Articles

Ganoderma lucidum extract decreases motility and lamellipodia formation of triple‐negative metastatic breast cancer

Breast cancer (BC) is the leading cause of cancer among women in the United States and Puerto Rico. Triple‐negative breast cancer (TNBC) is characterized by the absence of estrogen and progesterone receptors, and lack of HER2 overexpression. Approximately, 15% of all women with TNBC typically display shorter overall survival and an early peak of distant recurrences at 3y after diagnosis. Treatments for TNBC are limited; currently, there are no approved targeted therapies besides non‐targeted chemotherapy. The mortality of TNBC could result from the lack of effective treatments to decrease the migration of cancer cells to other tissues. Hence, it its highly important to understand these migration processes and to find effective therapies. Therefore, we aimed to test the anti‐migration effects of the medicinal mushroom Ganoderma lucidumextract (GLE) on TNBC cell motility and on the regulation of proteins involved in this process. First, MDA‐MB‐231 cells were treated with various GLE concentrations and evaluated for cell viability and a washout assay. To study the effect of GLE on cell migration, cells were treated with vehicle, 0.25 or 0.96mg/mL (IC50) of GLE for 24h, followed by wound healing and invasion assays. The activity of Rac was tested by a pulldown activity assay and immunoblotting. Furthermore, we tested the expression of proteins important in the dynamics of cell motility Cdc42, Lamellipodin (Lpd), FAK, WAVE and Rho. The localization and expression of Lpd was visualized by immunocytochemistry. Results showed that GLE significantly decreased the cell viability on TNBC, with no cell recovery after a 72h period of treatment withdrawal. GLE significantly decreased MDA‐MB‐231 cell migration and invasion, while reducing the activity of Rac. The expression of Lpd, FAK, WAVE, c‐myc, and Cdc 42 were also modulated upon GLE treatment. GLE decreased Lpd localization in ruffled edges of cancer cells, as well as changes in nuclear and cell morphology. In conclusion, our results suggest the potential of GLE as a natural anti‐TNBC migration agent.Support or Funding InformationThis work was supported by grants from the National Institutes of Health GM111171 (MMM), MD007583 (MMM), GM103475 INBRE‐Supplies for Graduate Students (GOS), from the Puerto Rico Science, Technology and Research Trust (PRSTRT) SGRP 2017‐043, PRSTRT Hurricane Relief Funds, and from the U.S. Dept of Education Title‐V‐PPOHA P031M105050 and Title‐V‐Cooperative P031S130068. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Puerto Rico Science, Technology and Research Trust or the U.S. Department of Education.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Morphological and mechanical properties of blades of Saccharina latissima

Interactions between water flow and aquatic vegetation strongly depend on morphological and biomechanical characteristics of vegetation. Although any physical or numerical model that aims to replicate flow-vegetation interactions requires these characteristics, information on morphology and mechanics of vegetation living in coastal waters remains insufficient. The present study investigates the mechanical properties of blades of Saccharina latissima, a seaweed species spread along the shores of the UK and North East Atlantic. More than 50 seaweed samples with lengths spanning from 150 mm to 650 mm were collected from Loch Fyne (Scotland) and tested. Seaweed blades had a natural ‘stretched droplet’ shape with bullations in the central fascia and ruffled edges in the area close to the stipe. Their morphological features showed high variability for samples longer than 400 mm. The blades were almost neutrally buoyant, their material was found to be very flexible and ductile, being stiffer in longer blades. The laboratory tests showed that estimates of tensile Young's modulus appeared to be similar to bending Young's modulus suggesting a reasonable degree of isotropy in studied seaweed tissues.

Predicting cell shapes in melanomas

Predicting cell shapes in melanomas

Localisation of DivIVA by targeting to negatively curved membranes

DivIVA is a conserved protein in Gram-positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis, DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

Lamellae lead the way

![Graphic][1] The localization of Arp2/3 (white) in lamellipodia (top) is disrupted by tropomyosin (bottom).Open any cell biology text and it is clear that lamellipodia, the ruffled edges of motile cells, initiate movement. So it may come as a surprise that Gupton et al. can disrupt

Segregation of CD4 and CXCR4 into distinct lipid microdomains in T lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus.

Recent evidence has suggested that plasma membrane sphingolipids and cholesterol spontaneously coalesce into raft-like microdomains and that specific proteins, including CD4 and some other T-cell signaling molecules, sequester into these rafts. In agreement with these results, we found that CD4 and the associated Lck tyrosine kinase of peripheral blood mononuclear cells and H9 leukemic T cells were selectively and highly enriched in a low-density lipid fraction that was resistant at 0 degrees C to the neutral detergent Triton X-100 but was disrupted by extraction of cholesterol with filipin or methyl-beta-cyclodextrin. In contrast, the CXCR4 chemokine receptor, a coreceptor for X4 strains of human immunodeficiency virus type 1 (HIV-1), was almost completely excluded from the detergent-resistant raft fraction. Accordingly, as determined by immunofluorescence with confocal microscopy, CD4 and CXCR4 did not coaggregate into antibody-induced cell surface patches or into patches of CXCR4 that formed naturally at the ruffled edges of adherent cells. The CXCR4 fluorescent patches were extracted with cold 1% Triton X-100, whereas the CD4 patches were resistant. In stringent support of these data, CD4 colocalized with patches of cholera toxin bound to the raft-associated sphingoglycolipid GM1, whereas CXCR4 did not. Addition of the CXCR4-activating chemokine SDF-1 alpha did not induce CXCR4 movement into rafts. Moreover, binding of purified monomeric gp120 envelope glycoproteins from strains of HIV-1 that use this coreceptor did not stimulate detectable redistributions of CD4 or CXCR4 between their separate membrane domains. However, adsorption of multivalent gp120-containing HIV-1 virion particles appeared to destabilize the local CD4-containing rafts. Indeed, adsorbed HIV-1 virions were detected by immunofluorescence microscopy and were almost all situated in nonraft regions of the cell surface. We conclude that HIV-1 initially binds to CD4 in a raft domain and that its secondary associations with CXCR4 require shifts of proteins and associated lipids away from their preferred lipid microenvironments. Our evidence suggests that these changes in protein-lipid interactions destabilize the plasma membrane microenvironment underlying the virus by at least several kilocalories per mole, and we propose that this makes an important contribution to fusion of the viral and cellular membranes during infection. Thus, binding of HIV-1 may be favored by the presence of CD4 in rafts, but the rafts may then disperse prior to the membrane fusion reaction.

Localization of myosin Va is dependent on the cytoskeletal organization in the cell

Myosin V plays an important role in membrane trafficking events. Its implication in the transport of pigment granules in melanocytes and synaptic vesicles in neurons is now well established. However, less is known about its function(s) in other cell types. Finding a common function is complicated by the diversity of myosin V expression in different tissues and organisms and by its association with different subcellular compartments. Here we show that myosin V is present in a variety of cells. Within the same cell type under different physiological conditions, we observed two main cellular locations for myosin V that were dependent on the dynamics of the plasma membrane: in cells with highly dynamic membranes, myosin V was specifically concentrated at the leading edge in membrane ruffles, whereas in cells with less dynamic membranes, myosin V was enriched around the microtubule-organizing center. The presence of myosin V in the leading ruffling edge of the cell was induced by growth factor stimulation and was dependent on the presence of a functional motor domain. Moreover, myosin V localization at the microtubule-organizing center was dependent on the integrity of the microtubules. In polarized epithelial cells (WIF-B), where the microtubule-organizing region is close to the actin-rich apical surface, one single pool of myosin V, sensitive to the integrity of both microtubules and actin filaments, was observed.

The p16(INK4a) tumour suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of alphavbeta3 to focal contacts.

Expression of full-length p16(INK4a) blocks alphavbeta3 integrin-dependent cell spreading on vitronectin but not collagen IV. Similarly, G1-associated cell cycle kinases (CDK) inhibitory (CKI) synthetic peptides derived from p16(INK4a), p18(INK4c) and p21(Cip1/Waf1), which can be delivered directly into cells from the tissue culture medium, do not affect non-alphavbeta3-dependent spreading on collagen IV, laminin and fibronectin at concentrations that inhibit cell cycle progression in late G1. The alphavbeta3 heterodimer remains intact after CKI peptide treatment but is immediately dissociated from the focal adhesion contacts. Treatment with phorbol 12-myristate 13-acetate (PMA) allows alphavbeta3 to locate to the focal adhesion contacts and the cells to spread on vitronectin in the presence of CKI peptides. The cdk6 protein is found to suppress p16(INK4a)-mediated inhibition of spreading and is also shown to localize to the ruffling edge of spreading cells, indicating a function for cdk6 in controlling matrix-dependent cell spreading. These results demonstrate a novel G1 CDK-associated integrin regulatory pathway that acts upstream of alphavbeta3-dependent activation of PKC as well as a novel function for the p16(INK4a) tumour suppressor protein in regulating matrix-dependent cell migration.

Merlin differentially associates with the microtubule and actin cytoskeleton.

The neurofibromatosis 2 (NF2) suppressor gene encodes a protein termed merlin (or schwannomin) with sequence similarity to a family of proteins that link the actin cytoskeleton to cell surface glycoproteins. Members of this ERM family of proteins include ezrin, radixin, and moesin. These proteins contain a carboxyl (C-) terminus actin binding site. In contrast to the ERM proteins, merlin lacks the conventional C-terminal actin binding site, but still localizes to the ruffling edge of plasma membranes. In this study, we investigate the ability of merlin to interact with actin through a nonconventional actin binding domain. We demonstrate for the first time that merlin can associate with polymerized actin in vitro by virtue of an amino (N-) terminal actin binding domain including residues 178-367. Merlin actin binding is not affected by several naturally-occurring NF2 patient mutations or alternatively spliced isoforms. These results suggest that merlin, like other ERM proteins, can directly interact with the actin cytoskeleton. In addition, merlin associates with polymerized microtubules in vitro using a novel microtubule binding region in the N-terminal region of merlin that is masked in the full-length merlin molecule, such that wild-type functional merlin in the "closed" conformation fails to bind polymerized microtubules. These microtubule association results confirm the notion that merlin exists in "open" and "closed" conformations relevant to its function as a negative growth regulator.

The 14 kDa beta-galactoside binding lectin in myoblast and myotube cultures: localization by confocal microscopy.

The endogenous 14 kDa beta-galactoside-specific lectin has been localised in myoblasts and myotubes by indirect immunofluorescence using confocal microscopy. The antibodies used in these experiments suggest that in myoblast cultures the lectin is abundant on the cell surface and concentrated at ruffled edges of migrating cells. The quantity and distribution of cell surface lectin is independent of the nature of the substratum and undiminished by brief trypsinisation or lactose-washing of the cell suspension. The lectin is also abundant intracellularly, apparently relatively free in the cytoplasm until the cells approach confluency, when the lectin is concentrated into particular areas of cytoplasm resulting in a 'patchy' appearance of stained cells. After fusion to form multinucleate myotubes, intracellular lectin is less abundant and concentrated at the periphery of myotubes, from where lectin appears to be released in vesicles packed with lectin which 'bud off' from the myotube surface. In recolonising cultures, lectin is apparently more abundant and lectin-packed vesicles can also be seen 'budding off' from myoblasts. In differentiated cultures, extracellular lectin is detected both on the myotube surface and in fibrillar arrays of extracellular material.

Immunolocalization of the gamma isoform of nonmuscle actin in cultured cells.

In many vertebrate nonmuscle cells, the microfilament subunit protein, actin, exists as two isoforms, called beta and gamma, whose sequences differ only in their amino-terminal regions. We have prepared a peptide antibody specifically reactive with the amino-terminal sequence of gamma actin. This antibody reacted with nonmuscle actin as determined by Western blots of SDS gels, and reacted with the gamma, but not the beta, nonmuscle actin isoform as shown by Western blots of isoelectric focusing gels. In immunofluorescence experiments, the gamma peptide antibody stained microfilament bundles, ruffled edges, and the contractile ring of a variety of cultured cells, including mouse L cells, which have previously been reported to contain only the beta actin isoform (Sakiyama, S., S. Fujimura, and H. Sakiyama, 1981, J. Biol. Chem., 256:31-33). Double immunofluorescence experiments using the gamma peptide antibody and an antibody reactive with all actin isoforms revealed no differences in isoform localization. Thus, at the level of resolution of light microscopy, we have detected the gamma actin isoform in all microfilament-containing structures in cultured cells, and have observed no subcellular sorting of the nonmuscle actin isoforms.

Vasoactive amines modulate actin cables (stress fibers) and surface area in cultured bovine endothelium.

Cultured bovine aortic endothelial cells were fixed and stained with NBD-phallicidin and quantitated with a digital image analyzer for changes in actin cables and surface area. Serotonin (5-HT), norepinephrine (NE), dopamine and histamine (all at 10(-4)M concentrations) were tested for their ability to induce cytoskeletal changes. Only 5-HT and NE increased actin cables significantly (p less than 0.01), 80.7% and 97.9%, respectively. Dopamine and histamine treated cells showed a 67.4% and 80.8% decrease in actin cables respectively (p less than 0.01). Stimulated increases of actin cables by 5-HT were inhibited by Ketanserin, and propranolol inhibited NE stimulation of actin cables. Treatment of cells with these blockers alone also decreased actin cables below control values (p less than 0.01). Pretreatment of cells with diphenhydramine, but not cimetidine, inhibited histamine-induced decreases in actin cables. Stimulation of surface area by 5-HT and NE was also observed, with 40.8% and 80.7% increases respectively, when compared with controls (p less than 0.01). The increases in actin cables were associated with a lack of ruffled edges that are indicative of motile cells. In contrast, induced decreases in actin cables resulted in cells with ruffled edges. Exogenous 5-HT and NE have been shown to prevent the increased permeability visible as extravasation of red blood cells from postcapillary venules in thrombocytopenic animals. The present data suggest that 5-HT and NE may be involved in maintaining the endothelial barrier function by a receptor-mediated stimulation of actin cables. Also, histamine-induced decreases in actin cables may be correlated with the amine's action in vivo as a mediator of increased inflammatory permeability.

Lysosome-associated membrane proteins: Characterization of LAMP-1 of macrophage P388 and mouse embryo 3T3 cultured cells

Lysosome-associated membrane protein (LAMP)-1, a major glycoprotein of mouse embryo 3T3 cells and specifically associated with the lysosomal membrane, has been identified in P388 macrophage cells and compared with the homologous glycoprotein of NIH 3T3 cells. Immunofluorescence microscopy with anti-LAMP-1 monoclonal antibodies shows that the antigen was distributed throughout P388 cells including the ruffled edges or pseudopodia, identical to the pattern of acridine orange accumulation. LAMP-1 was purified from P388 cells by affinity chromotography with 1D4B monoclonal antibody, yielding a homogenous glycoprotein comprising 0.1% of the total detergent-extracted cell protein. The apparent mass of P388 LAMP-1 was 130,000 to 150,000 compared to the 3T3 glycoprotein of 105,000 to 115,000. Analysis of tryptic peptides indicated that the two purified glycoproteins were highly homologous. Protein synthesis was analyzed in a variety of cell lines by pulse-chase labeling with [ 35S ]methionine; in every case, LAMP-1 was synthesized as a precursor of apparent M r 92,000, and then converted to heterogeneous mature forms differing in average M r from 110,000 to 140,000. The basis for these apparent differences in mass was examined by studies of the biosynthesis and oligosaccharide composition of the glycoprotein. Core polypeptides of 45,000 Da were obtained from both HaNIH and P388 cells by treating immunoprecipitates of [ 35S ]methionine pulse-labeled molecules with endoglycosidase H. Cells treated with monensin contained heterogeneous molecules of 80,000 to 85,000 Da. Isoelectric heterogeneity of mature LAMP-1 was markedly reduced by treatment with neuraminidase whereas there was little effect on the apparent molecular weight of the molecules or the differences between the various cell lines. β- d-Xyloside inhibition of glycosaminoglycan synthesis had little effect on the apparent mass of LAMP-1.

Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH.

An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic "map" of various spectroscopic parameters can be produced. The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm). Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles. The average cytoplasmic pH is 6.83 (+/- 0.38). However, cytoplasmic pH exhibits a nonunimodal distribution, the lower mean pH being 6.74 (+/- 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell. The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry. We also discuss several other applications of this ratio imaging technique in cell biology.

Observations on the marginal ruffles of an established fibroblast-like cell line.

LW13K2 cells, a clone of a spontaneously in vitro transformed derivative of embryonic Lewis rat fibroblastic cells, were studied by phase contrast cine-light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ruffles found at the advancing edge of cells grown on glass substrates in vitro form and recede in a period of less than one min if they do not make an attachment of the substrate. If they fail to make an attachment they may form pinocytotic channels near the leading edge as described by Price (1972) and/or collapse, generally backwards, towards the cell body. The "spines" which appear to reinforce the membranous ruffles are the last structures to disappear, and accumulate in an irregular array behind the ruffling edge; this area is behind that in which pinocytosis occurs. In comparison with the sparse numbers of ribosomes found in the trailing edge, they are present in notable concentrations near the leading, ruffling edge of the cell. No membrane vesicles have been found in or near the ruffling edges at the ruffle-spine concentration zone.