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Sample records for rpe cell migration

  1. Lutein inhibits the migration of retinal pigment epithelial cells via cytosolic and mitochondrial Akt pathways (lutein inhibits RPE cells migration).

    PubMed

    Su, Ching-Chieh; Chan, Chi-Ming; Chen, Han-Min; Wu, Chia-Chun; Hsiao, Chien-Yu; Lee, Pei-Lan; Lin, Victor Chia-Hsiang; Hung, Chi-Feng

    2014-08-08

    During the course of proliferative vitreoretinopathy (PVR), the retinal pigment epithelium (RPE) cells will de-differentiate, proliferate, and migrate onto the surfaces of the sensory retina. Several studies have shown that platelet-derived growth factor (PDGF) can induce migration of RPE cells via an Akt-related pathway. In this study, the effect of lutein on PDGF-BB-induced RPE cells migration was examined using transwell migration assays and Western blot analyses. We found that both phosphorylation of Akt and mitochondrial translocation of Akt in RPE cells induced by PDGF-BB stimulation were suppressed by lutein. Furthermore, the increased migration observed in RPE cells with overexpressed mitochondrial Akt could also be suppressed by lutein. Our results demonstrate that lutein can inhibit PDGF-BB induced RPE cells migration through the inhibition of both cytoplasmic and mitochondrial Akt activation.

  2. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    SciTech Connect

    Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  3. Isolation, culture and characterization of primary mouse RPE cells.

    PubMed

    Fernandez-Godino, Rosario; Garland, Donita L; Pierce, Eric A

    2016-07-01

    Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD). PMID:27281648

  4. RPE Cell and Sheet Properties in Normal and Diseased Eyes.

    PubMed

    Rashid, Alia; Bhatia, Shagun K; Mazzitello, Karina I; Chrenek, Micah A; Zhang, Qing; Boatright, Jeffrey H; Grossniklaus, Hans E; Jiang, Yi; Nickerson, John M

    2016-01-01

    Previous studies of human retinal pigment epithelium (RPE) morphology found spatial differences in density: a high density of cells in the macula, decreasing peripherally. Because the RPE sheet is not perfectly regular, we anticipate that there will be differences between conditions and when and where damage is most likely to begin. The purpose of this study is to establish relationships among RPE morphometrics in age, cell location, and disease of normal human and AMD eyes that highlight irregularities reflecting damage. Cadaveric eyes from 11 normal and 3 age-related macular degeneration (AMD) human donors ranging from 29 to 82 years of age were used. Borders of RPE cells were identified with phalloidin. RPE segmentation and analysis were conducted with CellProfiler. Exploration of spatial point patterns was conducted using the "spatstat" package of R. In the normal human eye, with increasing age, cell size increased, and cells lost their regular hexagonal shape. Cell density was higher in the macula versus periphery. AMD resulted in greater variability in size and shape of the RPE cell. Spatial point analysis revealed an ordered distribution of cells in normal and high spatial disorder in AMD eyes. Morphometrics of the RPE cell readily discriminate among young vs. old and normal vs. diseased in the human eye. The normal RPE sheet is organized in a regular array of cells, but AMD exhibited strong spatial irregularity. These findings reflect on the robust recovery of the RPE sheet after wounding and the circumstances under which it cannot recover. PMID:26427486

  5. Heterotypic RPE-choroidal endothelial cell contact increases choroidal endothelial cell transmigration via PI 3-kinase and Rac1

    PubMed Central

    Peterson, Lynda J.; Wittchen, Erika S.; Geisen, Pete; Burridge, Keith; Hartnett, M. Elizabeth

    2008-01-01

    Age-related macular degeneration (AMD) is the major cause of non-preventable blindness. Severe forms of AMD involve breaching of the retinal pigment epithelial (RPE) barrier by underlying choroidal endothelial cells (CECs), followed by migration into, and subsequent neovascularization of the neurosensory retina. However, little is known about the interactions between RPE and CECs and the signaling events leading to CEC transmigration. While soluble chemotactic factors secreted from RPE can contribute to inappropriate CEC transmigration, other unidentified stimuli may play an additional role. Using a coculture model that maintains the natural structural orientation of CECs to the basal aspect of RPE, we show that “contact” with RPE and/or RPE extracellular matrix increases CEC transmigration of the RPE barrier. From a biochemical standpoint, contact between CECs and RPE results in an increase in the activity of the GTPase Rac1 within the CECs; this increase is dependent on upstream activation of PI 3-K and Akt1. To confirm a link between these signaling molecules and increased CEC transmigration, we performed transmigration assays while inhibiting both PI 3-K and Rac1 activity, and observed that both decreased CEC transmigration. We hypothesize that contact between CECs and RPE stimulates a signaling pathway involving PI 3-K, Akt1, and Rac1 that facilitates CEC transmigration across the RPE barrier, an important step in the development of neovascular AMD. PMID:17292356

  6. Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture.

    PubMed

    Mazzoni, Francesca; Safa, Hussein; Finnemann, Silvia C

    2014-09-01

    RPE cells are the most actively phagocytic cells in the human body. In the eye, RPE cells face rod and cone photoreceptor outer segments at all times but contribute to shedding and clearance phagocytosis of distal outer segment tips only once a day. Analysis of RPE phagocytosis in situ has succeeded in identifying key players of the RPE phagocytic mechanism. Phagocytic processes comprise three distinct phases, recognition/binding, internalization, and digestion, each of which is regulated separately by phagocytes. Studies of phagocytosis by RPE cells in culture allow specifically analyzing and manipulating these distinct phases to identify their molecular mechanisms. Here, we compare similarities and differences of primary, immortalized, and stem cell-derived RPE cells in culture to RPE cells in situ with respect to phagocytic function. We discuss in particular potential pitfalls of RPE cell culture phagocytosis assays. Finally, we point out considerations for phagocytosis assay development for future studies.

  7. Signaling pathways involved in PDGF-evoked cellular responses in human RPE cells

    SciTech Connect

    Hollborn, Margrit . E-mail: hollbm@medizin.uni-leipzig.de; Bringmann, Andreas; Faude, Frank; Wiedemann, Peter; Kohen, Leon

    2006-06-09

    We examined whether PDGF may directly stimulate the expression of VEGF by retinal pigment epithelial (RPE) cells in vitro, and the involvement of three signal transduction pathways in the regulation of PDGF-evoked cell proliferation, migration, and production of VEGF-A was investigated. PDGF stimulated the gene and protein expression of VEGF-A by RPE cells, and increased cell proliferation and chemotaxis. PDGF activated all signaling pathways investigated, as determined by increased phosphorylation levels of ERK1/2, p38, and Akt proteins. The three signaling pathways were involved in the mediation of PDGF-evoked cell proliferation, while p38 and PI3K mediated cell migration, and PI3K mediated secretion of VEGF-A. In addition to VEGF-A, the cells expressed mRNAs for various members of the VEGF family and for their receptors, including VEGF-B, -C, -D, flt-1, and KDR. The data indicate that PDGF selectively stimulates the expression of VEGF-A in RPE cells. PDGF evokes at least three signal transduction pathways which are differentially involved in various cellular responses.

  8. RPE cell surface proteins in normal and dystrophic rats

    SciTech Connect

    Clark, V.M.; Hall, M.O.

    1986-02-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

  9. Actin-dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assays.

    PubMed

    McNeil, E L; Tacelosky, D; Basciano, P; Biallas, B; Williams, R; Damiani, P; Deacon, S; Fox, C; Stewart, B; Petruzzi, N; Osborn, C; Klinger, K; Sellers, J R; Smith, C King

    2004-06-01

    Melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells of fish and amphibians undergo massive migrations in response to light conditions to control light flux to the retina. Previous research has shown that melanosome motility within apical projections of dissociated fish RPE cells requires an intact actin cytoskeleton, but the mechanisms and motors involved in melanosome transport in RPE have not been identified. Two in vitro motility assays, the Nitella assay and the sliding filament assay, were used to characterize actin-dependent motor activity of RPE melanosomes. Melanosomes applied to dissected filets of the Characean alga, Nitella, moved along actin cables at a mean rate of 2 microm/min, similar to the rate of melanosome motility in dissociated, cultured RPE cells. Path lengths of motile melanosomes ranged from 9 to 37 microm. Melanosome motility in the sliding filament assay was much more variable, ranging from 0.4-33 microm/min; 70% of velocities ranged from 1-15 microm/min. Latex beads coated with skeletal muscle myosin II and added to Nitella filets moved in the same direction as RPE melanosomes, indicating that the motility is barbed-end directed. Immunoblotting using antibodies against myosin VIIa and rab27a revealed that both proteins are enriched on melanosome membranes, suggesting that they could play a role in melanosome transport within apical projections of fish RPE.

  10. TGF-β1 Induced Transdifferentiation of RPE Cells is Mediated by TAK1

    PubMed Central

    Dvashi, Zeev; Goldberg, Mordechai; Adir, Orit; Shapira, Michal; Pollack, Ayala

    2015-01-01

    Background and Aim Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells. Methodology ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis. Results This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent. Conclusions/Significance This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment. PMID

  11. Plasma membrane protein polarity and trafficking in RPE cells: Past, present and future

    PubMed Central

    Lehmann, Guillermo L.; Benedicto, Ignacio; Philp, Nancy J.; Rodriguez-Boulan, Enrique

    2015-01-01

    The retinal pigment epithelium (RPE) comprises a monolayer of polarized pigmented epithelial cells that is strategically interposed between the neural retina and the fenestrated choroid capillaries. The RPE performs a variety of vectorial transport functions (water, ions, metabolites, nutrients and waste products) that regulate the composition of the subretinal space and support the functions of photoreceptors (PRs) and other cells in the neural retina. To this end, RPE cells display a polarized distribution of channels, transporters and receptors in their plasma membrane (PM) that is remarkably different from that found in conventional extra-ocular epithelia, e.g. intestine, kidney, and gall bladder. This characteristic PM protein polarity of RPE cells depends on the interplay of sorting signals in the RPE PM proteins and sorting mechanisms and biosynthetic/recycling trafficking routes in the RPE cell. Although considerable progress has been made in our understanding of the RPE trafficking machinery, most available data have been obtained from immortalized RPE cell lines that only partially maintain the RPE phenotype and by extrapolation of data obtained in the prototype Madin–Darby Canine Kidney (MDCK) cell line. The increasing availability of RPE cell cultures that more closely resemble the RPE in vivo together with the advent of advanced live imaging microscopy techniques provides a platform and an opportunity to rapidly expand our understanding of how polarized protein trafficking contributes to RPE PM polarity. PMID:25152359

  12. Hypoxia‐inducible factor expression in human RPE cells

    PubMed Central

    Forooghian, Farzin; Razavi, Rozita; Timms, Lee

    2007-01-01

    Background Hypoxia‐inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease. Methods ARPE‐19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme‐linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real‐time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques. Results HIF‐1α was readily produced by ARPE‐19 cells under hypoxia, but HIF‐2α and HIF‐3α could not be detected even after HIF‐1α silencing. HIF‐1α protein levels showed an increasing trend for the first 24 h while HIF‐1α mRNA levels fluctuated during this time. After 36 h HIF‐1α protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF‐1α significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level. Conclusions HIF‐1α appears to be the main isoform of HIF functioning in ARPE‐19 cells. Under hypoxia, HIF‐1α levels are likely self‐regulated by a feedback loop that involves both transcriptional and post‐translational mechanisms. VEGF production by human RPE cells is regulated by HIF‐1α. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina

  13. Comparative study between amniotic-fluid mesenchymal stem cells and retinal pigmented epithelium (RPE) stem cells ability to differentiate towards RPE cells.

    PubMed

    Mariotti, Cesare; Lazzarini, Raffaella; Nicolai, Michele; Saitta, Andrea; Orsini, Emanuele; Orciani, Monia; Di Primio, Roberto

    2015-10-01

    Dysfunction of the retinal pigmented epithelium (RPE) is one of the first effects of dry age-related macular degeneration (AMD) with consequent blindness. Hence, patients affected by this retinal disorder could benefit from a cell-based transplantation strategy for RPE. Actually, an effective protocol to approach this problem is lacking, though recently, it has been postulated the existence of a subpopulation of RPE stem cells (RPESCs) derived from adult RPE and able to reconstitute a functional RPE. On the other hand, the evidence related to the differentiative potential of human mesenchymal stem cells (MSCs) is continuously increasing. Among others, amniotic fluid-derived MSCs (AF-MSCs) may be a promising candidate, since these cells are characterized by high proliferation and differentiative potential. In this study, AF-MSCs and RPESCs were isolated, characterized to assay their stemness and induced to neuronal/retinal differentiation; specific RPE markers were then analyzed. Our results indicate that RPESCs are more suitable candidates for RPE replacement than AF-MSCs.

  14. The Role of Bcl-xL in Mouse RPE Cell Survival

    PubMed Central

    Medearis, Sarah; Han, Ian C.; Huang, Jessica K.; Yang, Ping

    2011-01-01

    Purpose. Retinal pigment epithelial (RPE) cell survival plays a critical role in normal physiology and in retinal diseases, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). We have previously demonstrated that Bcl-xL is an important cell survival protein in human RPE (hRPE) cells. Herein, we determined the role of Bcl-xL as a survival protein in mouse RPE (mRPE) cells. Methods. Survival factor gene expression and Bcl-xL protein distribution were determined using qRT-PCR and immunohistochemistry, respectively. Cultured mRPE cells were transfected with two modified 2′-O-methoxyethoxy antisense oligonucleotides (ASOs): Bcl-xL–mismatched control and Bcl-xL–specific. Bcl-xL protein levels were analyzed using Western blot. To determine the effects of survival factor regulation in mRPE cells, cultured cells were treated for 24 hours with mouse TNF-α, human IL-1β, and human TNF-α. Results. Bcl-xL was the most highly expressed survival factor in both mouse eyecup and cultured mRPE cells, whereas Bax was the most highly expressed antisurvival factor. Bcl-xL was expressed in the RPE layer, and the distribution among the retinal layers was similar to that observed in human eyecups. IL-1β and TNF-α had minimal effect on Bcl-xL and Bax expression and strongly upregulated Traf-1. Transfection with Bcl-xL–specific ASO resulted in markedly diminished Bcl-xL gene expression, Bcl-xL protein levels, and cell number. Conclusions. Bcl-xL is the most highly expressed survival gene in mRPE cells and is essential for mRPE cell survival. Our data suggest that mouse tissue is an appropriate model for investigations of RPE survival factor genes. PMID:21724914

  15. Blockade of MerTK Activation by AMPK Inhibits RPE Cell Phagocytosis.

    PubMed

    Qin, Suofu

    2016-01-01

    Timely removal of shed photoreceptor outer segments by retinal pigment epithelial cells (RPE) plays a key role in biological renewal of these highly peroxidizable structures and in maintenance of retina health. How environmental stress cause RPE cell dysfunction is undefined however. AMP-activated protein kinase (AMPK), a heterotrimer of a catalytic α subunit and regulatory β and γ subunits, maintains energy homeostasis by limiting energy utilization and/or promoting energy production when energy supply is compromised. Intriguingly, AMPK has been shown to be important in functions of RPE cells. In this mini-review, the role and mechanisms of AMPK in controlling RPE cell phagocytosis are discussed.

  16. Human RPE Stem Cells Grown into Polarized RPE Monolayers on a Polyester Matrix Are Maintained after Grafting into Rabbit Subretinal Space

    PubMed Central

    Stanzel, Boris V.; Liu, Zengping; Somboonthanakij, Sudawadee; Wongsawad, Warapat; Brinken, Ralf; Eter, Nicole; Corneo, Barbara; Holz, Frank G.; Temple, Sally; Stern, Jeffrey H.; Blenkinsop, Timothy A.

    2014-01-01

    Summary Transplantation of the retinal pigment epithelium (RPE) is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC) as an alternative RPE source. Polarized monolayers of adult hRPESC-derived RPE grown on polyester (PET) membranes had near-native characteristics. Trephined pieces of RPE monolayers on PET were transplanted subretinally in the rabbit, a large-eyed animal model. After 4 days, retinal edema was observed above the implant, detected by spectral domain optical coherence tomography (SD-OCT) and fundoscopy. At 1 week, retinal atrophy overlying the fetal or adult transplant was observed, remaining stable thereafter. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. Taken together, the xeno-RPE survived with retained characteristics in the subretinal space. These experiments support that adult hRPESC-derived RPE are a potential source for transplantation therapies. PMID:24511471

  17. The Mitochondrial-Derived Peptide Humanin Protects RPE Cells From Oxidative Stress, Senescence, and Mitochondrial Dysfunction

    PubMed Central

    Sreekumar, Parameswaran G.; Ishikawa, Keijiro; Spee, Chris; Mehta, Hemal H.; Wan, Junxiang; Yen, Kelvin; Cohen, Pinchas; Kannan, Ram; Hinton, David R.

    2016-01-01

    Purpose To investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress–induced cell death, mitochondrial bioenergetics, and senescence. Methods Humanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5–10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress–induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment. Results A prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress–induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress–induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death. Conclusions Humanin protected RPE cells against oxidative stress–induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD. PMID:26990160

  18. Cell Migration

    PubMed Central

    Trepat, Xavier; Chen, Zaozao; Jacobson, Ken

    2015-01-01

    Cell migration is fundamental to establishing and maintaining the proper organization of multicellular organisms. Morphogenesis can be viewed as a consequence, in part, of cell locomotion, from large-scale migrations of epithelial sheets during gastrulation, to the movement of individual cells during development of the nervous system. In an adult organism, cell migration is essential for proper immune response, wound repair, and tissue homeostasis, while aberrant cell migration is found in various pathologies. Indeed, as our knowledge of migration increases, we can look forward to, for example, abating the spread of highly malignant cancer cells, retarding the invasion of white cells in the inflammatory process, or enhancing the healing of wounds. This article is organized in two main sections. The first section is devoted to the single-cell migrating in isolation such as occurs when leukocytes migrate during the immune response or when fibroblasts squeeze through connective tissue. The second section is devoted to cells collectively migrating as part of multicellular clusters or sheets. This second type of migration is prevalent in development, wound healing, and in some forms of cancer metastasis. PMID:23720251

  19. Trypan Blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis.

    PubMed

    Srivastava, Girish K; Reinoso, Roberto; Singh, Amar K; Fernandez-Bueno, Ivan; Hileeto, Denise; Martino, Mario; Garcia-Gutierrez, Maria T; Merino, Jose M Pigazo; Alonso, Nieves Fernández; Corell, Alfredo; Pastor, J Carlos

    2011-12-01

    Retinal pigment epithelial (RPE) cells are currently in the "spotlight" of cell therapy approaches to some retinal diseases. The analysis of the expressed proteins of RPE primary cells is an essential step for many of these approaches. But the emission of autofluorescence by RPE cells produces higher background noise interference thereby creating an impediment to this analysis. Trypan Blue (TB), a routinely used counterstain, has the capacity to quench this autofluorescence, if it is used in optimized concentration. The results from the method developed in our study indicate that incubation of the cultured RPE cells with 20 μg/ml of TB after immunolabelling (post-treatment) as well as incubation of the retinal tissue specimens with same concentration before paraffin embedding, sectioning and immunolabelling (pre-treatment) can be applied to effectively quench the autofluorescence of RPE cells. Thus it can facilitate the evaluation of expressed cellular proteins in experimental as well as in pathological conditions, fulfilling the current requirement for developing a method which can serve to eliminate the autofluorescence of the cells, not only in cell cultures but also in tissues samples. This method should significantly increase the quality and value of RPE cell protein analysis, as well as other cell protein analysis performed by Flow cytometry (FC) and Immunohistochemistry (IHC) techniques.

  20. Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo

    PubMed Central

    Johnen, Sandra; Djalali-Talab, Yassin; Kazanskaya, Olga; Möller, Theresa; Harmening, Nina; Kropp, Martina; Izsvák, Zsuzsanna; Walter, Peter; Thumann, Gabriele

    2015-01-01

    Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth. PMID:26697494

  1. Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo.

    PubMed

    Johnen, Sandra; Djalali-Talab, Yassin; Kazanskaya, Olga; Möller, Theresa; Harmening, Nina; Kropp, Martina; Izsvák, Zsuzsanna; Walter, Peter; Thumann, Gabriele

    2015-01-01

    Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth. PMID:26697494

  2. A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases

    PubMed Central

    Heller, Janosch P.; Kwok, Jessica C. F.; Vecino, Elena; Martin, Keith R.; Fawcett, James W.

    2015-01-01

    Diseases such as age-related macular degeneration (AMD) affect the retinal pigment epithelium (RPE) and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 min yielded 4 × 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases. PMID:26635529

  3. Taurine suppresses the spread of cell death in electrically coupled RPE cells

    PubMed Central

    Udawatte, Chandani; Qian, Haohua; Mangini, Nancy J.; Kennedy, Brian G.

    2008-01-01

    Purpose To determine whether taurine exerts a protective effect on retinal pigment epithelium (RPE) cells exposed to a cytotoxic agent, cytochrome C (cyC), shown previously to induce apoptosis and produce cell death in electrically coupled neighboring cells. Methods Monolayer cultures of confluent human RPE (ARPE-19) cells, which express gap-junctional proteins, were incubated in culture medium with or without taurine. After scrape loading cyC into the cells, we assayed these cells for caspase 3 activity and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to determine the spread of apoptosis. Results We found that cyC, too large a molecule to traverse gap junctional channels, produced apoptosis in cells injured by the scrape as well as those distant from the site of the scrape, presumably by the intercellular transmission of a toxic agent through the gap junctions that couple these cells. Incubation in taurine, or the gap-junction blocker, octanol, before application of cyC, reduced significantly the fraction of cells undergoing apoptosis. Voltage clamp recordings from electrically coupled Xenopus oocytes transfected with Cx43 showed that junctional communication was unaffected by taurine. Conclusions Our results indicate that taurine can serve to suppress cell death in RPE cells independent of any effect on gap junctions. We have considered various avenues by which taurine can exert its protective effect, but the precise mechanism involved under these experimental conditions has yet to be identified. PMID:18958305

  4. The influence of sublethal blue light exposure on human RPE cells

    PubMed Central

    Schaller, Annette; Knels, Lilla; Funk, Richard H.W.

    2009-01-01

    Purpose To evaluate the in vitro response of retinal pigment epithelial (RPE) cells to a nonlethal dose of blue light. Methods The human RPE cell line ARPE-19 was irradiated with blue light (405 nm) at an output power of 1 mW/cm2 or 0.3 mW/cm2. The following parameters were studied: metabolic activity; apoptosis; reactive oxygen species (ROS) production; mitochondrial membrane potential (MMP); ultrastructural changes of mitochondria; production of advanced glycation endproducts (AGEs); and stress-related cellular proteins. Results Nonlethal doses of blue light irradiation significantly reduced ARPE-19 metabolic activity and MMP while increasing intracellular ROS levels and expression of stress-related proteins heme oxygenase-1 (HO-1), osteopontin, heat shock protein 27 (Hsp-27), manganese superoxide dismutase (SOD-Mn), and cathepsin D. Blue light irradiation also induced ultrastructural conformation changes in mitochondria, resulting in the appearance of giant mitochondria after 72 h. We further found enhanced formation of AGEs, particularly Nε-(carboxymethyl) lysine (CML) modifications, and a delay in the cell cycle. Conclusions ARPE-19 cells avoid cell death and recover from blue light irradiation by activating a host of defense mechanisms while simultaneously triggering cellular stress responses that may be involved in RPE disease development. Continuous light exposure can therefore detrimentally affect metabolically stressed RPE cells. This may have implications for pathogenesis of age-related macular degeneration. PMID:19784391

  5. Humanin Protects RPE Cells from Endoplasmic Reticulum Stress-Induced Apoptosis by Upregulation of Mitochondrial Glutathione

    PubMed Central

    Matsunaga, Douglas; Sreekumar, Parameswaran G.; Ishikawa, Keijiro; Terasaki, Hiroto; Barron, Ernesto; Cohen, Pinchas

    2016-01-01

    Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1–20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress. PMID

  6. Oxidative Stress Induces Senescence in Cultured RPE Cells

    PubMed Central

    Aryan, Nona; Betts-Obregon, Brandi S.; Perry, George; Tsin, Andrew T.

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence. PMID:27651846

  7. Oxidative Stress Induces Senescence in Cultured RPE Cells.

    PubMed

    Aryan, Nona; Betts-Obregon, Brandi S; Perry, George; Tsin, Andrew T

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence. PMID:27651846

  8. Oxidative Stress Induces Senescence in Cultured RPE Cells

    PubMed Central

    Aryan, Nona; Betts-Obregon, Brandi S.; Perry, George; Tsin, Andrew T.

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.

  9. Oxidative Stress Induces Mitochondrial Dysfunction and a Protective Unfolded Protein Response in RPE cells

    PubMed Central

    Cano, Marisol; Wang, Lei; Wan, Jun; Barnett, Bradley P.; Ebrahimi, Katayoon; Qian, Jiang; Handa, James T.

    2014-01-01

    How cells degenerate from oxidative stress in aging-related disease is incompletely understood. The study’s intent was to identify key cytoprotective pathways activated by oxidative stress, and determine the extent of their protection. Using an unbiased strategy with microarray analysis, retinal pigmented epithelial (RPE) cells treated with cigarette smoke extract (CSE) had over-represented genes involved in the antioxidant and unfolded protein response (UPR). Differentially expressed antioxidant genes were predominantly located in the cytoplasm, with no induction of genes that neutralize superoxide and H2O2 in the mitochondria, resulting in accumulation of superoxide and decreased ATP production. Simultaneously, CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, including CHOP, which was cytoprotective because CHOP knockdown decreased cell viability. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP, and developed epithelial-mesenchymal transition, as suggested by decreased LRAT abundance, altered ZO1 immunolabeling, and dysmorphic cell shape. Mildly degenerated RPE from early AMD samples had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling. While oxidative stress is thought to induce an antioxidant response with cooperation between the mitochondria and ER, herein, we show that mitochondria become impaired sufficiently to induce epithelial-mesenchymal transition despite a protective UPR. With similar responses in early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress despite a protective UPR during early phases of aging-related disease. PMID:24434119

  10. RPE, a plant gene involved in early developmental steps of nematode feeding cells.

    PubMed Central

    Favery, B; Lecomte, P; Gil, N; Bechtold, N; Bouchez, D; Dalmasso, A; Abad, P

    1998-01-01

    Sedentary plant-parasitic nematodes are able to induce the redifferentiation of root cells into multinucleate nematode feeding sites (NFSs). We have isolated by promoter trapping an Arabidopsis thaliana gene that is essential for the early steps of NFS formation induced by the root-knot nematode Meloidogyne incognita. Its pattern of expression is similar to that of key regulators of the cell cycle, but it is not observed with the cyst nematode. Later in NFS development, this gene is induced by both root-knot and cyst nematodes. It encodes a protein similar to the D-ribulose-5-phosphate 3-epimerase (RPE) (EC 5.1.3.1), a key enzyme in the reductive Calvin cycle and the oxidative pentose phosphate pathway (OPPP). Quantitative RT-PCR showed the accumulation of RPE transcripts in potato, as in Arabidopsis NFS. Homozygous rpe plants have a germination mutant phenotype that can be rescued in dwarf plants on sucrose-supplemented medium. During root development, this gene is expressed in the meristems and initiation sites of lateral roots. These results suggest that the genetic control of NFSs and the first stages of meristem formation share common steps and confirms the previous cytological observations which indicate that root cells undergo metabolic reprogramming when they turn into NFSs. PMID:9843485

  11. [Study of blue light induced DNA damage of retinal pigment epithelium(RPE) cells and the protection of vitamin C].

    PubMed

    Zhou, Jian Wei; Ren, Guo Liang; Zhang, Xiao Ming; Zhu, Xi; Lin, Hai Yan; Zhou, Ji Lin

    2003-10-01

    To evaluate protection of vitamin C on blue light-induced DNA damage of human retinal pigment epithelium (RPE) cells. The cultured RPE cells were divided into 3 groups: Control group (no blue light exposure), blue light exposure group (blue light exposure for 20 minutes) and blue light exposure + vitamin C group (blue light exposure + 100 mumol/L vitamin C). Travigen's comet assay kit and Euclid comet assay software were used to assay the DNA damage levels. The DNA percentage in the tail of electrophoretogram in the three groups were 18.44%, 54.42% and 32.43% respectively (p < 0.01). Tail moments were 8.2, 48.3, and 18.4 respectively (p < 0.01). Blue light could induce DNA damage to RPE cells but vitamin C could protect the RPE cells from the blue light-induced DNA damage.

  12. Ginsenoside Rg-1 Protects Retinal Pigment Epithelium (RPE) Cells from Cobalt Chloride (CoCl2) and Hypoxia Assaults

    PubMed Central

    Yao, Jin; Zhao, Yu-xia; Duan, Jing; Cao, Cong; Jiang, Qin

    2013-01-01

    Severe retinal ischemia causes persistent visual impairments in eye diseases. Retinal pigment epithelium (RPE) cells are located near the choroidal capillaries, and are easily affected by ischemic or hypoxia. Ginsenoside Rg-1 has shown significant neuroprotective effects. This study was performed to test the cytoprotective effect of ginsenoside Rg-1 in RPE cells against hypoxia and cobalt chloride (CoCl2) assaults, and to understand the underlying mechanisms. We found that Rg-1 pre-administration significantly inhibited CoCl2- and hypoxia-induced RPE cell death and apoptosis. Reactive oxygen specisis (ROS)-dependent p38 and c-Jun NH(2)-terminal kinases (JNK) MAPK activation was required for CoCl2-induced RPE cell death, and Rg-1 pre-treatment significantly inhibited ROS production and following p38/JNK activation. Further, CoCl2 suppressed pro-survival mTOR complex 1 (mTORC1) activation in RPE cells through activating of AMP-activated protein kinase (AMPK), while Rg-1 restored mTORC1 activity through inhibiting AMPK activation. CoCl2-induced AMPK activation was also dependent on ROS production, and anti-oxidant N-acetylcysteine (NAC) prevented AMPK activation and RPE cell death by CoCl2. Our results indicated that Rg-1 could be further investigated as a novel cell-protective agent for retinal ischemia. PMID:24386346

  13. Neurogenin1 effectively reprograms cultured chick RPE cells to differentiate towards photoreceptors

    PubMed Central

    Yan, Run-Tao; Liang, Lina; Ma, Wenxin; Li, Xiumei; Xie, Wenlian; Wang, Shu-Zhen

    2009-01-01

    Photoreceptors are highly specialized sensory neurons in the retina, and their degeneration results in blindness. Replacement with developing photoreceptor cells promises to be an effective therapy, but it requires a supply of new photoreceptors, because the neural retina in human eyes lacks regeneration capability. We report efficient generation of differentiating, photoreceptor-like neurons from chick retinal pigment epithelial (RPE) cells propagated in culture through reprogramming with neurogenin1 (ngn1). In reprogrammed culture, a large number of the cells (85.0 ± 5.9%) began to differentiate towards photoreceptors. Reprogrammed cells expressed transcription factors that set in motion photoreceptor differentiation, including Crx, Nr2E3, NeuroD, and RXRγ, and phototransduction pathway components, including transducin, cGMP-gated channel, and red opsin of cone photoreceptors (equivalent to rhodopsin of rod photoreceptors). They developed inner segments rich in mitochondria. Furthermore, they responded to light by decreasing their cellular free calcium (Ca2+) levels and responded to 9-cis-retinal by increasing their Ca2+ levels after photobleaching, hallmarks of photoreceptor physiology. The high efficiency and the advanced photoreceptor differentiation indicate ngn1 as a gene of choice to reprogram RPE progeny cells to differentiate into photoreceptor neurons in future cell replacement studies. PMID:20029995

  14. Live-Cell Imaging of Phagosome Motility in Primary Mouse RPE Cells.

    PubMed

    Hazim, Roni; Jiang, Mei; Esteve-Rudd, Julian; Diemer, Tanja; Lopes, Vanda S; Williams, David S

    2016-01-01

    The retinal pigment epithelium (RPE) is a post-mitotic epithelial monolayer situated between the light-sensitive photoreceptors and the choriocapillaris. Given its vital functions for healthy vision, the RPE is a primary target for insults that result in blinding diseases, including age-related macular degeneration (AMD). One such function is the phagocytosis and digestion of shed photoreceptor outer segments. In the present study, we examined the process of trafficking of outer segment disk membranes in live cultures of primary mouse RPE, using high speed spinning disk confocal microscopy. This approach has enabled us to track phagosomes, and determine parameters of their motility, which are important for their efficient degradation.

  15. p27KIP1 loss promotes proliferation and phagocytosis but prevents epithelial–mesenchymal transition in RPE cells after photoreceptor damage

    PubMed Central

    ul Quraish, Reeshan; Sudou, Norihiro; Nomura-Komoike, Kaori; Sato, Fumi

    2016-01-01

    Purpose p27KIP1 (p27), originally identified as a cell cycle inhibitor, is now known to have multifaceted roles beyond cell cycle regulation. p27 is required for the normal histogenesis of the RPE, but the role of p27 in the mature RPE remains elusive. To define the role of p27 in the maintenance and function of the RPE, we investigated the effects of p27 deletion on the responses of the RPE after photoreceptor damage. Methods Photoreceptor damage was induced in wild-type (WT) and p27 knockout (KO) mice with N-methyl-N-nitrosourea (MNU) treatment. Damage-induced responses of the RPE were investigated with bromodeoxyuridine (BrdU) incorporation assays, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays at different stages after MNU treatment. Subcellular localization of p27 in the WT RPE was also analyzed in vivo and in vitro. Results MNU treatment induced photoreceptor-specific degeneration in the WT and KO retinas. BrdU incorporation assays revealed virtually no proliferation of RPE cells in the WT retinas while, in the KO retinas, approximately 16% of the RPE cells incorporated BrdU at day 2 after MNU treatment. The RPE in the KO retinas developed aberrant protrusions into the outer nuclear layer in response to photoreceptor damage and engulfed outer segment debris, as well as TUNEL-positive photoreceptor cells. Increased phosphorylation of myosin light chains and their association with rhodopsin-positive phagosomes were observed in the mutant RPE, suggesting possible deregulation of cytoskeletal dynamics. In addition, WT RPE cells exhibited evidence of the epithelial–mesenchymal transition (EMT), including morphological changes, induction of α-smooth muscle actin expression, and attenuated expression of tight junction protein ZO-1 while these changes were absent in the KO retinas. In the normal WT retinas, p27 was localized to the nuclei of RPE cells while nuclear and cytoplasmic p27 was detected in RPE cells

  16. Exogenous NAD(+) decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy.

    PubMed

    Zhu, Ying; Zhao, Ke-Ke; Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

    2016-05-31

    Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD(+)) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD(+) administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD(+) administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD(+) administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD(+) against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD(+) administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD(+) administration might be potential value for the treatment of AMD.

  17. Exogenous NAD+ decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy

    PubMed Central

    Zhu, Ying; Zhao, Ke-ke; Tong, Yao; Zhou, Ya-li; Wang, Yi-xiao; Zhao, Pei-quan; Wang, Zhao-yang

    2016-01-01

    Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD+) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD+ administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD+ administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD+ administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD+ against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD+ administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD+ administration might be potential value for the treatment of AMD. PMID:27240523

  18. Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

    PubMed Central

    Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

    2009-01-01

    Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

  19. Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

    NASA Astrophysics Data System (ADS)

    Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

    2001-07-01

    Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

  20. Aquaporins and cell migration.

    PubMed

    Papadopoulos, M C; Saadoun, S; Verkman, A S

    2008-07-01

    Aquaporin (AQP) water channels are expressed primarily in cell plasma membranes. In this paper, we review recent evidence that AQPs facilitate cell migration. AQP-dependent cell migration has been found in a variety of cell types in vitro and in mice in vivo. AQP1 deletion reduces endothelial cell migration, limiting tumor angiogenesis and growth. AQP4 deletion slows the migration of reactive astrocytes, impairing glial scarring after brain stab injury. AQP1-expressing tumor cells have enhanced metastatic potential and local infiltration. Impaired cell migration has also been seen in AQP1-deficient proximal tubule epithelial cells, and AQP3-deficient corneal epithelial cells, enterocytes, and skin keratinocytes. The mechanisms by which AQPs enhance cell migration are under investigation. We propose that, as a consequence of actin polymerization/depolymerization and transmembrane ionic fluxes, the cytoplasm adjacent to the leading edge of migrating cells undergoes rapid changes in osmolality. AQPs could thus facilitate osmotic water flow across the plasma membrane in cell protrusions that form during migration. AQP-dependent cell migration has potentially broad implications in angiogenesis, tumor metastasis, wound healing, glial scarring, and other events requiring rapid, directed cell movement. AQP inhibitors may thus have therapeutic potential in modulating these events, such as slowing tumor growth and spread, and reducing glial scarring after injury to allow neuronal regeneration. PMID:17968585

  1. Analysis of the RPE sheet in the rd10 retinal degeneration model

    SciTech Connect

    Jiang, Yi

    2011-01-04

    The normal RPE sheet in the C57Bl/6J mouse is subclassified into two major tiling patterns: A regular generally hexagonal array covering most of the surface and a 'soft network' near the ciliary body made of irregularly shaped cells. Physics models predict these two patterns based on contractility and elasticity of the RPE cell, and strength of cellular adhesion between cells. We hypothesized and identified major changes in RPE regular hexagonal tiling pattern in rdl0 compared to C57BL/6J mice. RPE sheet damage was extensive but occurred in rd10 later than expected, after most retinal degeneration. RPE sheet changes occur in zones with a bullseye pattern. In the posterior zone around the optic nerve RPE cells take on larger irregular and varied shapes to form an intact monolayer. In mid periphery, there is a higher than normal density of cells that progress into involuted layers of RPE under the retina. The periphery remains mostly normal until late stages of degeneration. The number of neighboring cells varies widely depending on zone and progression. RPE morphology continues to deteriorate long after the photoreceptors have degenerated. The RPE cells are bystanders to the rd10 degeneration within photo receptors, and the collateral damage to the RPE sheet resembles stimulation of migration or chemotaxis. Quantitative measures of the tiling patterns and histopathology detected here, scripted in a pipeline written in Perl and Cell Profiler (an open source Matlab plugin), are directly applicable to RPE sheet images from noninvasive fundus autofluorescence (FAF), adaptive optics confocal scanning laser ophthalmoscope (AO-cSLO), and spectral domain optical coherence tomography (SD-OCT) of patients with early stage AMD or RP.

  2. Epithelial-to-Mesenchymal Transition of RPE Cells In Vitro Confers Increased β1,6-N-Glycosylation and Increased Susceptibility to Galectin-3 Binding

    PubMed Central

    Priglinger, Claudia S.; Obermann, Jara; Szober, Christoph M.; Merl-Pham, Juliane; Ohmayer, Uli; Behler, Jennifer; Gruhn, Fabian; Kreutzer, Thomas C.; Wertheimer, Christian; Geerlof, Arie; Priglinger, Siegfried G.; Hauck, Stefanie M.

    2016-01-01

    Epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells is a crucial event in the onset of proliferative vitreoretinopathy (PVR), the most common reason for treatment failure in retinal detachment surgery. We studied alterations in the cell surface glycan expression profile upon EMT of RPE cells and focused on its relevance for the interaction with galectin-3 (Gal-3), a carbohydrate binding protein, which can inhibit attachment and spreading of human RPE cells in a dose- and carbohydrate-dependent manner, and thus bares the potential to counteract PVR-associated cellular events. Lectin blot analysis revealed that EMT of RPE cells in vitro confers a glycomic shift towards an abundance of Thomsen-Friedenreich antigen, poly-N-acetyllactosamine chains, and complex-type branched N-glycans. Using inhibitors of glycosylation we found that both, binding of Gal-3 to the RPE cell surface and Gal-3-mediated inhibition of RPE attachment and spreading, strongly depend on the interaction of Gal-3 with tri- or tetra-antennary complex type N-glycans and sialylation of glycans but not on complex-type O-glycans. Importantly, we found that β1,6 N-acetylglucosaminyltransferase V (Mgat5), the key enzyme catalyzing the synthesis of tetra- or tri-antennary complex type N-glycans, is increased upon EMT of RPE cells. Silencing of Mgat5 by siRNA and CRISPR-Cas9 genome editing resulted in reduced Gal-3 binding. We conclude from these data that binding of recombinant Gal-3 to the RPE cell surface and inhibitory effects on RPE attachment and spreading largely dependent on interaction with Mgat5 modified N-glycans, which are more abundant on dedifferentiated than on the healthy, native RPE cells. Based on these findings we hypothesize that EMT of RPE cells in vitro confers glycomic changes, which account for high affinity binding of recombinant Gal-3, particularly to the cell surface of myofibroblastic RPE. From a future perspective recombinant Gal-3 may disclose a

  3. The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4

    SciTech Connect

    Vatsyayan, Rit; Lelsani, Poorna Chandra Rao; Chaudhary, Pankaj; Kumar, Sushil; Awasthi, Sanjay; Awasthi, Yogesh C.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Low concentration of HNE (0.1-1.0 {mu}M) induced secretion of VEGF in RPE cells. Black-Right-Pointing-Pointer VEGF secreted medium of RPE cells promoted proliferation of endothelial cells. Black-Right-Pointing-Pointer VEGFR2 expression was attenuated with increasing concentrations of HNE. Black-Right-Pointing-Pointer These effects of HNE could be blocked by the over expression of GSTA4-4 in cells. -- Abstract: It is well established that 4-hydroxynonenal (HNE) plays a major role in oxidative stress-induced signaling and the toxicity of oxidants. Surprisingly our recent studies also demonstrate that low levels of HNE generated during oxidative stress promote cell survival mechanisms and proliferation. Since the expression and secretion of VEGF is known to be affected by Oxidative stress, during present studies, we have examined dose dependent effect of HNE on VEGF expression and secretion in a model of retinal pigment epithelial (RPE) cells in culture. Results of these studies showed that while inclusion of 0.1 {mu}M HNE in the medium caused increased secretion of VEGF, its secretion and expression was significantly suppressed in the presence of >5 {mu}M HNE in the media. These concentration dependent hormetic effects of HNE on VEGF secretion could be blocked by the over expression of GSTA4-4 indicating that these effects were specifically attributed to HNE and regulated by GSTA4-4. VEGF secreted into the media showed angiogenic properties as indicated by increased migration and tube formation of HUVEC in matrigel when grown in media from RPE cells treated with 1 {mu}M HNE. The corresponding media from GSTA4-4 over expressing RPE cells had no effect on migration and tube formation of HUVEC in matrigel. These results are consistent with earlier studies showing that at low concentrations, HNE promotes proliferative mechanisms and suggest that HNE induces VEGF secretion from RPE cells that acts in a paracrine fashion to induce

  4. A Novel Inhibitor of 5-Lipoxygenase (5-LOX) Prevents Oxidative Stress–Induced Cell Death of Retinal Pigment Epithelium (RPE) Cells

    PubMed Central

    Subramanian, Preeti; Mendez, Emily F.; Becerra, S. Patricia

    2016-01-01

    Purpose 5-Lipoxygenase (5-LOX) oxygenates arachidonic acid to form 5-hydroperoxyeicosatetraenoic acid, which is further converted into biologically detrimental leukotrienes, such as leukotriene B4 (LTB4). The RPE and retina express the PNPLA2 gene for pigment epithelium–derived factor receptor (PEDF-R), a lipase involved in cell survival. The purpose here was to investigate the role of PEDF-R on the 5-LOX pathway in oxidative stress of RPE. Methods Lipoxygenase activity assays were performed with soybean and potato lipoxygenase. Binding was evaluated by peptide-affinity chromatography and pull-down assays with PEDF-R–derived synthetic peptides or recombinant protein. Oxidative stress was induced in human ARPE-19 and primary pig RPE cells with indicated concentrations of H2O2/TNF-α. Reverse transcription–PCR of ALOX5 and PNPLA2 genes was performed. Cell viability and death rates were determined using respective biomarkers. Leukotriene B4 levels were measured by ELISA. Results Among five peptides spanning between positions Leu159 and Met325 of human PEDF-R polypeptide, only two overlapping peptides, E5b and P1, bound and inhibited lipoxygenase activity. Human recombinant 5-LOX bound specifically to peptide P1 and to His6/Xpress-tagged PEDF-R via ionic interactions. The two inhibitor peptides E5b and P1 promoted cell viability and decreased cell death of RPE cells undergoing oxidative stress. Oxidative stress decreased the levels of PNPLA2 transcripts with no effect on ALOX5 expression. Exogenous additions of P1 peptide or overexpression of the PNPLA2 gene decreased both LTB4 levels and death of RPE cells undergoing oxidative stress. Conclusions A novel peptide region of PEDF-R inhibits 5-LOX, which intersects with RPE cell death pathways induced by oxidative stress. PMID:27635633

  5. γδ T Cells as a Major Source of IL-17 Production During Age-Dependent RPE Degeneration

    PubMed Central

    Zhao, Zhenyang; Xu, Pei; Jie, Zuliang; Zuo, Yiqin; Yu, Bo; Soong, Lynn; Sun, Jiaren; Chen, Yan; Cai, Jiyang

    2014-01-01

    Purpose. Chronic inflammation is a key factor contributing to the progression of age-related macular degeneration (AMD). The goals of the current study were to develop an improved mouse model with retinal pathologic features similar to those of AMD and to characterize the immunoreactive cells in the outer retina and choroid during degeneration of the retinal pigment epithelium (RPE). Methods. Mice deficient in nuclear erythroid 2-related factor 2 (Nrf2) at 12 months of age were fed a high-fat, cholesterol-rich diet for up to 16 weeks. Ocular phenotype was monitored by optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) in live animals, and was further validated by retinal histopathology. Immunofluorescence staining of either cryosections or RPE flat mounts was used to define immunoreactive cells. Flow cytometry analyses were further performed to define the subsets of intraocular T lymphocytes. Results. After 16 weeks on a high-fat (HF) diet, 58% of the eyes from Nrf2−/− mice had progression of retinal lesions. Major histocompatibility complex class II (MHC II)-positive microglia, FoxP3+ regulatory T cells (Tregs), and CD3+ IL-17-producing T cells were detected in either the retina or sub-RPE space. Flow cytometry analyses further revealed that most of the IL-17-producing cells were CD3+ CD4− TCRγδ+ cells. Conclusions. The results suggest that the T cell-mediated immune responses played important roles in controlling the progression of AMD-like phenotype in Nrf2-deficient mice. PMID:25212781

  6. IAPs and cell migration.

    PubMed

    Dubrez, Laurence; Rajalingam, Krishnaraj

    2015-03-01

    Inhibitors of apoptosis (IAPs) constitute a family of cell signaling regulators controlling several fundamental biological processes such as innate immunity, inflammation, cell death, cell proliferation, and cell differentiation. Increasing evidence from in vivo and in vitro studies indicate a function for IAPs in the modulation of invasive and migratory properties of cells. Here, we present and discuss the mechanisms whereby IAPs can control cell migration.

  7. High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression.

    PubMed

    Farnoodian, Mitra; Halbach, Caroline; Slinger, Cassidy; Pattnaik, Bikash R; Sorenson, Christine M; Sheibani, Nader

    2016-09-01

    Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis. PMID:27440660

  8. Transcriptional Reactivation of OTX2, RX1 and SIX3 during Reprogramming Contributes to the Generation of RPE Cells from Human iPSCs

    PubMed Central

    Li, Peng; Sun, Xiaofeng; Ma, Zhizhong; Liu, Yinan; Jin, Ying; Ge, Ruimin; Hao, Limin; Ma, Yanling; Han, Shuo; Sun, Haojie; Zhang, Mingzhi; Li, Ruizhi; Li, Tao; Shen, Li

    2016-01-01

    Directed differentiation of human induced pluripotent stem cells (iPSCs) into retinal pigmented epithelium (RPE) holds great promise in cell replacement therapy for patients suffering from degenerative eye diseases, including age-related macular degeneration (AMD). In this study, we generated iPSCs from human dermal fibroblasts (HDFs) by electroporation with episomal plasmid vectors encoding OCT4, SOX2, KLF4, L-MYC together with p53 suppression. Intriguingly, cell reprogramming resulted in a metastable transcriptional activation and selective demethylation of neural and retinal specification-associated genes, such as OTX2, RX1 and SIX3. In contrast, RPE progenitor genes were transcriptionally silent in HDFs and descendant iPSCs. Overexpression of OCT4 and SOX2 directly stimulated the expression of OTX2, RX1 and SIX3 in HDFs and iPSCs. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified an OCT4- and two SOX2-binding sites located in the proximal promoter of OTX2. Histone acetylation and methylation on the local promoter also participated in the reactivation of OTX2. The transcriptional conversion of RX1 and SIX3 genes partially attributed to DNA demethylation. Subsequently, iPSCs were induced into the RPE cells displaying the characteristics of polygonal shapes and pigments, and expressing typical RPE cell markers. Taken together, our results establish readily efficient and safe protocols to produce iPSCs and iPSC-derived RPE cells, and underline that the reactivation of anterior neural transcription factor OTX2, eye field transcription factor RX1 and SIX3 in iPSCs is a feature of pluripotency acquisition and predetermines the potential of RPE differentiation. PMID:27019633

  9. Transcriptional Reactivation of OTX2, RX1 and SIX3 during Reprogramming Contributes to the Generation of RPE Cells from Human iPSCs.

    PubMed

    Li, Peng; Sun, Xiaofeng; Ma, Zhizhong; Liu, Yinan; Jin, Ying; Ge, Ruimin; Hao, Limin; Ma, Yanling; Han, Shuo; Sun, Haojie; Zhang, Mingzhi; Li, Ruizhi; Li, Tao; Shen, Li

    2016-01-01

    Directed differentiation of human induced pluripotent stem cells (iPSCs) into retinal pigmented epithelium (RPE) holds great promise in cell replacement therapy for patients suffering from degenerative eye diseases, including age-related macular degeneration (AMD). In this study, we generated iPSCs from human dermal fibroblasts (HDFs) by electroporation with episomal plasmid vectors encoding OCT4, SOX2, KLF4, L-MYC together with p53 suppression. Intriguingly, cell reprogramming resulted in a metastable transcriptional activation and selective demethylation of neural and retinal specification-associated genes, such as OTX2, RX1 and SIX3. In contrast, RPE progenitor genes were transcriptionally silent in HDFs and descendant iPSCs. Overexpression of OCT4 and SOX2 directly stimulated the expression of OTX2, RX1 and SIX3 in HDFs and iPSCs. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified an OCT4- and two SOX2-binding sites located in the proximal promoter of OTX2. Histone acetylation and methylation on the local promoter also participated in the reactivation of OTX2. The transcriptional conversion of RX1 and SIX3 genes partially attributed to DNA demethylation. Subsequently, iPSCs were induced into the RPE cells displaying the characteristics of polygonal shapes and pigments, and expressing typical RPE cell markers. Taken together, our results establish readily efficient and safe protocols to produce iPSCs and iPSC-derived RPE cells, and underline that the reactivation of anterior neural transcription factor OTX2, eye field transcription factor RX1 and SIX3 in iPSCs is a feature of pluripotency acquisition and predetermines the potential of RPE differentiation. PMID:27019633

  10. Tumorigenicity studies of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration.

    PubMed

    Kanemura, Hoshimi; Go, Masahiro J; Shikamura, Masayuki; Nishishita, Naoki; Sakai, Noriko; Kamao, Hiroyuki; Mandai, Michiko; Morinaga, Chikako; Takahashi, Masayo; Kawamata, Shin

    2014-01-01

    Basic studies of human pluripotential stem cells have advanced rapidly and stem cell products are now seeing therapeutic applications. However, questions remain regarding the tumorigenic potential of such cells. Here, we report the tumorigenic potential of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of wet-type, age-related macular degeneration (AMD). First, immunodeficient mouse strains (nude, SCID, NOD-SCID and NOG) were tested for HeLa cells' tumor-forming capacity by transplanting various cell doses subcutaneously with or without Matrigel. The 50% Tumor Producing Dose (TPD50 value) is the minimal dose of transplanted cells that generated tumors in 50% of animals. For HeLa cells, the TPD50 was the lowest when cells were embedded in Matrigel and transplanted into NOG mice (TPD50 = 10(1.1), n = 75). The TPD50 for undifferentiated iPSCs transplanted subcutaneously to NOG mice in Matrigel was 10(2.12); (n = 30). Based on these experiments, 1×10(6) iPSC-derived RPE were transplanted subcutaneously with Matrigel, and no tumor was found during 15 months of monitoring (n = 65). Next, to model clinical application, we assessed the tumor-forming potential of HeLa cells and iPSC 201B7 cells following subretinal transplantation of nude rats. The TPD50 for iPSCs was 10(4.73) (n = 20) and for HeLa cells 10(1.32) (n = 37) respectively. Next, the tumorigenicity of iPSC-derived RPE was tested in the subretinal space of nude rats by transplanting 0.8-1.5×10(4) iPSC-derived RPE in a collagen-lined (1 mm×1 mm) sheet. No tumor was found with iPSC-derived RPE sheets during 6-12 months of monitoring (n = 26). Considering the number of rodents used, the monitoring period, the sensitivity of detecting tumors via subcutaneous and subretinal administration routes and the incidence of tumor formation from the iPSC-derived RPE, we conclude that the tumorigenic potential of the iPSC-derived RPE was negligible.

  11. Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

    SciTech Connect

    Zhang, Wenjie; Zhang, Xiaomei; Lu, Hong; Matsukura, Makoto; Zhao, Jien; Shinohara, Makoto

    2013-05-10

    Highlights: •HO-1 is highly induced in RPE cells by hypoxia. •Inhibition of HO-1 activity and knockdown of HO-1 expression inhibit VEGF expression in RPE cells under hypoxia. •Knockdown of HO-1 in RPE cells inhibits angiogenesis of endothelial cells in vitro. -- Abstract: Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cell HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.

  12. Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4−/− Mice: Pharmacologic Approaches and Functional Recovery

    PubMed Central

    Liu, Ji; Lu, Wennan; Reigada, David; Nguyen, Jonathan; Laties, Alan M.; Mitchell, Claire H.

    2008-01-01

    Purpose Degradation of engulfed material is primarily mediated by lysosomal enzymes that function optimally within a narrow range of acidic pH values. RPE cells are responsible for daily degradation of photoreceptor outer segments and are thus particularly susceptible to perturbations in lysosomal pH. The authors hypothesized that elevated lysosomal pH levels could slow enzyme activity and encourage accumulation of partially digested material. Consequently, treatment to lower perturbed lysosomal pH levels may enhance degradative activity. Methods A high-throughput screening assay was developed to quantify the lysosomal pH of fresh mouse and cultured ARPE-19 cells. The effect of lysosomal pH on outer segment clearance was determined. Results Lysosomal pH is elevated in RPE cells from ABCA4 knockout mice and in cultured human ARPE-19 cells exposed to N-retinylidene-N-retinylethanolamine (A2E), tamoxifen, or chloroquine. The lysosomal pH of fresh RPE cells from ABCA4−/− mice and of chemically compromised RPE cells was reacidified by elevating intracellular cAMP directly. Compromised lysosomal pH was also restored by stimulating A2A adenosine or β-adrenergic receptors, consistent with Gs-protein coupling of these receptors. Restoring lysosomal pH with these treatments enhanced photoreceptor outer segment clearance, demonstrating functional relevance consistent with an enhancement of degradative enzyme activity. Conclusions Elevation of lysosomal pH in RPE cells interferes with the degradation of outer segments and may contribute to the pathologies associated with A2E. Pharmacologic elevation of cAMP can restore an acid pH and improve degradative function. PMID:18235027

  13. Reprogramming Progeny Cells of Embryonic RPE to Produce Photoreceptors: Development of Advanced Photoreceptor Traits under the Induction of neuroD

    PubMed Central

    Liang, Lina; Yan, Run-Tao; Li, Xiumei; Chimento, Melissa; Wang, Shu-Zhen

    2008-01-01

    PURPOSE In examining the prospect of producing functional photoreceptors by reprogramming the differentiation of RPE progeny cells, this study was conducted to investigate whether reprogrammed cells can develop highly specialized ultrastructural and physiological traits that characterize retinal photoreceptors. METHODS Cultured chick RPE cells were reprogrammed to differentiate along the photoreceptor pathway by ectopic expression of neuroD. Cellular ultrastructure was examined with electron microscopy. Cellular physiology was studied by monitoring cellular free calcium (Ca2+) levels in dark-adapted cells in response to light and in light-bleached cells in response to 9-cis-retinal. RESULTS Reprogrammed cells were found to localize red opsin protein appropriately to the apex. These cells developed inner segments rich in mitochondria, and while in culture, some formed rudimentary outer segments, analogous to those of developing photoreceptors in the retina. In response to light, reprogrammed cells reduced their Ca2+ levels, as observed with developing retinal photoreceptors in culture. Further, on exposure to 9-cis-retinal, the light-bleached, reprogrammed cells increased their Ca2+ levels, reminiscent of visual cycle recovery. CONCLUSIONS These results indicate the potential of reprogrammed cells to develop advanced ultrastructural and physiological traits of photoreceptors and point to reprogramming progeny cells of embryonic RPE as a possible alternative in producing developing photoreceptors. PMID:18469196

  14. In vitro measurements of oxygen consumption rates in hTERT-RPE cells exposed to low levels of red light

    NASA Astrophysics Data System (ADS)

    Wigle, Jeffrey C.; Castellanos, Cherry C.

    2016-03-01

    Exposure to 2.88 J/cm2 of red light induces an adaptive response against a lethal pulse of 2.0 μm laser radiation in hTERT-RPE cells in vitro, but not in a knockdown mutant for vascular endothelial growth factor c (VEGF-C). The generally accepted initiation sequence for photobiomodulation is that absorption of red light by cytochome c oxidase (CCOX) of the electron transport chain increases the binding affinity of CCOX for O2 vs. nitric oxide (NO). This results in displacement of NO by O2 in the active site of CCOX, thereby increasing cellular respiration and intracellular ATP. We've previously reported that red-light exposure induces a small, but consistently reproducible, increase in NO levels in these cells. But the relative importance of NO and oxidative phosphorylation is unclear because little is known about the relative contributions of NO and ATP to the response. However, if NO dissociation from CCOX actually increases oxidative phosphorylation, one should see a corresponding increase in oxygen consumption. A Seahorse Extracellular Flux Analyzer was used to measure oxygen consumption rates (OCR) in normal and mutant cells as a proxy for oxidative phosphorylation. Both basal respiration and maximum respiration rates in normal cells are significantly higher than in the mutant. The normal cells have a significant amount of "excess capacity," whereas the VEGF-C(KD) have little or none. The OCR in exposed normal cells is lower than in unexposed cells when measured immediately after exposure. The exposures used for these experiments had no effect on the OCR in mutant cells.

  15. Inhibition of the oxidative stress-induced miR-23a protects the human retinal pigment epithelium (RPE) cells from apoptosis through the upregulation of glutaminase and glutamine uptake.

    PubMed

    Li, Dan-Dan; Zhong, Bin-Wu; Zhang, Hai-Xia; Zhou, Hong-Yan; Luo, Jie; Liu, Yang; Xu, Gui-Chun; Luan, Chun-Sheng; Fang, Jun

    2016-10-01

    The degeneration of retinal pigment epithelium (RPE) cells in the sub retinal pigment epithelial space and choroid is an initial pathological characteristic for the age-related macular degeneration which is the leading cause of severe vision loss in old people. Moreover, oxidative stress is implicated as a major inducer of RPE cell death. Here, we assessed the correlation between the H2O2-induced RPE cell death and glutamine metabolism. We found under low glutamine supply (20 %), the ARPE-19 cells were more susceptive to H2O2-induced apoptosis. Moreover, the glutamine uptake and the glutaminase (GLS) were suppressed by H2O2 treatments. Moreover, we observed miR-23a was upregulated by H2O2 treatments and overexpression of miR-23a significantly sensitized ARPE-19 cells to H2O2. Importantly, Western blotting and luciferase assay demonstrated GLS1 is a direct target of miR-23a in RPE cells. Inhibition of the H2O2-induced miR-23a by antagomiR protected the RPE cells from the oxidative stress-induced cell death. In addition, recovery of GLS1 expression in miR-23a overexpressed RPE cells rescued the H2O2-induced cell death. This study illustrated a mechanism for the protection of the oxidative-induced RPE cell death through the recovery of glutamine metabolism by inhibition of miR-23a, contributing to the discovery of novel targets and the developments of therapeutic strategies for the prevention of RPE cells from oxidative stress.

  16. Inhibition of the oxidative stress-induced miR-23a protects the human retinal pigment epithelium (RPE) cells from apoptosis through the upregulation of glutaminase and glutamine uptake.

    PubMed

    Li, Dan-Dan; Zhong, Bin-Wu; Zhang, Hai-Xia; Zhou, Hong-Yan; Luo, Jie; Liu, Yang; Xu, Gui-Chun; Luan, Chun-Sheng; Fang, Jun

    2016-10-01

    The degeneration of retinal pigment epithelium (RPE) cells in the sub retinal pigment epithelial space and choroid is an initial pathological characteristic for the age-related macular degeneration which is the leading cause of severe vision loss in old people. Moreover, oxidative stress is implicated as a major inducer of RPE cell death. Here, we assessed the correlation between the H2O2-induced RPE cell death and glutamine metabolism. We found under low glutamine supply (20 %), the ARPE-19 cells were more susceptive to H2O2-induced apoptosis. Moreover, the glutamine uptake and the glutaminase (GLS) were suppressed by H2O2 treatments. Moreover, we observed miR-23a was upregulated by H2O2 treatments and overexpression of miR-23a significantly sensitized ARPE-19 cells to H2O2. Importantly, Western blotting and luciferase assay demonstrated GLS1 is a direct target of miR-23a in RPE cells. Inhibition of the H2O2-induced miR-23a by antagomiR protected the RPE cells from the oxidative stress-induced cell death. In addition, recovery of GLS1 expression in miR-23a overexpressed RPE cells rescued the H2O2-induced cell death. This study illustrated a mechanism for the protection of the oxidative-induced RPE cell death through the recovery of glutamine metabolism by inhibition of miR-23a, contributing to the discovery of novel targets and the developments of therapeutic strategies for the prevention of RPE cells from oxidative stress. PMID:27411920

  17. Analysis of RPE morphometry in human eyes

    PubMed Central

    Bhatia, Shagun K.; Rashid, Alia; Chrenek, Micah A.; Zhang, Qing; Bruce, Beau B.; Klein, Mitchel; Boatright, Jeffrey H.; Jiang, Yi; Grossniklaus, Hans E.

    2016-01-01

    Purpose To describe the RPE morphometry of healthy human eyes regarding age and topographic location using modern computational methods with high accuracy and objectivity. We tested whether there were regional and age-related differences in RPE cell area and shape. Methods Human cadaver donor eyes of varying ages were dissected, and the RPE flatmounts were immunostained for F-actin with AF635-phalloidin, nuclei stained with propidium iodide, and imaged with confocal microscopy. Image analysis was performed using ImageJ (NIH) and CellProfiler software. Quantitative parameters, including cell density, cell area, polygonality of cells, number of neighboring cells, and measures of cell shape, were obtained from these analyses to characterize individual and groups of RPE cells. Measurements were taken from selected areas spanning the length of the temporal retina through the macula and the mid-periphery to the far periphery. Results Nineteen eyes from 14 Caucasian donors of varying ages ranging from 29 to 80 years were used. Along a horizontal nasal to temporal meridian, there were differences in several cell shape and size characteristics. Generally, the cell area and shape was relatively constant and regular except in the far periphery. In the outer third of the retina, the cell area and shape differed from the inner two-thirds statistically significantly. In the macula and the far periphery, an overall decreasing trend in RPE cell density, percent hexagonal cells, and form factor was observed with increasing age. We also found a trend toward increasing cell area and eccentricity with age in the macula and the far periphery. When individuals were divided into two age groups, <60 years and ≥60 years, there was a higher cell density, lower cell area, lower eccentricity, and higher form factor in the younger group in the macula and the far periphery (p<0.05 for all measurements). No statistically significant differences in RPE morphometry between age groups were found

  18. A Discrete Cell Migration Model

    SciTech Connect

    Nutaro, James J; Kruse, Kara L; Ward, Richard C; O'Quinn, Elizabeth; Woerner, Matthew M; Beckerman, Barbara G

    2007-01-01

    Migration of vascular smooth muscle cells is a fundamental process in the development of intimal hyperplasia, a precursor to development of cardiovascular disease and a potential response to injury of an arterial wall. Boyden chamber experiments are used to quantify the motion of cell populations in response to a chemoattractant gradient (i.e., cell chemotaxis). We are developing a mathematical model of cell migration within the Boyden chamber, while simultaneously conducting experiments to obtain parameter values for the migration process. In the future, the model and parameters will be used as building blocks for a detailed model of the process that causes intimal hyperplasia. The cell migration model presented in this paper is based on the notion of a cell as a moving sensor that responds to an evolving chemoattractant gradient. We compare the results of our three-dimensional hybrid model with results from a one-dimensional continuum model. Some preliminary experimental data that is being used to refine the model is also presented.

  19. Predicting the Pathogenicity of RPE65 Mutations

    PubMed Central

    Philp, A.R.; Jin, M.; Li, S.; Schindler, E.I.; Iannaccone, A.; Lam, B.L.; Weleber, R.G.; Fishman, G.A.; Jacobson, S.G.; Mullins, R.F.; Travis, G.H.; Stone, E.M.

    2009-01-01

    To assist in distinguishing disease-causing mutations from non-pathogenic polymorphisms, we developed an objective algorithm to calculate an “estimate of pathogenic probability” (EPP) based on the prevalence of a specific variation, its segregation within families, and its predicted effects on protein structure. Eleven missense variations in the RPE65 gene were evaluated in patients with Leber congenital amaurosis (LCA) using the EPP algorithm. The accuracy of the EPP algorithm was evaluated using a cell-culture assay of RPE65-isomerase activity The variations were engineered into plasmids containing a human RPE65 cDNA and the retinoid isomerase activity of each variant was determined in cultured cells. The EPP algorithm predicted eight substitution mutations to be disease-causing variants. The isomerase catalytic activities of these RPE65 variants were all less than 6% of wild-type. In contrast, the EPP algorithm predicted the other three substitutions to be non-disease-causing, with isomerase activities of 68%, 127% and 110% of wild-type, respectively. We observed complete concordance between the predicted pathogenicities of missense variations in the RPE65 gene and retinoid isomerase activities measured in a functional assay. These results suggest that the EPP algorithm may be useful to evaluate the pathogenicity of missense variations in other disease genes where functional assays are not available. PMID:19431183

  20. Zebrafish germ cells: motility and guided migration.

    PubMed

    Paksa, Azadeh; Raz, Erez

    2015-10-01

    In the course of embryonic development, the process of cell migration is critical for establishment of the embryonic body plan, for morphogenesis and for organ function. Investigating the molecular mechanisms underlying cell migration is thus crucial for understanding developmental processes and clinical conditions resulting from abnormal cell migration such as cancer metastasis. The long-range migration of primordial germ cells toward the region at which the gonad develops occurs in embryos of various species and thus constitutes a useful in vivo model for single-cell migration. Recent studies employing zebrafish embryos have greatly contributed to the understanding of the mechanisms facilitating the migration of these cells en route to their target.

  1. Endothelial cells enhance migration of meniscus cells

    PubMed Central

    Yuan, Xiaoning; Eng, George M.; Arkonac, Derya E.; Chao, Pen-hsiu Grace; Vunjak-Novakovic, Gordana

    2014-01-01

    Objective To study the interactions between vascular endothelial cells and meniscal fibrochondrocytes from the inner avascular and outer vascular regions of the meniscus, and identify angiogenic factors that enhance cell migration and integrative repair. Methods Bovine meniscal fibrochondrocytes (bMFCs) from the inner and outer regions of meniscus were cultured for seven days with and without human umbilical vein endothelial cells (HUVECs) in a micropatterned three-dimensional hydrogel system for cell migration. Angiogenic factors secreted by HUVECs were probed for their role in paracrine mechanisms governing bMFC migration, and applied to a full-thickness defect model of meniscal repair in explants from the inner and outer regions over four weeks. Results Endothelial cells enhanced migration of inner and outer bMFCs in the micropatterned system via endothelin-1 (ET-1) signaling. Supplementation of ET-1 significantly enhanced integration strength of full-thickness defects in inner and outer explants, and cell migration at the macro-scale, compared to controls without ET-1 treatment. Conclusion We report for the first time that bMFCs from both the avascular and vascular regions respond to the presence of endothelial cells with increased migration. Paracrine signaling by endothelial cells regulates the bMFCs differentially by region, but we identify ET-1 as an angiogenic factor that stimulates migration of inner and outer cells at the micro-scale, and integrative repair of inner and outer explants at the macro-scale. These findings reveal the regional interactions between vasculature and MFCs, and suggest ET-1 as a potential new treatment modality for avascular meniscal injuries, in order to prevent the development of osteoarthritis. PMID:25307081

  2. TGF-β2 secretion from RPE decreases with polarization and becomes apically oriented.

    PubMed

    Hirsch, Louis; Nazari, Hossein; Sreekumar, Parameswaran G; Kannan, Ram; Dustin, Laurie; Zhu, Danhong; Barron, Ernesto; Hinton, David R

    2015-02-01

    Retinal pigmented epithelium (RPE) secretes transforming growth factor beta 1 and 2 (TGF-β1 and -β2) cytokines involved in fibrosis, immune privilege, and proliferative vitreoretinopathy (PVR). Since RPE cell polarity may be altered in various disease conditions including PVR and age-related macular degeneration, we determined levels of TGF-β from polarized human RPE (hRPE) and human stem cell derived RPE (hESC-RPE) as compared to nonpolarized cells. TGF-β2 was the predominant isoform in all cell culture conditions. Nonpolarized cells secreted significantly more TGF-β2 supporting the contention that loss of polarity of RPE in PVR leads to rise of intravitreal TGF-β2. Active TGF-β2, secreted mainly from apical side of polarized RPE, represented 6-10% of total TGF-β2. In conclusion, polarity is an important determinant of TGF-β2 secretion in RPE. Low levels of apically secreted active TGF-β2 may play a role in the normal physiology of the subretinal space. Comparable secretion of TGF-β from polarized hESC-RPE and hRPE supports the potential for hESC-RPE in RPE replacement therapies.

  3. A novel RPE65 hypomorph expands the clinical phenotype of RPE65 mutations. A comprehensive clinical and biochemical functional study

    PubMed Central

    Lorenz, Birgit; Poliakov, Eugenia; Schambeck, Maria; Friedburg, Christoph; Preising, Markus N.; Redmond, T. Michael

    2009-01-01

    Purpose Later onset and progression of retinal dystrophy occur with some RPE65 missense mutations. We correlate the functional consequences of the novel P25L RPE65 mutation with its early childhood phenotype and compare it with other pathogenic missense mutations. Methods In addition to typical clinical tests, fundus autofluorescence (FAF), optical coherence tomography (OCT), and 2-color-threshold perimetry (2CTP) were measured. RPE65 mutations were screened by SSCP and direct sequencing. Isomerase activity of mutant RPE65 was assayed in 293F cells and quantified by HPLC analysis of retinoids. Results A very mild phenotype was detected in a now 7-y old boy homozygous for the P25L mutation in RPE65. Though abnormal dark adaptation was noticed early, best corrected visual acuity was 20/20 at age 5-y and 20/30 at age 7-y. Nystagmus was absent. Cone electroretinogram (ERG) was measurable, rod ERG severely reduced, and FAF very low. 2CTP detected mainly cone-mediated answers under scotopic conditions, light-adapted cone answers were about 1.5 log units below normal. High resolution spectral domain OCT revealed morphological changes. Isomerase activity in 293F cells transfected with RPE65/P25L was reduced to 7.7% of wildtype RPE65-transfected cells, while RPE65/L22P-transfected cells had 13.5%. Conclusions The mild clinical phenotype observed is consistent with the residual activity of a severely hypomorphic mutant RPE65. Reduction to < 10% of wildtype RPE65 activity by homozygous P25L correlates with almost complete rod function loss and cone amplitude reduction. We conclude that functional survival of cones is possible in patients with residual RPE65 isomerase activity. This patient should profit most from gene therapy. PMID:18599565

  4. Approaches for detecting lysosomal alkalinization and impaired degradation in fresh and cultured RPE cells: evidence for a role in retinal degenerations

    PubMed Central

    Guha, Sonia; Coffey, Erin E.; Lu, Wennan; Lim, Jason C.; Beckel, Jonathan M.; Laties, Alan M.; Boesze-Battaglia, Kathleen; Mitchell, Claire H.

    2014-01-01

    Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TFEB and of the vesicular proton pump vHATPase in the RPE/choroid of mice. An elevated lysosomal pH with upregulation of TFEB and vHATPase resembles the pathology in fibroblasts of patients with mutant presenilin 1 (PS1), suggesting a common link between age-related macular degeneration (AMD) and Alzheimer’s disease. While the absolute rise in pH is often small, elevations of only a few tenths of a pH unit can have a major impact on both lysosomal function and the accumulation of waste over decades. Accurate measurement of lysosomal pH can be complex, and imprecise measurements have clouded the field. Protocols to optimize pH measurement from fresh and cultured cells are discussed, and indirect measurements to confirm changes in lysosomal pH and degradative capacity are addressed. The ability of reacidifying treatments to restore degradative function confirms the central role of lysosomal pH in these functions and identifies potential approaches to treat diseases of accumulation like AMD and Alzheimer’s disease. In summary, various

  5. Reprogramming RPE to differentiate towards retinal neurons with Sox2

    PubMed Central

    Ma, Wenxin; Yan, Run-Tao; Li, Xiumei; Wang, Shu-Zhen

    2009-01-01

    Guiding non-neural, retinal pigment epithelium (RPE) to produce retinal neurons may offer a source of developing neurons for cell-replacement. Sox2 plays important roles in maintaining neural progenitor/stem cell properties and in converting fibroblasts into pluripotent stem cells. This study tests the possibility of using Sox2 to reprogram RPE to differentiate towards retinal neurons in vivo and in vitro. Expression of Sox2 in the chick retina was detected in progenitor cells, in cells at a discrete location in the layers of amacrine and ganglion cells, and in Műller glia. Overexpression of Sox2 in the developing eye resulted in hypo-pigmentation of the RPE. In the affected regions, expression of retinal ganglion cell markers became apparent in the RPE layer. In RPE cell culture, Sox2 promoted the expression of retinal ganglion and amacrine markers and suppressed the expression of genes associate with RPE properties. Mechanistic investigation using the developing retina revealed a co-expression of Sox2 and bFGF, a growth factor commonly used in stem cell culture and capable of inducing RPE-to-retina transdifferentiation (or reprogramming) during early development. Similar patterns of changes in Sox2 expression and in bFGF expression were observed in atrophic retina and in injured retina. In RPE cell culture, Sox2 and bFGF mutually enhanced one another's expression. Up-regulation of bFGF expression by Sox2 also occurred in the retina. These results suggest that Sox2 can initiate a reprogramming of RPE cells to differentiate towards retinal neurons and may engage bFGF during the process. PMID:19489100

  6. Lycopene inhibits PDGF-BB-induced retinal pigment epithelial cell migration by suppression of PI3K/Akt and MAPK pathways

    SciTech Connect

    Chan, Chi-Ming; Fang, Jia-You; Lin, Hsin-Huang; Yang, Chi-Yea; Hung, Chi-Feng

    2009-10-09

    Retinal pigment epithelial (RPE) cells play a dominant role in the development of proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal reattachment surgery. Several studies have shown that platelet-derived growth factor (PDGF) exhibits chemotaxis and proliferation effects on RPE cells in PVR. In this study, the inhibitory effect of lycopene on PDGF-BB-induced ARPE19 cell migration is examined. In electric cell-substrate impedance sensing (ECIS) and Transwell migration assays, significant suppression of PDGF-BB-induced ARPE19 cell migration by lycopene is observed. Cell viability assays show no cytotoxicity of lycopene on RPE cells. Lycopene shows no effect on ARPE19 cell adhesion and is found to inhibit PDGF-BB-induced tyrosine phosphorylation and the underlying signaling pathways of PI3K, Akt, ERK and p38 activation. However, PDGF-BB and lycopene show no effects on JNK activation. Taken together, our results demonstrate that lycopene inhibits PDGF-BB-induced ARPE19 cell migration through inhibition of PI3K/Akt, ERK and p38 activation.

  7. Characterization of Collective Cell Migration Dynamics

    NASA Astrophysics Data System (ADS)

    Lee, Rachel; Yue, Haicen; Rappel, Wouter-Jan; Losert, Wolfgang

    2015-03-01

    During cancer progression, tumor cells invade the surrounding tissue and migrate throughout the body, forming clinically dangerous secondary tumors. This metastatic process begins when cells leave the primary tumor, either as individual cells or collectively migrating groups. Here we present data on the migration dynamics of epithelial sheets composed of many cells. Using quantitative image analysis techniques, we are able to extract motion information from time-lapse images of cell lines with varying malignancy. Adapting metrics originally used to study fluid flows we are able to characterize the migration dynamics of these cell lines. By describing the migration dynamics in great detail, we are able to make a clear comparison of our results to a simulation of collective cell migration. Specifically, we explore whether leader cells are required to describe our expanding sheets of cells and whether the answer depends on individual cell activity.

  8. Schwann cells promote endothelial cell migration

    PubMed Central

    Ramos, Tiago; Ahmed, Maqsood; Wieringa, Paul; Moroni, Lorenzo

    2015-01-01

    Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration. PMID:26491999

  9. Factors controlling cardiac neural crest cell migration

    PubMed Central

    Hutson, Mary R

    2010-01-01

    Cardiac neural crest cells originate as part of the postotic caudal rhombencephalic neural crest stream. Ectomesenchymal cells in this stream migrate to the circumpharyngeal ridge and then into the caudal pharyngeal arches where they condense to form first a sheath and then the smooth muscle tunics of the persisting pharyngeal arch arteries. A subset of the cells continues migrating into the cardiac outflow tract where they will condense to form the aorticopulmonary septum. Cell signaling, extracellular matrix and cell-cell contacts are all critical for the initial migration, pauses, continued migration and condensation of these cells. This Review elucidates what is currently known about these factors. PMID:20890117

  10. Transplantation stimulates interstitial cell migration in hydra

    SciTech Connect

    Fujisawa, T.; David, C.N.; Bosch, T.C. )

    1990-04-01

    Migration of interstitial cells and nerve cell precursors was analyzed in Hydra magnipapillata and Hydra vulgaris (formerly Hydra attenuata). Axial grafts were made between ({sup 3}H)thymidine-labeled donor and unlabeled host tissue. Migration of labeled cells into the unlabeled half was followed for 4 days. The results indicate that the rate of migration was initially high and then slowed on Days 2-4. Regrafting fresh donor tissue on Days 2-4 maintained high levels of migration. Thus, migration appears to be stimulated by the grafting procedure itself.

  11. Methodologies for analysis of patterning in the mouse RPE sheet

    PubMed Central

    Boatright, Jeffrey H.; Dalal, Nupur; Chrenek, Micah A.; Gardner, Christopher; Ziesel, Alison; Jiang, Yi; Grossniklaus, Hans E.

    2015-01-01

    Purpose Our goal was to optimize procedures for assessing shapes, sizes, and other quantitative metrics of retinal pigment epithelium (RPE) cells and contact- and noncontact-mediated cell-to-cell interactions across a large series of flatmount RPE images. Methods The two principal methodological advances of this study were optimization of a mouse RPE flatmount preparation and refinement of open-access software to rapidly analyze large numbers of flatmount images. Mouse eyes were harvested, and extra-orbital fat and muscles were removed. Eyes were fixed for 10 min, and dissected by puncturing the cornea with a sharp needle or a stab knife. Four radial cuts were made with iridectomy scissors from the puncture to near the optic nerve head. The lens, iris, and the neural retina were removed, leaving the RPE sheet exposed. The dissection and outcomes were monitored and evaluated by video recording. The RPE sheet was imaged under fluorescence confocal microscopy after staining for ZO-1 to identify RPE cell boundaries. Photoshop, Java, Perl, and Matlab scripts, as well as CellProfiler, were used to quantify selected parameters. Data were exported into Excel spreadsheets for further analysis. Results A simplified dissection procedure afforded a consistent source of images that could be processed by computer. The dissection and flatmounting techniques were illustrated in a video recording. Almost all of the sheet could be routinely imaged, and substantial fractions of the RPE sheet (usually 20–50% of the sheet) could be analyzed. Several common technical problems were noted and workarounds developed. The software-based analysis merged 25 to 36 images into one and adjusted settings to record an image suitable for large-scale identification of cell-to-cell boundaries, and then obtained quantitative descriptors of the shape of each cell, its neighbors, and interactions beyond direct cell–cell contact in the sheet. To validate the software, human- and computer

  12. Rho GTPase signalling in cell migration

    PubMed Central

    Ridley, Anne J

    2015-01-01

    Cells migrate in multiple different ways depending on their environment, which includes the extracellular matrix composition, interactions with other cells, and chemical stimuli. For all types of cell migration, Rho GTPases play a central role, although the relative contribution of each Rho GTPase depends on the environment and cell type. Here, I review recent advances in our understanding of how Rho GTPases contribute to different types of migration, comparing lamellipodium-driven versus bleb-driven migration modes. I also describe how cells migrate across the endothelium. In addition to Rho, Rac and Cdc42, which are well known to regulate migration, I discuss the roles of other less-well characterized members of the Rho family. PMID:26363959

  13. Multiscale Cues Drive Collective Cell Migration

    NASA Astrophysics Data System (ADS)

    Nam, Ki-Hwan; Kim, Peter; Wood, David K.; Kwon, Sunghoon; Provenzano, Paolo P.; Kim, Deok-Ho

    2016-07-01

    To investigate complex biophysical relationships driving directed cell migration, we developed a biomimetic platform that allows perturbation of microscale geometric constraints with concomitant nanoscale contact guidance architectures. This permits us to elucidate the influence, and parse out the relative contribution, of multiscale features, and define how these physical inputs are jointly processed with oncogenic signaling. We demonstrate that collective cell migration is profoundly enhanced by the addition of contract guidance cues when not otherwise constrained. However, while nanoscale cues promoted migration in all cases, microscale directed migration cues are dominant as the geometric constraint narrows, a behavior that is well explained by stochastic diffusion anisotropy modeling. Further, oncogene activation (i.e. mutant PIK3CA) resulted in profoundly increased migration where extracellular multiscale directed migration cues and intrinsic signaling synergistically conspire to greatly outperform normal cells or any extracellular guidance cues in isolation.

  14. Multiscale Cues Drive Collective Cell Migration

    PubMed Central

    Nam, Ki-Hwan; Kim, Peter; Wood, David K.; Kwon, Sunghoon; Provenzano, Paolo P.; Kim, Deok-Ho

    2016-01-01

    To investigate complex biophysical relationships driving directed cell migration, we developed a biomimetic platform that allows perturbation of microscale geometric constraints with concomitant nanoscale contact guidance architectures. This permits us to elucidate the influence, and parse out the relative contribution, of multiscale features, and define how these physical inputs are jointly processed with oncogenic signaling. We demonstrate that collective cell migration is profoundly enhanced by the addition of contract guidance cues when not otherwise constrained. However, while nanoscale cues promoted migration in all cases, microscale directed migration cues are dominant as the geometric constraint narrows, a behavior that is well explained by stochastic diffusion anisotropy modeling. Further, oncogene activation (i.e. mutant PIK3CA) resulted in profoundly increased migration where extracellular multiscale directed migration cues and intrinsic signaling synergistically conspire to greatly outperform normal cells or any extracellular guidance cues in isolation. PMID:27460294

  15. Cyclin D1 functions in cell migration.

    PubMed

    Li, Zhiping; Wang, Chenguang; Prendergast, George C; Pestell, Richard G

    2006-11-01

    Cell migration is essential for developmental morphogenesis, tissue repair, and tumor metastasis. A recent study reveals that cyclin D1 acts to promote cell migration by inhibiting Rho/ROCK signaling and expression of thrombospondin-1 (TSP-1), an extracellular matrix protein that regulates cell migration in many settings including cancer. Given the frequent overexpression of cyclin D1 in cancer cells, due to its upregulation by Ras, Rho, Src, and other genes that drive malignant development, the new findings suggest that cyclin D1 may have a central role in mediating invasion and metastasis of cancer cells by controlling Rho/ROCK signaling and matrix deposition of TSP-1.

  16. Centrosome Positioning in 1D Cell Migration

    NASA Astrophysics Data System (ADS)

    Adlerz, Katrina; Aranda-Espinoza, Helim

    During cell migration, the positioning of the centrosome and nucleus define a cell's polarity. For a cell migrating on a two-dimensional substrate the centrosome is positioned in front of the nucleus. Under one-dimensional confinement, however, the centrosome is positioned behind the nucleus in 60% of cells. It is known that the centrosome is positioned by CDC42 and dynein for cells moving on a 2D substrate in a wound-healing assay. It is currently unknown, however, if this is also true for cells moving under 1D confinement, where the centrosome position is often reversed. Therefore, centrosome positioning was studied in cells migrating under 1D confinement, which mimics cells migrating through 3D matrices. 3 to 5 μm fibronectin lines were stamped onto a glass substrate and cells with fluorescently labeled nuclei and centrosomes migrated on the lines. Our results show that when a cell changes directions the centrosome position is maintained. That is, when the centrosome is between the nucleus and the cell's trailing edge and the cell changes direction, the centrosome will be translocated across the nucleus to the back of the cell again. A dynein inhibitor did have an influence on centrosome positioning in 1D migration and change of directions.

  17. Nitric oxide measurements in hTERT-RPE cells and subcellular fractions exposed to low levels of red light

    NASA Astrophysics Data System (ADS)

    Wigle, Jeffrey C.; Castellanos, Cherry C.; Denton, Michael L.; Holwitt, Eric A.

    2014-02-01

    Cells in a tissue culture model for laser eye injury exhibit increased resistance to a lethal pulse of 2.0-μm laser radiation if the cells are first exposed to 2.88 J/cm2 of red light 24 hr prior to the lethal laser exposure. Changes in expression of various genes associated with apoptosis have been observed, but the biochemical link between light absorption and gene expression remains unknown. Cytochome c oxidase (CCOX), in the electron transport chain, is the currentlyhypothesized absorber. Absorption of the red light by CCOX is thought to facilitate displacement of nitric oxide (NO) by O2 in the active site, increasing cellular respiration and intracellular ATP. However, NO is also an important regulator and mediator of numerous physiological processes in a variety of cell and tissue types that is synthesized from l-arginine by NO synthases. In an effort to determine the relative NO contributions from these competing pathways, we measured NO levels in whole cells and subcellular fractions, with and without exposure to red light, using DAF-FM, a fluorescent dye that stoichiometrically reacts with NO. Red light induced a small, but consistently reproducible, increase in fluorescence intensity in whole cells and some subcellular fractions. Whole cells exhibited the highest overall fluorescence intensity followed by (in order) cytosolic proteins, microsomes, then nuclei and mitochondria.

  18. Epithelial phenotype and the RPE: Is the answer blowing in the Wnt?

    PubMed Central

    Burke, Janice M.

    2008-01-01

    Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell–cell adhesion and melanization are linked by a common signaling pathway: the Wnt/β-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis. Also discussed is the effect of aging on RPE phenotype. Preliminary evidence is provided to suggest that light-induced sub-lethal oxidative stress to cultured ARPE-19 cells impairs organelle motility. Organelle translocation, which is mediated by stress-susceptible cytoskeletal scaffolds, is an essential process in cell phenotype development and retention. The observation of impaired organelle motility therefore raises the possibility that low levels of stress, which are believed to accompany RPE aging, may produce subtle disruptions of cell phenotype. Over time these would be expected to diminish the support functions performed by the RPE on behalf of

  19. Migration of Airway Smooth Muscle Cells

    PubMed Central

    Gerthoffer, William T.

    2008-01-01

    Migration of smooth muscle cells is a process fundamental to development of hollow organs, including blood vessels and the airways. Migration is also thought to be part of the response to tissue injury. It has also been suggested to contribute to airways remodeling triggered by chronic inflammation. In both nonmuscle and smooth muscle cells numerous external signaling molecules and internal signal transduction pathways contribute to cell migration. The review includes evidence for the functional significance of airway smooth muscle migration, a summary of promigratory and antimigratory agents, and summaries of important signaling pathways mediating migration. Important signaling pathways and effector proteins described include small G proteins, phosphatidylinositol 3-kinases (PI3-K), Rho activated protein kinase (ROCK), p21-activated protein kinases (PAK), Src family tyrosine kinases, and mitogen-activated protein kinases (MAPK). These signaling modules control multiple critical effector proteins including actin nucleating, capping and severing proteins, myosin motors, and proteins that remodel microtubules. Actin filament remodeling, focal contact remodeling and propulsive force of molecular motors are all coordinated to move cells along gradients of chemical cues, matrix adhesiveness, or matrix stiffness. Airway smooth muscle cell migration can be modulated in vitro by drugs commonly used in pulmonary medicine including β-adrenergic agonists and corticosteroids. Future studies of airway smooth muscle cell migration may uncover novel targets for drugs aimed at modifying airway remodeling. PMID:18094091

  20. Emergence of oligarchy in collective cell migration

    NASA Astrophysics Data System (ADS)

    Schumacher, Linus; Maini, Philip; Baker, Ruth

    Identifying the principles of collective cell migration has the potential to help prevent birth defects, improve regenerative therapies and develop model systems for cancer metastasis. In collaboration with experimental biologists, we use computational simulations of a hybrid model, comprising individual-based stochastic cell movement coupled to a reaction-diffusion equation for a chemoattractant, to explore the role of cell specialisation in the guidance of collective cell migration. In the neural crest, an important migratory cell population in vertebrate embryo development, we present evidence that just a few cells are guiding group migration in a cell-induced chemoattractant gradient that determines the switch between ``leader'' and ``follower'' behaviour in individual cells. This leads us to more generally consider under what conditions cell specialisation might become advantageous for collective migration. Alternatively, individual cell responses to locally different microenvironmental conditions could create the (artefactual) appearance of heterogeneity in a population of otherwise identical cellular agents. We explore these questions using a self-propelled particle model as a minimal description for collective cell migration in two and three dimensions.

  1. Entropy measures of collective cell migration

    NASA Astrophysics Data System (ADS)

    Whitby, Ariadne; Parrinello, Simona; Faisal, Aldo

    2015-03-01

    Collective cell migration is a critical process during tissue formation and repair. To this end there is a need to develop tools to quantitatively measure the dynamics of collective cell migration obtained from microscopy data. Drawing on statistical physics we use entropy of velocity fields derived from dense optic flow to quantitatively measure collective migration. Using peripheral nerve repair after injury as experimental system, we study how Schwann cells, guided by fibroblasts, migrate in cord-like structures across the cut, paving a highway for neurons. This process of emergence of organised behaviour is key for successful repair, yet the emergence of leader cells and transition from a random to ordered state is not understood. We find fibroblasts induce correlated directionality in migrating Schwann cells as measured by a decrease in the entropy of motion vector. We show our method is robust with respect to image resolution in time and space, giving a principled assessment of how various molecular mechanisms affect macroscopic features of collective cell migration. Finally, the generality of our method allows us to process both simulated cell movement and microscopic data, enabling principled fitting and comparison of in silico to in vitro. ICCS, Imperial College London & MRC Clinical Sciences Centre.

  2. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  3. Chemistry and biology of the compounds that modulate cell migration.

    PubMed

    Tashiro, Etsu; Imoto, Masaya

    2016-03-01

    Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. Extensive studies have attempted to reveal the molecular mechanisms behind cell migration; however, they remain largely unclear. Bioactive compounds that modulate cell migration show promise as not only extremely powerful tools for studying the mechanisms behind cell migration but also as drug seeds for chemotherapy against tumor metastasis. Therefore, we have screened cell migration inhibitors and analyzed their mechanisms for the inhibition of cell migration. In this mini-review, we introduce our chemical and biological studies of three cell migration inhibitors: moverastin, UTKO1, and BU-4664L.

  4. Cell migration in the postnatal subventricular zone.

    PubMed

    Menezes, J R L; Marins, M; Alves, J A J; Froes, M M; Hedin-Pereira, C

    2002-12-01

    New neurons are constantly added to the olfactory bulb of rodents from birth to adulthood. This accretion is not only dependent on sustained neurogenesis, but also on the migration of neuroblasts and immature neurons from the cortical and striatal subventricular zone (SVZ) to the olfactory bulb. Migration along this long tangential pathway, known as the rostral migratory stream (RMS), is in many ways opposite to the classical radial migration of immature neurons: it is faster, spans a longer distance, does not require radial glial guidance, and is not limited to postmitotic neurons. In recent years many molecules have been found to be expressed specifically in this pathway and to directly affect this migration. Soluble factors with inhibitory, attractive and inductive roles in migration have been described, as well as molecules mediating cell-to-cell and cell-substrate interactions. However, it is still unclear how the various molecules and cells interact to account for the special migratory behavior in the RMS. Here we will propose some candidate mechanisms for roles in initiating and stopping SVZ/RMS migration.

  5. Visualizing T Cell Migration in situ

    PubMed Central

    Benechet, Alexandre P.; Menon, Manisha; Khanna, Kamal M.

    2014-01-01

    Mounting a protective immune response is critically dependent on the orchestrated movement of cells within lymphoid tissues. The structure of secondary lymphoid organs regulates immune responses by promoting optimal cell–cell and cell–extracellular matrix interactions. Naïve T cells are initially activated by antigen presenting cells in secondary lymphoid organs. Following priming, effector T cells migrate to the site of infection to exert their functions. Majority of the effector cells die while a small population of antigen-specific T cells persists as memory cells in distinct anatomical locations. The persistence and location of memory cells in lymphoid and non-lymphoid tissues is critical to protect the host from re-infection. The localization of memory T cells is carefully regulated by several factors including the highly organized secondary lymphoid structure, the cellular expression of chemokine receptors and compartmentalized secretion of their cognate ligands. This balance between the anatomy and the ordered expression of cell surface and soluble proteins regulates the subtle choreography of T cell migration. In recent years, our understanding of cellular dynamics of T cells has been advanced by the development of new imaging techniques allowing in situ visualization of T cell responses. Here, we review the past and more recent studies that have utilized sophisticated imaging technologies to investigate the migration dynamics of naïve, effector, and memory T cells. PMID:25120547

  6. The Status of RPE65 Gene Therapy Trials: Safety and Efficacy.

    PubMed

    Pierce, Eric A; Bennett, Jean

    2015-01-01

    Several groups have reported the results of clinical trials of gene augmentation therapy for Leber congenital amaurosis (LCA) because of mutations in the RPE65 gene. These studies have used subretinal injection of adeno-associated virus (AAV) vectors to deliver the human RPE65 cDNA to the retinal pigment epithelial (RPE) cells of the treated eyes. In all of the studies reported to date, this approach has been shown to be both safe and effective. The successful clinical trials of gene augmentation therapy for retinal degeneration caused by mutations in the RPE65 gene sets the stage for broad application of gene therapy to treat retinal degenerative disorders.

  7. Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

    PubMed

    Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

    2014-02-01

    Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

  8. PGC-1α Induces Human RPE Oxidative Metabolism and Antioxidant Capacity

    PubMed Central

    Iacovelli, Jared; Rowe, Glenn C.; Khadka, Arogya; Diaz-Aguilar, Daniel; Spencer, Carrie; Arany, Zoltan; Saint-Geniez, Magali

    2016-01-01

    Purpose Oxidative stress and metabolic dysregulation of the RPE have been implicated in AMD; however, the molecular regulation of RPE metabolism remains unclear. The transcriptional coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) is a powerful mediator of mitochondrial function. This study examines the ability of PGC-1α to regulate RPE metabolic program and oxidative stress response. Methods Primary human fetal RPE (hfRPE) and ARPE-19 were matured in vitro using standard culture conditions. Mitochondrial mass of RPE was measured using MitoTracker staining and citrate synthase activity. Expression of PGC-1 isoforms, RPE-specific genes, oxidative metabolism proteins, and antioxidant enzymes was analyzed by quantitative PCR and Western blot. Mitochondrial respiration and fatty-acid oxidation were monitored using the Seahorse extracellular flux analyzer. Expression of PGC-1α was increased using adenoviral delivery. ARPE-19 were exposed to hydrogen peroxide to induce oxidative stress. Reactive oxygen species were measured by CM-H2DCFDA fluorescence. Cell death was analyzed by LDH release. Results Maturation of ARPE-19 and hfRPE was associated with significant increase in mitochondrial mass, expression of oxidative phosphorylation (OXPHOS) genes, and PGC-1α gene expression. Overexpression of PGC-1α increased expression of OXPHOS and fatty-acid β-oxidation genes, ultimately leading to the potent induction of mitochondrial respiration and fatty-acid oxidation. PGC-1α gain of function also strongly induced numerous antioxidant genes and, importantly, protected RPE from oxidant-mediated cell death without altering RPE functions. Conclusions This study provides important insights into the metabolic changes associated with RPE functional maturation and identifies PGC-1α as a potent driver of RPE mitochondrial function and antioxidant capacity. PMID:26962700

  9. Primordial Germ Cell Specification and Migration

    PubMed Central

    Marlow, Florence

    2015-01-01

    Primordial germ cells are the progenitor cells that give rise to the gametes. In some animals, the germline is induced by zygotic transcription factors, whereas in others, primordial germ cell specification occurs via inheritance of maternally provided gene products known as germ plasm. Once specified, the primordial germ cells of some animals must acquire motility and migrate to the gonad in order to survive. In all animals examined, perinuclear structures called germ granules form within germ cells. This review focuses on some of the recent studies, conducted by several groups using diverse systems, from invertebrates to vertebrates, which have provided mechanistic insight into the molecular regulation of germ cell specification and migration. PMID:26918157

  10. Animal Models, in "The Quest to Decipher RPE Phagocytosis".

    PubMed

    Nandrot, Emeline F

    2014-01-01

    Renewal and elimination of aged photoreceptor outer segment (POS) tips by cells from the retinal pigment epithelial (RPE) is a daily rhythmic process that is crucial for long-term vision. Anomalies can arise during any of the sequential steps required for completion of this phagocytic function, from POS recognition to complete digestion of POS components. During the past 15 years, many animal models helped us characterize the molecular machinery implicated in RPE phagocytosis as well as understand associated defects leading to various retinal pathologies. Depending on which part of the machinery is flawed, phenotypes can either appear early in life, such as retinitis pigmentosa or Usher syndrome, or develop with aging of the individual, like age-related macular degeneration, affecting first either the peripheral or the central retina. This chapter describes mouse and rat models related to defective phagocytosis, and how they have been a tremendous help for us to comprehend RPE phagocytosis, its rhythm, and its failures.

  11. Bursts of activity in collective cell migration

    PubMed Central

    Chepizhko, Oleksandr; Giampietro, Costanza; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stéphane; Alava, Mikko J.; Zapperi, Stefano; La Porta, Caterina A. M.

    2016-01-01

    Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media, and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems. PMID:27681632

  12. Networking galore: intermediate filaments and cell migration.

    PubMed

    Chung, Byung-Min; Rotty, Jeremy D; Coulombe, Pierre A

    2013-10-01

    Intermediate filaments (IFs) are assembled from a diverse group of evolutionarily conserved proteins and are specified in a tissue-dependent, cell type-dependent, and context-dependent fashion in the body. IFs are involved in multiple cellular processes that are crucial for the maintenance of cell and tissue integrity and the response and adaptation to various stresses, as conveyed by the broad array of crippling clinical disorders caused by inherited mutations in IF coding sequences. Accordingly, the expression, assembly, and organization of IFs are tightly regulated. Migration is a fitting example of a cell-based phenomenon in which IFs participate as both effectors and regulators. With a particular focus on vimentin and keratin, we here review how the contributions of IFs to the cell's mechanical properties, to cytoarchitecture and adhesion, and to regulatory pathways collectively exert a significant impact on cell migration.

  13. Impact of jamming on collective cell migration

    NASA Astrophysics Data System (ADS)

    Nnetu, Kenechukwu David; Knorr, Melanie; Pawlizak, Steve; Fuhs, Thomas; Zink, Mareike; KäS, Josef A.

    2012-02-01

    Multi-cellular migration plays an important role in physiological processes such as embryogenesis, cancer metastasis and tissue repair. During migration, single cells undergo cycles of extension, adhesion and retraction resulting in morphological changes. In a confluent monolayer, there are inter-cellular interactions and crowding, however, the impact of these interactions on the dynamics and elasticity of the monolayer at the multi-cellular and single cell level is not well understood. Here we study the dynamics of a confluent epithelial monolayer by simultaneously measuring cell motion at the multi-cellular and single cell level for various cell densities and tensile elasticity. At the multi-cellular level, the system exhibited spatial kinetic transitions from isotropic to anisotropic migration on long times and the velocity of the monolayer decreased with increasing cell density. Moreover, the dynamics was spatially and temporally heterogeneous. Interestingly, the dynamics was also heterogeneous in wound-healing assays and the correlation length was fitted by compressed exponential. On the single cell scale, we observed transient caging effects with increasing cage rearrangement times as the system age due to an increase in density. Also, the density dependent elastic modulus of the monolayer scaled as a weak power law. Together, these findings suggest that caging effects at the single cell level initiates a slow and heterogeneous dynamics at the multi-cellular level which is similar to the glassy dynamics of deformable colloidal systems.

  14. Contractile forces in tumor cell migration.

    PubMed

    Mierke, Claudia Tanja; Rösel, Daniel; Fabry, Ben; Brábek, Jan

    2008-09-01

    Cancer is a deadly disease primarily because of the ability of tumor cells to spread from the primary tumor, to invade into the connective tissue, and to form metastases at distant sites. In contrast to cell migration on a planar surface where large cell tractions and contractile forces are not essential, tractions and forces are thought to be crucial for overcoming the resistance and steric hindrance of a dense three-dimensional connective tissue matrix. In this review, we describe recently developed biophysical tools, including 2-D and 3-D traction microscopy to measure contractile forces of cells. We discuss evidence indicating that tumor cell invasiveness is associated with increased contractile force generation.

  15. Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration

    PubMed Central

    Wang, Haibo; Han, Xiaokun; Kunz, Eric; Hartnett, M. Elizabeth

    2016-01-01

    Purpose This study addresses the hypothesis that age-related stresses upregulate Thy-1 in choroidal endothelial cells (CECs) and contribute to CEC activation and migration, processes important in choroidal neovascularization (CNV). Methods Measurements were made of Thy-1 protein (Western blot) in CECs and Thy-1 mRNA (real time quantitative PCR) in CECs treated with VEGF, CCL11, or PBS or in RPE/choroids from young or old donors or lasered or nonlasered mice. Immunolabeled Thy-1 in ocular sections was compared from young versus old human donor eyes or those with or without neovascular AMD or from lasered versus nonlasered mice. Choroidal endothelial cells transfected with Thy-1 or control siRNA or pretreated with Thy-1 blocking peptide or control were stimulated with VEGF or 7-ketocholesterol (7-KC). Choroidal endothelial cell migration, proliferation, cytoskeletal stress fibers, Rac1 activation, and phosphorylated VEGF receptor 2 (VEGFR2), integrin β3, and Src were measured. Statistics were performed using ANOVA. Results Thy-1 was expressed in retinal ganglion cells and in vascular endothelial-cadherin–labeled choroid and localized to human or mouse laser-induced CNV lesions. Thy-1 protein and mRNA were significantly increased in CECs treated with VEGF or CCL11 and in RPE/choroids from aged versus young donor eyes or from lasered mice versus nonlasered controls. Knockdown or inhibition of Thy-1 in CECs significantly reduced VEGF-induced CEC migration and proliferation, stress fiber formation and VEGFR2, Src, integrin β3 and Rac1 activation, and 7-KC–induced Rac1 and Src activation. Conclusions Thy-1 in CECs regulates VEGF-induced CEC activation and migration and links extracellular 7-KC to intracellular signaling. Future studies elucidating Thy-1 mechanisms in neovascular AMD are warranted. PMID:27768790

  16. T cell migration, search strategies and mechanisms.

    PubMed

    Krummel, Matthew F; Bartumeus, Frederic; Gérard, Audrey

    2016-03-01

    T cell migration is essential for T cell responses; it allows for the detection of cognate antigen at the surface of antigen-presenting cells and for interactions with other cells involved in the immune response. Although appearing random, growing evidence suggests that T cell motility patterns are strategic and governed by mechanisms that are optimized for both the activation stage of the cell and for environment-specific cues. In this Opinion article, we discuss how the combined effects of T cell-intrinsic and -extrinsic forces influence T cell motility patterns in the context of highly complex tissues that are filled with other cells involved in parallel motility. In particular, we examine how insights from 'search theory' can be used to describe T cell movement across an 'exploitation-exploration trade-off' in the context of activation versus effector function and lymph nodes versus peripheral tissues. PMID:26852928

  17. Cadmium migration in aerospace nickel cadmium cells

    NASA Technical Reports Server (NTRS)

    Mcdermott, P. P.

    1976-01-01

    The effects of temperature, the nature of separator material, charge and discharge, carbonate contamination, and the mode of storage are studied with respect to the migration of active material from the negative toward the positive plate. A theoretical model is proposed which takes into account the solubility of cadmium in various concentrations of hydroxide and carbonate at different temperatures, the generation of the cadmiate ion, Cd(OH)3(-), during discharge, the migration of the cadmiate ion and particulate Cd(OH)2 due to electrophoretic effects and the movement of electrolyte in and out of the negative plate and, finally, the recrystallization of cadmiate ion in the separator as Cd(OH)2. Application of the theoretical model to observations of cadmium migration in cycled cells is also discussed.

  18. Nestin(+) cells direct inflammatory cell migration in atherosclerosis.

    PubMed

    Del Toro, Raquel; Chèvre, Raphael; Rodríguez, Cristina; Ordóñez, Antonio; Martínez-González, José; Andrés, Vicente; Méndez-Ferrer, Simón

    2016-01-01

    Atherosclerosis is a leading death cause. Endothelial and smooth muscle cells participate in atherogenesis, but it is unclear whether other mesenchymal cells contribute to this process. Bone marrow (BM) nestin(+) cells cooperate with endothelial cells in directing monocyte egress to bloodstream in response to infections. However, it remains unknown whether nestin(+) cells regulate inflammatory cells in chronic inflammatory diseases, such as atherosclerosis. Here, we show that nestin(+) cells direct inflammatory cell migration during chronic inflammation. In Apolipoprotein E (ApoE) knockout mice fed with high-fat diet, BM nestin(+) cells regulate the egress of inflammatory monocytes and neutrophils. In the aorta, nestin(+) stromal cells increase ∼30 times and contribute to the atheroma plaque. Mcp1 deletion in nestin(+) cells-but not in endothelial cells only- increases circulating inflammatory cells, but decreases their aortic infiltration, delaying atheroma plaque formation and aortic valve calcification. Therefore, nestin expression marks cells that regulate inflammatory cell migration during atherosclerosis. PMID:27586429

  19. Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion

    PubMed Central

    Chen, Nan-Peng; Uddin, Borhan; Voit, Renate; Schiebel, Elmar

    2016-01-01

    Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14APD) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14APD cells. hCDC14APD cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14APD cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast. PMID:26747605

  20. Cell migration during heart regeneration in zebrafish.

    PubMed

    Tahara, Naoyuki; Brush, Michael; Kawakami, Yasuhiko

    2016-07-01

    Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, pre-existing cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration. Developmental Dynamics 245:774-787, 2016. © 2016 Wiley Periodicals, Inc. PMID:27085002

  1. Nestin+ cells direct inflammatory cell migration in atherosclerosis

    PubMed Central

    del Toro, Raquel; Chèvre, Raphael; Rodríguez, Cristina; Ordóñez, Antonio; Martínez-González, José; Andrés, Vicente; Méndez-Ferrer, Simón

    2016-01-01

    Atherosclerosis is a leading death cause. Endothelial and smooth muscle cells participate in atherogenesis, but it is unclear whether other mesenchymal cells contribute to this process. Bone marrow (BM) nestin+ cells cooperate with endothelial cells in directing monocyte egress to bloodstream in response to infections. However, it remains unknown whether nestin+ cells regulate inflammatory cells in chronic inflammatory diseases, such as atherosclerosis. Here, we show that nestin+ cells direct inflammatory cell migration during chronic inflammation. In Apolipoprotein E (ApoE) knockout mice fed with high-fat diet, BM nestin+ cells regulate the egress of inflammatory monocytes and neutrophils. In the aorta, nestin+ stromal cells increase ∼30 times and contribute to the atheroma plaque. Mcp1 deletion in nestin+ cells—but not in endothelial cells only— increases circulating inflammatory cells, but decreases their aortic infiltration, delaying atheroma plaque formation and aortic valve calcification. Therefore, nestin expression marks cells that regulate inflammatory cell migration during atherosclerosis. PMID:27586429

  2. Taking Aim at Moving Targets in Computational Cell Migration.

    PubMed

    Masuzzo, Paola; Van Troys, Marleen; Ampe, Christophe; Martens, Lennart

    2016-02-01

    Cell migration is central to the development and maintenance of multicellular organisms. Fundamental understanding of cell migration can, for example, direct novel therapeutic strategies to control invasive tumor cells. However, the study of cell migration yields an overabundance of experimental data that require demanding processing and analysis for results extraction. Computational methods and tools have therefore become essential in the quantification and modeling of cell migration data. We review computational approaches for the key tasks in the quantification of in vitro cell migration: image pre-processing, motion estimation and feature extraction. Moreover, we summarize the current state-of-the-art for in silico modeling of cell migration. Finally, we provide a list of available software tools for cell migration to assist researchers in choosing the most appropriate solution for their needs.

  3. Dynamic contact guidance of migrating cells

    NASA Astrophysics Data System (ADS)

    Losert, Wolfgang; Sun, Xiaoyu; Guven, Can; Driscoll, Meghan; Fourkas, John

    2014-03-01

    We investigate the effects of nanotopographical surfaces on the cell migration and cell shape dynamics of the amoeba Dictyostelium discoideum. Amoeboid motion exhibits significant contact guidance along surfaces with nanoscale ridges or grooves. We show quantitatively that nanoridges spaced 1.5 μm apart exhibit the greatest contact guidance efficiency. Using principal component analysis, we characterize the dynamics of the cell shape modulated by the coupling between the cell membrane and ridges. We show that motion parallel to the ridges is enhanced, while the turning, at the largest spatial scales, is suppressed. Since protrusion dynamics are principally governed by actin dynamics, we imaged the actin polymerization of cells on ridges. We found that actin polymerization occurs preferentially along nanoridges in a ``monorail'' like fashion. The ridges then provide us with a tool to study actin dynamics in an effectively reduced dimensional system.

  4. Localized RPE Removal with a Novel Instrument Aided by Viscoelastics in Rabbits

    PubMed Central

    Thieltges, Fabian; Liu, Zengping; Brinken, Ralf; Braun, Norbert; Wongsawad, Warapat; Somboonthanakij, Sudawadee; Herwig, Martina; Holz, Frank G.; Stanzel, Boris V.

    2016-01-01

    Purpose We developed a surgical method for localized and atraumatic removal of the retinal pigment epithelium (RPE) with a novel instrument. Methods Bleb retinal detachments (bRD) were raised with balanced salt solution (BSS) following vitrectomy in 27 rabbits. The RPE was scraped with 3 loop variants (polypropylene [PP], 0.1 mm; PP, 0.06 mm; metal, 0.1 mm) of a custom-made instrument. Stabilization of bRDs with BSS or various concentrations (0.1%–0.5%) of hyaluronic acid (HA) was video analyzed. Perfusion-fixed samples of scraped areas and controls were studied by light and transmission electron microscopy. Results The bRDs were sufficiently stabilized by ≥0.25% HA. Using the PP 0.1 mm loop with a single forward/backward stroke, an area of ca. 2.5 × 1.5 mm was nearly devoid of RPE, yet did show occasional Bruch's membrane (BM) defects combined with choriocapillaris hemorrhages in 13% of the bRDs. A single scrape with PP 0.06 mm resulted in unsatisfactory RPE denudement, while repeated scraping maneuvers caused more BM defects and hemorrhages. The metal loop resulted in incomplete RPE removal and massive intraoperative subretinal hemorrhages. Histologically, intact photoreceptor outer segments (POS) were observed above the RPE wounds in bRDs. Controls with bRDs alone showed an intact RPE monolayer with microvilli, with few engulfed remains of POS. Conclusions Localized removal of RPE in HA stabilized bRD can be achieved by a PP 0.1 mm loop instrument. Translational Relevance Removal of degenerated RPE may aid RPE cell replacement strategies. PMID:27294010

  5. T Cell Migration in Rheumatoid Arthritis.

    PubMed

    Mellado, Mario; Martínez-Muñoz, Laura; Cascio, Graciela; Lucas, Pilar; Pablos, José L; Rodríguez-Frade, José Miguel

    2015-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints, associated with synovial hyperplasia and with bone and cartilage destruction. Although the primacy of T cell-related events early in the disease continues to be debated, there is strong evidence that autoantigen recognition by specific T cells is crucial to the pathophysiology of rheumatoid synovitis. In addition, T cells are key components of the immune cell infiltrate detected in the joints of RA patients. Initial analysis of the cytokines released into the synovial membrane showed an imbalance, with a predominance of proinflammatory mediators, indicating a deleterious effect of Th1 T cells. There is nonetheless evidence that Th17 cells also play an important role in RA. T cells migrate from the bloodstream to the synovial tissue via their interactions with the endothelial cells that line synovial postcapillary venules. At this stage, selectins, integrins, and chemokines have a central role in blood cell invasion of synovial tissue, and therefore in the intensity of the inflammatory response. In this review, we will focus on the mechanisms involved in T cell attraction to the joint, the proteins involved in their extravasation from blood vessels, and the signaling pathways activated. Knowledge of these processes will lead to a better understanding of the mechanism by which the systemic immune response causes local joint disorders and will help to provide a molecular basis for therapeutic strategies.

  6. T Cell Migration in Rheumatoid Arthritis

    PubMed Central

    Mellado, Mario; Martínez-Muñoz, Laura; Cascio, Graciela; Lucas, Pilar; Pablos, José L.; Rodríguez-Frade, José Miguel

    2015-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints, associated with synovial hyperplasia and with bone and cartilage destruction. Although the primacy of T cell-related events early in the disease continues to be debated, there is strong evidence that autoantigen recognition by specific T cells is crucial to the pathophysiology of rheumatoid synovitis. In addition, T cells are key components of the immune cell infiltrate detected in the joints of RA patients. Initial analysis of the cytokines released into the synovial membrane showed an imbalance, with a predominance of proinflammatory mediators, indicating a deleterious effect of Th1 T cells. There is nonetheless evidence that Th17 cells also play an important role in RA. T cells migrate from the bloodstream to the synovial tissue via their interactions with the endothelial cells that line synovial postcapillary venules. At this stage, selectins, integrins, and chemokines have a central role in blood cell invasion of synovial tissue, and therefore in the intensity of the inflammatory response. In this review, we will focus on the mechanisms involved in T cell attraction to the joint, the proteins involved in their extravasation from blood vessels, and the signaling pathways activated. Knowledge of these processes will lead to a better understanding of the mechanism by which the systemic immune response causes local joint disorders and will help to provide a molecular basis for therapeutic strategies. PMID:26284069

  7. Alignment of cell division axes in directed epithelial cell migration

    NASA Astrophysics Data System (ADS)

    Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

    2014-11-01

    Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

  8. A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers

    PubMed Central

    Toops, Kimberly A.; Tan, Li Xuan; Lakkaraju, Aparna

    2014-01-01

    The retinal pigment epithelium (RPE) performs numerous functions that are indispensable for photoreceptor health and vision. This monolayer of cells is also a major site of insult in inherited and age-related macular degenerations. In vitro models of primary RPE such as human fetal and adult RPE cultures have been invaluable for dissecting disease pathways at the cellular and molecular level. However, numerous studies show that it takes over four weeks for human RPE cell monolayers to become fully polarized after plating on semipermeable membrane supports. Poor persistence of transgene expression over this time period critically limits the applicability of human RPE cultures for live imaging studies required to follow dynamic processes like intracellular trafficking and organelle transport that occur over timescales of milliseconds. Here, we provide a detailed three-step protocol for live imaging of polarized primary RPE using high-speed spinning disk confocal microscopy. Step 1: establish porcine RPE monolayers that undergo differentiation within one week after plating on semipermeable membrane supports; step 2: transfect or transduce RPE using either of two different protocols that result in prolonged transgene expression; and step 3: perform multicolor high-speed live imaging of organelle transport in polarized RPE monolayers. Porcine RPE cells and photoreceptor outer segments were isolated from freshly harvested eyes and plated on collagen-coated Transwell® filters to generate polarized monolayers. After seven days, RPE monolayers were highly pigmented, had TER values ≥ 200 Ω.cm2 and cleared outer segments within 5 hours after phagocytosis. These cells expressed RPE65, localized ZO-1 to the tight junction, Na+,K+-ATPase to the apical membrane and acetylated tubulin to the primary cilium. There was an inverse relationship between initial plating density and the time to differentiation. We used nucleofection to express fluorescently tagged genes in RPE cells

  9. Flow-Driven Cell Migration under External Electric Fields

    NASA Astrophysics Data System (ADS)

    Li, Yizeng; Mori, Yoichiro; Sun, Sean X.

    2015-12-01

    Electric fields influence many aspects of cell physiology, including various forms of cell migration. Many cells are sensitive to electric fields, and they can migrate toward a cathode or an anode, depending on the cell type. In this Letter, we examine an actomyosin-independent mode of cell migration under electrical fields. Our theory considers a one-dimensional cell with water and ionic fluxes at the cell boundary. Water fluxes through the membrane are governed by the osmotic pressure difference across the cell membrane. Fluxes of cations and anions across the cell membrane are determined by the properties of the ion channels as well as the external electric field. Results show that without actin polymerization and myosin contraction, electric fields can also drive cell migration, even when the cell is not polarized. The direction of migration with respect to the electric field direction is influenced by the properties of ion channels, and are cell-type dependent.

  10. Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis

    PubMed Central

    Li, Songhua; Hu, Jane; Jin, Robin J.; Aiyar, Ashok; Jacobson, Samuel G.; Bok, Dean; Jin, Minghao

    2015-01-01

    RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood. Here, we show that three Leber congenital amaurosis -associated RPE65 mutants (R91W, Y249C and R515W) undergo rapid proteasomal degradation mediated by the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13) in cultured human retinal pigment epithelium (RPE) cells. These mutant proteins formed cytosolic inclusion bodies or high molecular weight complexes via disulfide bonds. The mutations are mapped on non-active sites but severely reduced isomerase activity of RPE65. At 30°C, however, the enzymatic function and membrane-association of the mutant RPE65s are significantly rescued possibly due to proper folding. In addition, PSMD13 displayed a drastically decreased effect on degradation of the mutant proteins in the cells grown at 30°C. These results suggest that PSMD13 plays a critical role in regulating pathogenicity of the mutations and the molecular basis for the PSMD13-mediated rapid degradation and loss of function of the mutants is misfolding of RPE65. PMID:25752820

  11. Migrating Oligodendrocyte Progenitor Cells Swell Prior to Soma Dislocation

    PubMed Central

    Happel, Patrick; Möller, Kerstin; Schwering, Nina K.; Dietzel, Irmgard D.

    2013-01-01

    The migration of oligodendrocyte progenitor cells (OPCs) to the white matter is an indispensable requirement for an intact brain function. The mechanism of cell migration in general is not yet completely understood. Nevertheless, evidence is accumulating that besides the coordinated rearrangement of the cytoskeleton, a finetuned interplay of ion and water fluxes across the cell membrane is essential for cell migration. One part of a general hypothesis is that a local volume increase towards the direction of movement triggers a mechano-activated calcium influx that regulates various procedures at the rear end of a migrating cell. Here, we investigated cell volume changes of migrating OPCs using scanning ion conductance microscopy. We found that during accelerated migration OPCs undergo an increase in the frontal cell body volume. These findings are supplemented with time lapse calcium imaging data that hint an increase in calcium content the frontal part of the cell soma. PMID:23657670

  12. Mechanisms guiding primordial germ cell migration: strategies from different organisms

    PubMed Central

    Richardson, Brian E.; Lehmann, Ruth

    2015-01-01

    Preface The regulated migration of cells is essential for development and tissue homeostasis, and aberrant cell migration can lead to an impaired immune response and the progression of cancer. Primordial germ cells (PGCs), precursors to sperm and eggs, have to migrate across the embryo to reach somatic gonadal precursors (SGPs) and fulfill their function. Studies of model organisms have revealed that, despite important differences, several features of PGC migration are conserved. PGCs require both an intrinsic motility program and external guidance cues to survive and successfully migrate. Proper guidance involves both attractive and repulsive cues mediated by protein and lipid signalling. PMID:20027186

  13. Light activated cell migration in synthetic extracellular matrices.

    PubMed

    Guo, Qiongyu; Wang, Xiaobo; Tibbitt, Mark W; Anseth, Kristi S; Montell, Denise J; Elisseeff, Jennifer H

    2012-11-01

    Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues. However no method exists to manipulate single cells within these matrices. It is desirable to develop such methods in order to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement within these matrices. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac, which is an intracellular signaling protein that plays a key role in cell migration. Photoactivated cell migration in synthetic hydrogels depended on mechanical and biological cues in the biomaterial. Real-time hydrogel photodegradation was employed to create geometrically defined channels and spaces in which cells could be photoactivated to migrate. Cell migration speed was significantly higher in the photo-etched channels and cells could easily change direction of movement compared to the bulk hydrogels.

  14. Differential migration and proliferation of geometrical ensembles of cell clusters

    SciTech Connect

    Kumar, Girish; Chen, Bo; Co, Carlos C.; Ho, Chia-Chi

    2011-06-10

    Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell-cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.

  15. Water permeation drives tumor cell migration in confined microenvironments.

    PubMed

    Stroka, Kimberly M; Jiang, Hongyuan; Chen, Shih-Hsun; Tong, Ziqiu; Wirtz, Denis; Sun, Sean X; Konstantopoulos, Konstantinos

    2014-04-24

    Cell migration is a critical process for diverse (patho)physiological phenomena. Intriguingly, cell migration through physically confined spaces can persist even when typical hallmarks of 2D planar migration, such as actin polymerization and myosin II-mediated contractility, are inhibited. Here, we present an integrated experimental and theoretical approach ("Osmotic Engine Model") and demonstrate that directed water permeation is a major mechanism of cell migration in confined microenvironments. Using microfluidic and imaging techniques along with mathematical modeling, we show that tumor cells confined in a narrow channel establish a polarized distribution of Na+/H+ pumps and aquaporins in the cell membrane, which creates a net inflow of water and ions at the cell leading edge and a net outflow of water and ions at the trailing edge, leading to net cell displacement. Collectively, this study presents an alternate mechanism of cell migration in confinement that depends on cell-volume regulation via water permeation. PMID:24726433

  16. Water Permeation Drives Tumor Cell Migration in Confined Microenvironments

    PubMed Central

    Stroka, Kimberly M.; Jiang, Hongyuan; Chen, Shih-Hsun; Tong, Ziqiu; Wirtz, Denis; Sun, Sean X.; Konstantopoulos, Konstantinos

    2014-01-01

    SUMMARY Cell migration is a critical process for diverse (patho) physiological phenomena. Intriguingly, cell migration through physically confined spaces can persist even when typical hallmarks of 2D planar migration, such as actin polymerization and myosin II-mediated contractility, are inhibited. Here, we present an integrated experimental and theoretical approach (“Osmotic Engine Model”) and demonstrate that directed water permeation is a major mechanism of cell migration in confined microenvironments. Using microfluidic and imaging techniques along with mathematical modeling, we show that tumor cells confined in a narrow channel establish a polarized distribution of Na+/H+ pumps and aquaporins in the cell membrane, which creates a net inflow of water and ions at the cell leading edge and a net outflow of water and ions at the trailing edge, leading to net cell displacement. Collectively, this study presents an alternate mechanism of cell migration in confinement that depends on cell-volume regulation via water permeation. PMID:24726433

  17. Proinflammatory cytokines decrease the expression of genes critical for RPE function

    PubMed Central

    Samuel, William; Boyce, Kaifa; Cherukuri, Aswini; Duncan, Todd; Jaworski, Cynthia; Nagineni, Chandrasekharam N.; Redmond, T. Michael

    2016-01-01

    Purpose Proinflammatory cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β) secreted by infiltrating lymphocytes or macrophages may play a role in triggering RPE dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways that, in turn, may dysregulate cellular gene expression. The purpose of the present study was to investigate whether IFN-γ, TNF-α, and IL-1β have an adverse effect on the expression of genes essential for RPE function, employing the RPE cell line ARPE-19 as a model system. Methods ARPE-19 cells were cultured for 3–4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. The differentiated cells were treated with IFN-γ, TNF-α, and/or IL-1β, and gene expression was analyzed with real-time PCR analysis. Western immunoblotting was employed for the detection of proteins. Results Proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) greatly increased the expression of chemokines and cytokines in cultured ARPE-19 cells that exhibited RPE characteristics. However, this response was accompanied by markedly decreased expression of genes important for RPE function, such as CDH1, RPE65, RDH5, RDH10, TYR, and MERTK. This was associated with decreased expression of the genes MITF, TRPM1, and TRPM3, as well as microRNAs miR-204 and miR-211, which are known to regulate RPE-specific gene expression. The decreased expression of the epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM, and CCND1) and epithelial–mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1). Conclusions RPE cells exposed to proinflammatory cytokines IFN-γ, TNF-α, and IL-1β showed decreased expression of key genes involved in the visual cycle, epithelial morphology

  18. Texture sensing of cytoskeletal dynamics in cell migration

    NASA Astrophysics Data System (ADS)

    Das, Satarupa; Lee, Rachel; Hourwitz, Matthew J.; Sun, Xiaoyu; Parent, Carole; Fourkas, John T.; Losert, Wolfgang

    Migrating cells can be directed towards a target by gradients in properties such as chemical concentration or mechanical properties of the surrounding microenvironment. In previous studies we have shown that micro/nanotopographical features on scales comparable to those of natural collagen fibers can guide fast migrating amoeboid cells by aligning actin polymerization waves to such nanostructures. We find that actin microfilaments and microtubules are aligned along the nanoridge topographies, modulating overall cell polarity and directional migration in epithelial cells. This work shows that topographic features on a biologically relevant length scale can modulate migration outcomes by affecting the texture sensing property of the cytoskeleton.

  19. Systems microscopy approaches to understand cancer cell migration and metastasis

    PubMed Central

    Le Dévédec, Sylvia E.; Yan, Kuan; de Bont, Hans; Ghotra, Veerander; Truong, Hoa; Danen, Erik H.; Verbeek, Fons

    2010-01-01

    Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration. PMID:20556632

  20. Cell migration does not produce membrane flow

    PubMed Central

    1990-01-01

    We have previously reported that rearward migration of surface particles on slowly moving cells is not driven by membrane flow (Sheetz, M. P., S. Turney, H. Qian, and E. L. Elson. 1989. Nature (Lond.). 340:284-288) and recent photobleaching measurements have ruled out any rapid rearward lipid flow (Lee, J., M. Gustafsson, D. E. Magnussen, and K. Jacobson. 1990. Science (Wash. DC.) 247:1229-1233). It was not possible, however, to conclude from those studies that a slower or tank-tread membrane lipid flow does not occur. Therefore, we have used the technology of single particle tracking to examine the movements of diffusing particles on rapidly locomoting fish keratocytes where the membrane current is likely to be greatest. The keratocytes had a smooth lamellipodial surface on which bound Con A-coated gold particles were observed either to track toward the nuclear region (velocity of 0.35 +/- 0.15 micron/s) or to diffuse randomly (apparent diffusion coefficient of [3.5 +/- 2.0] x 10(-10) cm2/s). We detected no systematic drift relative to the cell edge of particles undergoing random diffusion even after the cell had moved many micrometers. The average net particle displacement was 0.01 +/- 2.7% of the cell displacement. These results strongly suggest that neither the motions of membrane proteins driven by the cytoskeleton nor other possible factors produce a bulk flow of membrane lipid. PMID:2211827

  1. Leader Cells Define Directionality of Trunk, but Not Cranial, Neural Crest Cell Migration.

    PubMed

    Richardson, Jo; Gauert, Anton; Briones Montecinos, Luis; Fanlo, Lucía; Alhashem, Zainalabdeen Mohmammed; Assar, Rodrigo; Marti, Elisa; Kabla, Alexandre; Härtel, Steffen; Linker, Claudia

    2016-05-31

    Collective cell migration is fundamental for life and a hallmark of cancer. Neural crest (NC) cells migrate collectively, but the mechanisms governing this process remain controversial. Previous analyses in Xenopus indicate that cranial NC (CNC) cells are a homogeneous population relying on cell-cell interactions for directional migration, while chick embryo analyses suggest a heterogeneous population with leader cells instructing directionality. Our data in chick and zebrafish embryos show that CNC cells do not require leader cells for migration and all cells present similar migratory capacities. In contrast, laser ablation of trunk NC (TNC) cells shows that leader cells direct movement and cell-cell contacts are required for migration. Moreover, leader and follower identities are acquired before the initiation of migration and remain fixed thereafter. Thus, two distinct mechanisms establish the directionality of CNC cells and TNC cells. This implies the existence of multiple molecular mechanisms for collective cell migration.

  2. HIF-1α Promotes A Hypoxia-Independent Cell Migration.

    PubMed

    Li, Liyuan; Madu, Chikezie O; Lu, Andrew; Lu, Yi

    2010-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects.

  3. HIF-1α Promotes A Hypoxia-Independent Cell Migration

    PubMed Central

    Li, Liyuan; Madu, Chikezie O.; Lu, Andrew; Lu, Yi

    2010-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects. PMID:20882121

  4. 3D cancer cell migration in a confined matrix

    NASA Astrophysics Data System (ADS)

    Alobaidi, Amani; Sun, Bo

    Cancer cell migration is widely studied in 2D motion, which does not mimic the invasion processes in vivo. More recently, 3D cell migration studies have been performed. The ability of cancer cells to migrate within the extracellular matrix depends on the physical and biochemical features of the extracellular matrix. We present a model of cell motility in confined matrix geometry. The aim of the study is to study cancer migration in collagen matrix, as a soft tissue, to investigate their motility within the confined and surrounding collagen environment. Different collagen concentrations have been used to show the ability of these cancer cells to move through such a complex structure by measuring Cancer cell migration velocity as well as the displacement. Graduate student physics department.

  5. Physical role for the nucleus in cell migration.

    PubMed

    Fruleux, Antoine; Hawkins, Rhoda J

    2016-09-14

    Cell migration is important for the function of many eukaryotic cells. Recently the nucleus has been shown to play an important role in cell motility. After giving an overview of cell motility mechanisms we review what is currently known about the mechanical properties of the nucleus and the connections between it and the cytoskeleton. We also discuss connections to the extracellular matrix and mechanotransduction. We identify key physical roles of the nucleus in cell migration.

  6. Physical role for the nucleus in cell migration

    NASA Astrophysics Data System (ADS)

    Fruleux, Antoine; Hawkins, Rhoda J.

    2016-09-01

    Cell migration is important for the function of many eukaryotic cells. Recently the nucleus has been shown to play an important role in cell motility. After giving an overview of cell motility mechanisms we review what is currently known about the mechanical properties of the nucleus and the connections between it and the cytoskeleton. We also discuss connections to the extracellular matrix and mechanotransduction. We identify key physical roles of the nucleus in cell migration.

  7. Physical role for the nucleus in cell migration.

    PubMed

    Fruleux, Antoine; Hawkins, Rhoda J

    2016-09-14

    Cell migration is important for the function of many eukaryotic cells. Recently the nucleus has been shown to play an important role in cell motility. After giving an overview of cell motility mechanisms we review what is currently known about the mechanical properties of the nucleus and the connections between it and the cytoskeleton. We also discuss connections to the extracellular matrix and mechanotransduction. We identify key physical roles of the nucleus in cell migration. PMID:27406341

  8. How does cancer cell metabolism affect tumor migration and invasion?

    PubMed

    Han, Tianyu; Kang, De; Ji, Daokun; Wang, Xiaoyu; Zhan, Weihua; Fu, Minggui; Xin, Hong-Bo; Wang, Jian-Bin

    2013-01-01

    Cancer metastasis is the major cause of cancer-associated death. Accordingly, identification of the regulatory mechanisms that control whether or not tumor cells become "directed walkers" is a crucial issue of cancer research. The deregulation of cell migration during cancer progression determines the capacity of tumor cells to escape from the primary tumors and invade adjacent tissues to finally form metastases. The ability to switch from a predominantly oxidative metabolism to glycolysis and the production of lactate even when oxygen is plentiful is a key characteristic of cancer cells. This metabolic switch, known as the Warburg effect, was first described in 1920s, and affected not only tumor cell growth but also tumor cell migration. In this review, we will focus on the recent studies on how cancer cell metabolism affects tumor cell migration and invasion. Understanding the new aspects on molecular mechanisms and signaling pathways controlling tumor cell migration is critical for development of therapeutic strategies for cancer patients.

  9. Follow-the-leader cell migration requires biased cell-cell contact and local microenvironmental signals.

    PubMed

    Wynn, Michelle L; Rupp, Paul; Trainor, Paul A; Schnell, Santiago; Kulesa, Paul M

    2013-06-01

    Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.

  10. An open data ecosystem for cell migration research.

    PubMed

    Masuzzo, Paola; Martens, Lennart; Ampe, Christophe; Anderson, Kurt I; Barry, Joseph; De Wever, Olivier; Debeir, Olivier; Decaestecker, Christine; Dolznig, Helmut; Friedl, Peter; Gaggioli, Cedric; Geiger, Benjamin; Goldberg, Ilya G; Horn, Elias; Horwitz, Rick; Kam, Zvi; Le Dévédec, Sylvia E; Vignjevic, Danijela Matic; Moore, Josh; Olivo-Marin, Jean-Christophe; Sahai, Erik; Sansone, Susanna A; Sanz-Moreno, Victoria; Strömblad, Staffan; Swedlow, Jason; Textor, Johannes; Van Troys, Marleen; Zantl, Roman

    2015-02-01

    Cell migration research has recently become both a high content and a high throughput field thanks to technological, computational, and methodological advances. Simultaneously, however, urgent bioinformatics needs regarding data management, standardization, and dissemination have emerged. To address these concerns, we propose to establish an open data ecosystem for cell migration research.

  11. Screening of genes involved in cell migration in Dictyostelium.

    PubMed

    Nagasaki, Akira; Uyeda, Taro Q P

    2008-03-10

    A single cell of wild-type Dictyostelium discoideum forms a visible colony on a plastic dish in several days, but due to enhanced cell migration, amiB-null mutant cells scatter over a large area and do not form noticeable colonies. Here, with an aim to identify genes involved in cell migration, we isolated suppresser mutants of amiB-null mutants that restore the ability to form colonies. From REMI (restriction enzyme-mediated integration)-mutagenized pool of double-mutants, we identified 18 responsible genes from them. These genes can be categorized into several biological processes. One cell line, Sab16 (Suppressor of amiB) was chosen for further analysis, which had a disrupted phospholipase D pldB gene. To confirm the role of pldB gene in cell migration, we knocked out the pldB gene and over-expressed gfp-pldB in wild-type cells. GFP-PLDB localized to plasma membrane and on vesicles, and in migrating cells, at the protruding regions of pseudopodia. Migration speed of vegetative pldB-null cells was reduced to 73% of that of the wild-type. These results suggest that PLDB plays an important role in migration in Dictyostelium cells, and that our screening system is useful for the identification of genes involved in cell migration. PMID:18164290

  12. Biochemical genetics of the pentose phosphate cycle: human ribose 5-phosphate isomerase (RPI) and ribulose 5-phosphate 3-epimerase (RPE).

    PubMed

    Spencer, N; Hopkinson, D A

    1980-05-01

    1. Staining procedures are described for the detection after starch-gel electrophoresis of ribose-5-phosphate isomerase (RPI) and ribulose 5-phosphate 3-epimerase (RPE). 2. Both RPI and RPE were detected in all human tissues including red cells, lymphocytes and fibroblasts. 3. No evidence was found for more than one structural gene locus for either enzyme. 4. No allelic variants of either enzyme were found in erythrocyte lysates from over 200 unrelated individuals. 5. Preliminary data are presented which suggest that differences in tissue RPE isozyme patterns may be due to endogenous proteolytic activity. 6. Electrophoretic analysis of RPE and RPI isozyme patterns in extracts of man/mouse hybrid cells indicates that RPE is probably a dimer and RPI may also be polymeric.

  13. Osteoactivin Promotes Migration of Oral Squamous Cell Carcinomas.

    PubMed

    Arosarena, Oneida A; Dela Cadena, Raul A; Denny, Michael F; Bryant, Evan; Barr, Eric W; Thorpe, Ryan; Safadi, Fayez F

    2016-08-01

    Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration, and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. The aims were to determine how integrin interactions modulate OA-induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OA's interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co-immunoprecipitation. We investigated OA-mediated activation of mitogen-activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA-mediated cell migration were determined. We assessed effects of OA knock-down on cell migration and proliferation. OA is overexpressed in OSCC cell lines, and serves as a migration-promoting adhesion molecule. OA co-localized with integrin subunits, and co-immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the β1 subunit, inhibited cell adhesion (P = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin-neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock-down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. J. Cell. Physiol. 231: 1761-1770, 2016. © 2015 Wiley Periodicals, Inc. PMID:26636434

  14. Osteoactivin Promotes Migration of Oral Squamous Cell Carcinomas.

    PubMed

    Arosarena, Oneida A; Dela Cadena, Raul A; Denny, Michael F; Bryant, Evan; Barr, Eric W; Thorpe, Ryan; Safadi, Fayez F

    2016-08-01

    Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration, and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. The aims were to determine how integrin interactions modulate OA-induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OA's interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co-immunoprecipitation. We investigated OA-mediated activation of mitogen-activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA-mediated cell migration were determined. We assessed effects of OA knock-down on cell migration and proliferation. OA is overexpressed in OSCC cell lines, and serves as a migration-promoting adhesion molecule. OA co-localized with integrin subunits, and co-immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the β1 subunit, inhibited cell adhesion (P = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin-neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock-down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. J. Cell. Physiol. 231: 1761-1770, 2016. © 2015 Wiley Periodicals, Inc.

  15. GLUT1 activity contributes to the impairment of PEDF secretion by the RPE

    PubMed Central

    Calado, Sofia M.; Alves, Liliana S.; Simão, Sónia

    2016-01-01

    Purpose In this study, we aimed to understand whether glucose transporter 1 (GLUT1) activity affects the secretion capacity of antiangiogenic factor pigment epithelium-derived factor (PEDF) by the RPE cells, thus explaining the reduction in PEDF levels observed in patients with diabetic retinopathy (DR). Methods Analysis of GLUT1 expression, localization, and function was performed in vitro in RPE cells (D407) cultured with different glucose concentrations, corresponding to non-diabetic (5 mM of glucose) and diabetic (25 mM of glucose) conditions, further subjected to normoxia or hypoxia. The expression of PEDF was also evaluated in the secretome of the cells cultured in these conditions. Analysis of GLUT1 and PEDF expression was also performed in vivo in the RPE of Ins2Akita diabetic mice and age-matched wild-type (WT) controls. Results We observed an increase in GLUT1 under hypoxia in a glucose-dependent manner, which we found to be directly associated with the translocation and stabilization of GLUT1 in the cell membrane. This stabilization led to an increase in glucose uptake by RPE cells. This increase was followed by a decrease in PEDF expression in RPE cells cultured in conditions that simulated DR. Compared with non-diabetic WT mice, the RPE of Ins2Akita mice showed increased GLUT1 overexpression with a concomitant decrease in PEDF expression. Conclusions Collectively, our data show that expression of GLUT1 is stimulated by hyperglycemia and low oxygen supply, and this overexpression was associated with increased activity of GLUT1 in the cell membrane that contributes to the impairment of the RPE secretory function of PEDF. PMID:27440994

  16. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells. PMID:24635174

  17. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells.

  18. Cerebellar granule cell migration and the effects of alcohol.

    PubMed

    Jiang, Yulan; Kumada, Tatsuro; Cameron, D Bryant; Komuro, Hitoshi

    2008-01-01

    In the developing brain the majority of neurons migrate from their birthplace to their final destination. This active movement is essential for the formation of cortical layers and nuclei. The impairment of migration does not affect the viability of neurons but often results in abnormal differentiation. The proper migration of neurons requires the orchestrated activities of multiple cellular and molecular events, such as pathway selection, the activation of specific receptors and channels, and the assembly and disassembly of cytoskeletal components. The migration of neurons is very vulnerable to exposure to environmental toxins, such as alcohol. In this article, we will focus on recent developments in the migration of cerebellar granule cells. First, we will describe when, where and how granule cells migrate through different cortical layers to reach their final destination. Second, we will present how internal programs control the sequential changes in granule cell migration. Third, we will review the roles of external guidance cues and transmembrane signals in granule cell migration. Finally, we will reveal mechanisms by which alcohol exposure impairs granule cell migration. PMID:18075250

  19. Neuronal Neuregulin 1 type III directs Schwann cell migration

    PubMed Central

    Perlin, Julie R.; Lush, Mark E.; Stephens, W. Zac; Piotrowski, Tatjana; Talbot, William S.

    2011-01-01

    During peripheral nerve development, each segment of a myelinated axon is matched with a single Schwann cell. Tight regulation of Schwann cell movement, proliferation and differentiation is essential to ensure that these glial cells properly associate with axons. ErbB receptors are required for Schwann cell migration, but the operative ligand and its mechanism of action have remained unknown. We demonstrate that zebrafish Neuregulin 1 (Nrg1) type III, which signals through ErbB receptors, controls Schwann cell migration in addition to its previously known roles in proliferation and myelination. Chimera analyses indicate that ErbB receptors are required in all migrating Schwann cells, and that Nrg1 type III is required in neurons for migration. Surprisingly, expression of the ligand in a few axons is sufficient to induce migration along a chimeric nerve constituted largely of nrg1 type III mutant axons. These studies also reveal a mechanism that allows Schwann cells to fasciculate axons regardless of nrg1 type III expression. Time-lapse imaging of transgenic embryos demonstrated that misexpression of human NRG1 type III results in ectopic Schwann cell migration, allowing them to aberrantly enter the central nervous system. These results demonstrate that Nrg1 type III is an essential signal that controls Schwann cell migration to ensure that these glia are present in the correct numbers and positions in developing nerves. PMID:21965611

  20. Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland.

    PubMed

    Sanderson, Julie; Dartt, Darlene A; Trinkaus-Randall, Vickery; Pintor, Jesus; Civan, Mortimer M; Delamere, Nicholas A; Fletcher, Erica L; Salt, Thomas E; Grosche, Antje; Mitchell, Claire H

    2014-10-01

    This review highlights recent findings that describ how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca(2+) concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target

  1. Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland

    PubMed Central

    Sanderson, Julie; Dartt, Darlene A.; Trinkaus-Randall, Vickery; Pintor, Jesus; Civan, Mortimer M.; Delamere, Nicholas A.; Fletcher, Erica L.; Salt, Thomas E.; Grosche, Antje; Mitchell, Claire H.

    2014-01-01

    This review highlights recent findings that describe how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca2+ concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target

  2. Regulation of C6 glioma cell migration by thymol

    PubMed Central

    LEE, KANG PA; KIM, JAI-EUN; PARK, WON-HWAN; HONG, HEEOK

    2016-01-01

    Tumor cell motility exhibits a crucial role in tumor development. Therefore, the present study aimed to investigate whether thymol could reduce C6 glioma cell migration. Cell viability was determined using the EZ-Cytox Cell Viability kit. The scratch wound healing and Boyden chamber assays were performed to test C6 glioma cell migration in the presence of fetal bovine serum (FBS). Additionally, the study investigated whether signaling proteins relevant to C6 glioma cell migration, i.e., extracellular signal-regulated kinases (ERK)1/2, protein kinase Cα (PKCα), matrix metallopeptidase (MMP)9 and MMP2, were affected by thymol treatment. Up to 30 µM, thymol did not alter cell viability, whereas 100 µM thymol induced the death of ~20% of the cells. Furthermore, thymol (30 µM) significantly reduced FBS-induced migration. In the FBS-stimulated C6 glioma cells, thymol (30 µM) suppressed PKCα and ERK1/2 phosphorylation. MMP9 and MMP2 production was also significantly reduced by treatment with 30 µM thymol in the C6 glioma cells. Taken together, these results indicate that thymol attenuates C6 glioma cell migration. Additionally, the study suggests that the effect of thymol on the FBS-induced migration of C6 glioma cells affects PKCα and ERK1/2 signaling, and suppresses MMP9 and MMP2 production. PMID:27073528

  3. Silk Film Topography Directs Collective Epithelial Cell Migration

    PubMed Central

    Rosenblatt, Mark I.

    2012-01-01

    The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

  4. Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells

    PubMed Central

    Yang, Shuai; Yao, Haipei; Li, Min; Li, Hui; Wang, Fang

    2016-01-01

    Purpose To study the role of long non-coding RNA (lncRNA) MALAT1 in transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. Methods ARPE-19 cells were cultured and exposed to TGF-β1. The EMT of APRE-19 cells is confirmed by morphological change, as well as the increased expression of alpha-smooth muscle actin (αSMA) and fibronectin, and the down-regulation of E-cadherin and Zona occludin-1(ZO-1) at both mRNA and protein levels. The expression of lncRNA MALAT1 in RPE cells were detected by quantitative real-time PCR. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). The effect of inhibition of MALAT1 on EMT, migration, proliferation, and TGFβ signalings were observed. MALAT1 expression was also detected in primary RPE cells incubated with proliferative vitreoretinopathy (PVR) vitreous samples. Results The expression of MALAT1 is significantly increased in RPE cells incubated with TGFβ1. MALAT1 silencing attenuates TGFβ1-induced EMT, migration, and proliferation of RPE cells, at least partially through activating Smad2/3 signaling. MALAT1 is also significantly increased in primary RPE cells incubated with PVR vitreous samples. Conclusion LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provides new understandings for the pathogenesis of PVR. PMID:27019196

  5. Cell migration in the normal and pathological postnatal mammalian brain

    PubMed Central

    Canoll, Peter; Goldman, James E.

    2009-01-01

    In the developing brain, cell migration is a crucial process for structural organization, and is therefore highly regulated to allow the correct formation of complex networks, wiring neurons, and glia. In the early postnatal brain, late developmental processes such as the production and migration of astrocyte and oligodendrocyte progenitors still occur. Although the brain is completely formed and structured few weeks after birth, it maintains a degree of plasticity throughout life, including axonal remodeling, synaptogenesis, but also neural cell birth, migration and integration. The subventricular zone (SVZ) and the dentate gyrus of the hippocampus (DG) are the two main neurogenic niches in the adult brain. Neural stem cells reside in these structures and produce progenitors that migrate toward their ultimate location: the olfactory bulb and granular cell layer of the DG respectively. The aim of this review is to synthesize the increasing information concerning the organization, regulation and function of cell migration in a mature brain. In a normal brain, protein involved in cell-cell or cell-matrix interactions together with secreted proteins acting as chemoattractant or chemorepellant play key roles in the regulation of neural progenitor cell migration. In addition, recent data suggest that gliomas arise from the transformation of neural stem cells or progenitor cells and that glioma cell infiltration recapitulates key aspects of glial progenitor migration. Thus, we will consider glioma migration in the context of progenitor migration. Finally, many observations show that brain lesions and neurological diseases trigger neural stem/progenitor cell activation and migration towards altered structures. The factors involved in such cell migration/recruitment are just beginning to be understood. Inflammation which has long been considered as thoroughly disastrous for brain repair is now known to produce some positive effects on stem/progenitor cell recruitment via

  6. Single cell migration dynamics mediated by geometric confinement.

    PubMed

    Zhang, Hua; Hou, Ruixia; Xiao, Peng; Xing, Rubo; Chen, Tao; Han, Yanchun; Ren, Penggang; Fu, Jun

    2016-09-01

    The migration dynamics of cells plays a key role in tissue engineering and regenerative medicine. Previous studies mostly focus on regulating stem cell fate and phenotype by biophysical cues. In contrast, less is known about how the geometric cues mediate the migration dynamics of cells. Here, we fabricate graphene oxide (GO) microstripes on cell non-adhesive PEG substrate by using micromolding in capillary (MIMIC) method. Such micropatterns with alternating cell adhesion and cell resistance enable an effective control of selective adhesion and migration of single cells. The sharp contrast in cell adhesion minimizes the invasion of cells into the PEG patterns, and thereby strongly confines the cells on GO microstripes. As a result, the cells are forced to adapt highly polarized, elongated, and oriented geometry to fit the patterns. A series of pattern widths have been fabricated to modulate the extent of cell deformation and polarization. Under strong confinement, the cytoskeleton contractility, intracellular traction, and actin filament elongation are highly promoted, which result in enhanced cell migration along the patterns. This work provides an important insight into developing combinatorial graphene-based patterns for the control of cell migration dynamics, which is of great significance for tissue engineering and regenerative medicine. PMID:27137805

  7. Glass-like dynamics in collective cell migration

    NASA Astrophysics Data System (ADS)

    Angelini, Thomas; Weitz, David

    2011-03-01

    The collective movement of tissue cells is essential to fundamental biological processes in both health and disease, and occurs throughout embryonic development, during wound healing, and in cancerous tumor invasion. Most knowledge of cell migration, however, comes from single cell studies. Single cells migrate by executing cyclic processes of extension, adhesion, and retraction, during which the cell body fluctuates dramatically and the cell changes direction erratically. These sub-cellular motions must be coupled between neighbors in confluent layers, yet the influence of this coupling on collective migration is not known. In this talk we present a study of motion in confluent epithelial cell sheets. We measure collective migration and sub-cellular motions, covering a broad range of length-scales, time-scales, and cell densities. We find that that collective cell migration exhibits many behaviors characteristic of classical supercooled particulate fluids, including growing dynamic heterogeneities in the migration velocity field, non-Arrhenius relaxation behavior, and peaks in the density of states analogous to the Boson peak. These results provide a suggestive analogy between collective cell motion and the dynamics of supercooled fluids approaching a glass transition.

  8. Bioengineering Paradigms for Cell Migration in Confined Microenvironments

    PubMed Central

    Stroka, Kimberly M.; Gu, Zhizhan; Sun, Sean X.; Konstantopoulos, Konstantinos

    2014-01-01

    Cell migration is a fundamental process underlying diverse (patho)physiological phenomena. The classical understanding of the molecular mechanisms of cell migration has been based on in vitro studies on two-dimensional substrates. More recently, mounting evidence from intravital studies has shown that during metastasis, tumor cells must navigate complex microenvironments in vivo, including narrow, pre-existing microtracks created by anatomical structures. It is becoming apparent that unraveling the mechanisms of confined cell migration in this context requires a multi-disciplinary approach through integration of in vivo and in vitro studies, along with sophisticated bioengineering techniques and mathematical modeling. Here, we highlight such an approach that has led to discovery of a new model for cell migration in confined microenvironments (i.e., the Osmotic Engine Model). PMID:24973724

  9. Bioengineering paradigms for cell migration in confined microenvironments.

    PubMed

    Stroka, Kimberly M; Gu, Zhizhan; Sun, Sean X; Konstantopoulos, Konstantinos

    2014-10-01

    Cell migration is a fundamental process underlying diverse (patho)physiological phenomena. The classical understanding of the molecular mechanisms of cell migration has been based on in vitro studies on two-dimensional substrates. More recently, mounting evidence from intravital studies has shown that during metastasis, tumor cells must navigate complex microenvironments in vivo, including narrow, pre-existing microtracks created by anatomical structures. It is becoming apparent that unraveling the mechanisms of confined cell migration in this context requires a multi-disciplinary approach through integration of in vivo and in vitro studies, along with sophisticated bioengineering techniques and mathematical modeling. Here, we highlight such an approach that has led to discovery of a new model for cell migration in confined microenvironments (i.e., the Osmotic Engine Model). PMID:24973724

  10. Migration of adhesive glioma cells: Front propagation and fingering

    NASA Astrophysics Data System (ADS)

    Khain, Evgeniy; Katakowski, Mark; Charteris, Nicholas; Jiang, Feng; Chopp, Michael

    2012-07-01

    We investigate the migration of glioma cells as a front propagation phenomenon both theoretically (by using both discrete lattice modeling and a continuum approach) and experimentally. For small effective strength of cell-cell adhesion q, the front velocity does not depend on q. When q exceeds a critical threshold, a fingeringlike front propagation is observed due to cluster formation in the invasive zone. We show that the experiments correspond to the transient regime, before the regime of front propagation is established. We performed an additional experiment on cell migration. A detailed comparison with experimental observations showed that the theory correctly predicts the maximal migration distance but underestimates the migration of the main mass of cells.

  11. Fundulus deep cells: directional migration in response to epithelial wounding.

    PubMed

    Fink, R D; Trinkaus, J P

    1988-09-01

    During the normal embryogenesis of the killifish Fundulus heteroclitus deep cells migrate in an apparently random fashion throughout the subepithelial space of the yolk sac. These cells migrate by blebbing locomotion, and individual cells show tendencies for persistence in the directionality of their movement. Immediately after the wounding of the yolk sac epithelium (the enveloping layer), these deep cells reorient and migrate directionally toward the site of wound closure. This directional migration results in the aggregation of a large number of cells at the wound site. The response is both rapid and widespread; cells as far away as 800 micron respond as quickly as those nearby, and by 100 min after wounding up to 90% of the blebbing deep cells within this radius have clustered about the wound site. Then, cells begin to disperse, and by 150 min after wounding, it is almost impossible to tell where the wound had been made. Because of the transparency of the Fundulus yolk sac, this phenomenon can be utilized as a model system for observing details of in vivo directional cell movements. Time-lapse video micrography has revealed that the modes, rates, and overall cell morphologies during locomotion are identical for cells migrating in both unwounded and wounded embryos. What is different in the wounded embryos is that a single directionality is imposed upon a large population of cells, resulting in aggregation. Several aspects of the aggregation phenomenon suggest that a possible attractant originating at the wound site may travel through the subepithelial space by diffusion.

  12. Dynamic cell adhesion and migration on nanoscale grooved substrates.

    PubMed

    Lamers, E; te Riet, J; Domanski, M; Luttge, R; Figdor, C G; Gardeniers, J G E; Walboomers, X F; Jansen, J A

    2012-01-01

    Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.

  13. Live Imaging of Border Cell Migration in Drosophila.

    PubMed

    Dai, Wei; Montell, Denise J

    2016-01-01

    Border cells are a cluster of cells that migrate from the anterior tip of the Drosophila egg chamber to the border of the oocyte in stage 9. They serve as a useful model to study collective cell migration in a native tissue environment. Here we describe a protocol for preparing ex vivo egg chamber cultures from transgenic flies expressing fluorescent proteins in the border cells, and using confocal microscopy to take a multi-positional time-lapse movie. We include an image analysis method for tracking border cell cluster dynamics as well as tracking individual cell movements. PMID:27271901

  14. Live Imaging of Border Cell Migration in Drosophila.

    PubMed

    Dai, Wei; Montell, Denise J

    2016-01-01

    Border cells are a cluster of cells that migrate from the anterior tip of the Drosophila egg chamber to the border of the oocyte in stage 9. They serve as a useful model to study collective cell migration in a native tissue environment. Here we describe a protocol for preparing ex vivo egg chamber cultures from transgenic flies expressing fluorescent proteins in the border cells, and using confocal microscopy to take a multi-positional time-lapse movie. We include an image analysis method for tracking border cell cluster dynamics as well as tracking individual cell movements.

  15. The thioredoxin system in breast cancer cell invasion and migration.

    PubMed

    Bhatia, Maneet; McGrath, Kelly L; Di Trapani, Giovanna; Charoentong, Pornpimol; Shah, Fenil; King, Mallory M; Clarke, Frank M; Tonissen, Kathryn F

    2016-08-01

    Metastasis is the most life threatening aspect of breast cancer. It is a multi-step process involving invasion and migration of primary tumor cells with a subsequent colonization of these cells at a secondary location. The aim of the present study was to investigate the role of thioredoxin (Trx1) in the invasion and migration of breast cancer cells and to assess the strength of the association between high levels of Trx1 and thioredoxin reductase (TrxR1) expression with breast cancer patient survival. Our results indicate that the expression of both Trx1 and TrxR1 are statistically significantly increased in breast cancer patient cells compared with paired normal breast tissue from the same patient. Over-expression of Trx1 in MDA-MB-231 breast cancer cell lines enhanced cell invasion in in vitro assays while expression of a redox inactive mutant form of Trx1 (designated 1SS) or the antisense mRNA inhibited cell invasion. Addition of exogenous Trx1 also enhanced cell invasion, while addition of a specific monoclonal antibody that inhibits Trx1 redox function decreased cell invasion. Over-expression of intracellular Trx1 did not increase cell migration but expression of intracellular 1SS inhibited migration. Addition of exogenous Trx1 enhanced cell migration while 1SS had no effect. Treatment with auranofin inhibited TrxR activity, cell migration and clonogenic activity of MDA-MB-231 cells, while increasing reactive oxygen species (ROS) levels. Analysis of 25 independent cohorts with 5910 patients showed that Trx1 and TrxR1 were both associated with a poor patient prognosis in terms of overall survival, distant metastasis free survival and disease free survival. Therefore, targeting the Trx system with auranofin or other specific inhibitors may provide improved breast cancer patient outcomes through inhibition of cancer invasion and migration.

  16. The thioredoxin system in breast cancer cell invasion and migration.

    PubMed

    Bhatia, Maneet; McGrath, Kelly L; Di Trapani, Giovanna; Charoentong, Pornpimol; Shah, Fenil; King, Mallory M; Clarke, Frank M; Tonissen, Kathryn F

    2016-08-01

    Metastasis is the most life threatening aspect of breast cancer. It is a multi-step process involving invasion and migration of primary tumor cells with a subsequent colonization of these cells at a secondary location. The aim of the present study was to investigate the role of thioredoxin (Trx1) in the invasion and migration of breast cancer cells and to assess the strength of the association between high levels of Trx1 and thioredoxin reductase (TrxR1) expression with breast cancer patient survival. Our results indicate that the expression of both Trx1 and TrxR1 are statistically significantly increased in breast cancer patient cells compared with paired normal breast tissue from the same patient. Over-expression of Trx1 in MDA-MB-231 breast cancer cell lines enhanced cell invasion in in vitro assays while expression of a redox inactive mutant form of Trx1 (designated 1SS) or the antisense mRNA inhibited cell invasion. Addition of exogenous Trx1 also enhanced cell invasion, while addition of a specific monoclonal antibody that inhibits Trx1 redox function decreased cell invasion. Over-expression of intracellular Trx1 did not increase cell migration but expression of intracellular 1SS inhibited migration. Addition of exogenous Trx1 enhanced cell migration while 1SS had no effect. Treatment with auranofin inhibited TrxR activity, cell migration and clonogenic activity of MDA-MB-231 cells, while increasing reactive oxygen species (ROS) levels. Analysis of 25 independent cohorts with 5910 patients showed that Trx1 and TrxR1 were both associated with a poor patient prognosis in terms of overall survival, distant metastasis free survival and disease free survival. Therefore, targeting the Trx system with auranofin or other specific inhibitors may provide improved breast cancer patient outcomes through inhibition of cancer invasion and migration. PMID:26760912

  17. The thioredoxin system in breast cancer cell invasion and migration

    PubMed Central

    Bhatia, Maneet; McGrath, Kelly L.; Di Trapani, Giovanna; Charoentong, Pornpimol; Shah, Fenil; King, Mallory M.; Clarke, Frank M.; Tonissen, Kathryn F.

    2015-01-01

    Metastasis is the most life threatening aspect of breast cancer. It is a multi-step process involving invasion and migration of primary tumor cells with a subsequent colonization of these cells at a secondary location. The aim of the present study was to investigate the role of thioredoxin (Trx1) in the invasion and migration of breast cancer cells and to assess the strength of the association between high levels of Trx1 and thioredoxin reductase (TrxR1) expression with breast cancer patient survival. Our results indicate that the expression of both Trx1 and TrxR1 are statistically significantly increased in breast cancer patient cells compared with paired normal breast tissue from the same patient. Over-expression of Trx1 in MDA-MB-231 breast cancer cell lines enhanced cell invasion in in vitro assays while expression of a redox inactive mutant form of Trx1 (designated 1SS) or the antisense mRNA inhibited cell invasion. Addition of exogenous Trx1 also enhanced cell invasion, while addition of a specific monoclonal antibody that inhibits Trx1 redox function decreased cell invasion. Over-expression of intracellular Trx1 did not increase cell migration but expression of intracellular 1SS inhibited migration. Addition of exogenous Trx1 enhanced cell migration while 1SS had no effect. Treatment with auranofin inhibited TrxR activity, cell migration and clonogenic activity of MDA-MB-231 cells, while increasing reactive oxygen species (ROS) levels. Analysis of 25 independent cohorts with 5910 patients showed that Trx1 and TrxR1 were both associated with a poor patient prognosis in terms of overall survival, distant metastasis free survival and disease free survival. Therefore, targeting the Trx system with auranofin or other specific inhibitors may provide improved breast cancer patient outcomes through inhibition of cancer invasion and migration. PMID:26760912

  18. Collective dynamics of cell migration and cell rearrangements

    NASA Astrophysics Data System (ADS)

    Kabla, Alexandre

    Understanding multicellular processes such as embryo development or cancer metastasis requires to decipher the contributions of local cell autonomous behaviours and long range interactions with the tissue environment. A key question in this context concerns the emergence of large scale coordination in cell behaviours, a requirement for collective cell migration or convergent extension. I will present a few examples where physical and mechanical aspects play a significant role in driving tissue scale dynamics.

  1. Cell migration and division in amoeboid-like fission yeast

    PubMed Central

    Flor-Parra, Ignacio; Bernal, Manuel; Zhurinsky, Jacob; Daga, Rafael R.

    2014-01-01

    Summary Yeast cells are non-motile and are encased in a cell wall that supports high internal turgor pressure. The cell wall is also essential for cellular morphogenesis and cell division. Here, we report unexpected morphogenetic changes in a Schizosaccharomyces pombe mutant defective in cell wall biogenesis. These cells form dynamic cytoplasmic protrusions caused by internal turgor pressure and also exhibit amoeboid-like cell migration resulting from repeated protrusive cycles. The cytokinetic ring responsible for cell division in wild-type yeast often fails in these cells; however, they were still able to divide using a ring-independent alternative mechanism relying on extrusion of the cell body through a hole in the cell wall. This mechanism of cell division may resemble an ancestral mode of division in the absence of cytokinetic machinery. Our findings highlight how a single gene change can lead to the emergence of different modes of cell growth, migration and division. PMID:24357230

  2. Albumen Transport to Bruch's Membrane and RPE by Choriocapillaris Caveolae

    PubMed Central

    Nakanishi, Masataka; Grebe, Rhonda; Bhutto, Imran A.; Edwards, Malia; McLeod, D. Scott; Lutty, Gerard A.

    2016-01-01

    Purpose The choriocapillaris (CC), the capillary network of the choroid, is positioned adjacent to Bruch's membrane (BM) and the RPE. The aim of this study was to clarify the mechanism(s) for transport of serum albumen from CC lumen to RPE. Methods Alexa647 conjugated to BSA (BSA-A647) or PBS was administrated via the femoral vein to young and aged wild-type (WT; C57BL/6J) mice and Caveolin-1 knockout mice (Cav1−/−). Mice were perfused with PBS and killed at 30 minutes, 1 hour, and 4 hours after injection. Eyecups were cryopreserved, and cryosections were analyzed on a Zeiss 710 confocal microscope. Bovine serum albumin conjugated to gold nanoparticles (BSA-GNP) was administrated through the left common carotid artery. Mice were perfused with PBS and killed at 30 minutes after injection. Eyecups were embedded after fixation, and 70-nm-thick sections were analyzed on a Hitachi H7600 transmission electron microscope. Results In eyes of WT young mice, BSA-A647 was transported to the RPE at 30 minutes and diffused to the photoreceptor layer by 1 hour. In contrast, most BSA-A647 was found in the CC in Cav1−/− eyes. The majority of BSA-GNP found in the CC of young WT mice was on the luminal side in caveolae at 30 minutes after injection. In aged WT mice, BSA-GNPs were found in defective tight junctions between endothelial cells and appeared trapped at the diaphragm of fenestrations. Conclusions Normally, CC carefully regulates transport system of BSA from lumen to BM by caveolae-mediated transcytosis; however, endothelium cells of aged control WT mice have leaky tight junctions and lacked regulated BSA transport. PMID:27116549

  3. Glutamate involvement in calcium–dependent migration of astrocytoma cells

    PubMed Central

    2014-01-01

    Background Astrocytoma are known to have altered glutamate machinery that results in the release of large amounts of glutamate into the extracellular space but the precise role of glutamate in favoring cancer processes has not yet been fully established. Several studies suggested that glutamate might provoke active killing of neurons thereby producing space for cancer cells to proliferate and migrate. Previously, we observed that calcium promotes disassembly of integrin-containing focal adhesions in astrocytoma, thus providing a link between calcium signaling and cell migration. The aim of this study was to determine how calcium signaling and glutamate transmission cooperate to promote enhanced astrocytoma migration. Methods The wound-healing model was used to assay migration of human U87MG astrocytoma cells and allowed to monitor calcium signaling during the migration process. The effect of glutamate on calcium signaling was evaluated together with the amount of glutamate released by astrocytoma during cell migration. Results We observed that glutamate stimulates motility in serum-starved cells, whereas in the presence of serum, inhibitors of glutamate receptors reduce migration. Migration speed was also reduced in presence of an intracellular calcium chelator. During migration, cells displayed spontaneous Ca2+ transients. L-THA, an inhibitor of glutamate re-uptake increased the frequency of Ca2+ oscillations in oscillating cells and induced Ca2+ oscillations in quiescent cells. The frequency of migration-associated Ca2+ oscillations was reduced by prior incubation with glutamate receptor antagonists or with an anti-β1 integrin antibody. Application of glutamate induced increases in internal free Ca2+ concentration ([Ca2+]i). Finally we found that compounds known to increase [Ca2+]i in astrocytomas such as thapsigagin, ionomycin or the metabotropic glutamate receptor agonist t-ACPD, are able to induce glutamate release. Conclusion Our data demonstrate that

  4. Cerium migration during PEM fuel cell assembly and operation

    SciTech Connect

    Baker, Andrew M.; Torraco, Dennis; Judge, Elizabeth J.; Spernjak, Dusan; Mukundan, Rangachary; Borup, Rod L.; Advani, Suresh G.; Prasad, Ajay K.

    2015-10-02

    Cerium migration between PEM fuel cell components is influenced by potential-driven mobility, ionic diffusion, and gradients in water content. These factors were investigated in ex situ experiments and in operating fuel cells. Potential-induced migration was measured ex situ in hydrated window cells. Cerium-containing MEAs were also fabricated and tested under ASTs. MEA disassembly and subsequent XRF analysis were used to observe rapid cerium migration during cell assembly and operation. During MEA hot pressing, humidification, and low RH operation at OCV, ionic diffusion causes uniform migration from the membrane into the catalyst layers. During high RH operation at OCV, in-plane cerium gradients arise due to variations in water content. These gradients may diminish the scavenging efficacy of cerium by reducing its proximity to generated radicals.

  5. Cerium migration during PEM fuel cell assembly and operation

    DOE PAGES

    Baker, Andrew M.; Torraco, Dennis; Judge, Elizabeth J.; Spernjak, Dusan; Mukundan, Rangachary; Borup, Rod L.; Advani, Suresh G.; Prasad, Ajay K.

    2015-09-14

    Cerium migration between PEM fuel cell components is influenced by potential-driven mobility, ionic diffusion, and gradients in water content. These factors were investigated in ex situ experiments and in operating fuel cells. Potential-induced migration was measured ex situ in hydrated window cells. Cerium-containing MEAs were also fabricated and tested under ASTs. MEA disassembly and subsequent XRF analysis were used to observe rapid cerium migration during cell assembly and operation. During MEA hot pressing, humidification, and low RH operation at OCV, ionic diffusion causes uniform migration from the membrane into the catalyst layers. During high RH operation at OCV, in-plane ceriummore » gradients arise due to variations in water content. These gradients may diminish the scavenging efficacy of cerium by reducing its proximity to generated radicals.« less

  6. Cerium migration during PEM fuel cell assembly and operation

    SciTech Connect

    Baker, Andrew M.; Torraco, Dennis; Judge, Elizabeth J.; Spernjak, Dusan; Mukundan, Rangachary; Borup, Rod L.; Advani, Suresh G.; Prasad, Ajay K.

    2015-09-14

    Cerium migration between PEM fuel cell components is influenced by potential-driven mobility, ionic diffusion, and gradients in water content. These factors were investigated in ex situ experiments and in operating fuel cells. Potential-induced migration was measured ex situ in hydrated window cells. Cerium-containing MEAs were also fabricated and tested under ASTs. MEA disassembly and subsequent XRF analysis were used to observe rapid cerium migration during cell assembly and operation. During MEA hot pressing, humidification, and low RH operation at OCV, ionic diffusion causes uniform migration from the membrane into the catalyst layers. During high RH operation at OCV, in-plane cerium gradients arise due to variations in water content. These gradients may diminish the scavenging efficacy of cerium by reducing its proximity to generated radicals.

  7. Phosphorylation of actopaxin regulates cell spreading and migration

    PubMed Central

    Clarke, Dominic M.; Brown, Michael C.; LaLonde, David P.; Turner, Christopher E.

    2004-01-01

    Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration. PMID:15353548

  8. Interior decoration: tropomyosin in actin dynamics and cell migration.

    PubMed

    Lees, Justin G; Bach, Cuc T T; O'Neill, Geraldine M

    2011-01-01

    Cell migration and invasion requires the precise temporal and spatial orchestration of a variety of biological processes. Filaments of polymerized actin are critical players in these diverse processes, including the regulation of cell anchorage points (both cell-cell and cell-extracellular matrix), the uptake and delivery of molecules via endocytic pathways and the generation of force for both membrane protrusion and retraction. How the actin filaments are specialized for each of these discrete functions is yet to be comprehensively elucidated. The cytoskeletal tropomyosins are a family of actin associating proteins that form head-to-tail polymers which lay in the major groove of polymerized actin filaments. In the present review we summarize the emerging isoform-specific functions of tropomyosins in cell migration and invasion and discuss their potential roles in the specialization of actin filaments for the diverse cellular processes that together regulate cell migration and invasion.

  9. Estrogen Stimulation of Cell Migration Involves Multiple Signaling Pathway Interactions

    PubMed Central

    Li, Yan; Wang, Ji-Ping; Santen, Richard J.; Kim, Tae-Hyun; Park, Hoyong; Fan, Ping; Yue, Wei

    2010-01-01

    Hormone-dependent breast cancers respond to inhibitors of estrogen synthesis or action with tumor regression and with a reduction of new metastases. The mechanisms underlying the effects of estrogen on metastasis likely differ from those on tumor regression. Cell migration is a key first step in the metastatic process. Based on our prior work and other published data, we designed and tested a working model that suggested that estrogen receptor α, epidermal growth factor receptor, focal adhesion kinase (FAK), paxillin, phosphatidylinositol 3 kinase, p60 Src tyrosine kinase (c-Src), c-Jun N-terminal kinase, and MAPK interact to facilitate estradiol (E2)-induced cell migration. Accordingly, we examined the effect of E2 on activation of these pathways and demonstrated mechanistic effects by blocking each component and assessing cell migration as a biologic endpoint. Initial studies validated a robust cell migration assay characterized by highly reproducible, dose-dependent responses to E2. Examining various mechanisms involved in migration, we showed that E2 induced activation of c-Src, FAK, and paxillin with early peaks within 5–30 min and later peaks at 24 h. ERK and protein kinase B phosphorylation exhibited only early peaks. Blockade of various steps in these signaling pathways with use of small interfering RNA or specific inhibitors demonstrated mechanistic effects of these signaling molecules on cell migration. Our results suggest that the effects of E2 on cell migration involve multiple, interacting signaling pathways. Important effects are mediated by the MAPK, phosphatidylinositol 3 kinase, and c-Jun N-terminal kinase pathways and use FAK, paxillin, and c-Src for activation. Each pathway represents a potential target for blocking cell migration and metastasis of breast cancer cells. PMID:20861240

  10. Displacement measurement of the depth migration of transparent cells

    SciTech Connect

    Yoshida, Makoto; Ishimaru, Ichirou; Ishizaki, Katsumi; Yasokawa, Toshiki; Kuriyama, Shigeki; Masaki, Tsutomu; Nakai, Seiji; Takegawa, Kaoru; Tanaka, Naoyuki

    2006-12-11

    This letter reports a method for displacement measurement of the depth migration of transparent cells. This proposed optical spatial filtering method allows visualization of the transparent cells and determination of depth migration as a horizontal displacement positive or negative first order diffracted light on the detector surface. When the sample is displaced upward or downward from the focal plane, first and negative first order diffracted light form images at a different point as a light circle. The coordinates of these two light circles on the detector surface change places when the displacement of depth migration moves to the opposite direction.

  11. Mast cell heparin stimulates migration of capillary endothelial cells in vitro

    PubMed Central

    1980-01-01

    Migration of capillary endothelial cells is an important component of angiogenesis in vivo. Increased numbers of mast cells have been associated with several types of angiogenesis. We have used a quantitative assay in vitro to demonstrate that mast cells release a factor that significantly increases bovine capillary endothelial cell migration. The factor is present in medium conditioned by mast cells as well as lysates of mast cells. The stimulatory effect of mast cells on migration is specific for capillary endothelial cells. Furthermore, mast cells have no mitogenic activity for capillary endothelial cells. Of all the secretory products of mast cells tested, only heparin stimulated capillary endothelial cell migration in vitro. Heparin preparations from a variety of sources stimulated capillary endothelial cell migration to the same degree but did not stimulate migration of several other cell types. The migration activity of heparin and mast cell conditioned medium was blocked by specific antagonists of heparin (protamine and heparinase), but not by chondroitinase ABC. The migration activity of mast cell conditioned medium was resistant to heat (100 degrees C) and incubation with proteolytic enzymes. These results suggest that the role of mast cells in angiogenesis may be to enhance migration of the endothelial cells of growing capillaries. PMID:7420025

  12. Hedgehog does not guide migrating Drosophila germ cells

    PubMed Central

    Renault, Andrew D.; Ricardo, Sara; Kunwar, Prabhat S.; Santos, Ana; Starz-Gaiano, Michelle; Stein, Jennifer; Lehmann, Ruth

    2009-01-01

    In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies. PMID:19389345

  13. Membrane nanowaves in single and collective cell migration.

    PubMed

    Zouani, Omar F; Gocheva, Veronika; Durrieu, Marie-Christine

    2014-01-01

    We report the characterization of three-dimensional membrane waves for migrating single and collective cells and describe their propagation using wide-field optical profiling technique with nanometer resolution. We reveal the existence of small and large membrane waves the amplitudes of which are in the range of ∼ 3-7 nm to ∼ 16-25 nm respectively, through the cell. For migrating single-cells, the amplitude of these waves is about 30 nm near the cell edge. Two or more different directions of propagation of the membrane nanowaves inside the same cell can be observed. After increasing the migration velocity by BMP-2 treatment, only one wave direction of propagation exists with an increase in the average amplitude (more than 80 nm near the cell edge). Furthermore for collective-cell migration, these membrane nanowaves are attenuated on the leader cells and poor transmission of these nanowaves to follower cells was observed. After BMP-2 treatment, the membrane nanowaves are transmitted from the leader cell to several rows of follower cells. Surprisingly, the vast majority of the observed membrane nanowaves is shared between the adjacent cells. These results give a new view on how single and collective-cells modulate their motility. This work has significant implications for the therapeutic use of BMPs for the regeneration of skin tissue. PMID:24846182

  14. Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis

    PubMed Central

    Jiang, Mei; Esteve-Rudd, Julian; Lopes, Vanda S.; Diemer, Tanja; Lillo, Concepción; Rump, Agrani

    2015-01-01

    The degradation of phagosomes, derived from the ingestion of photoreceptor outer segment (POS) disk membranes, is a major role of the retinal pigment epithelium (RPE). Here, POS phagosomes were observed to associate with myosin-7a, and then kinesin-1, as they moved from the apical region of the RPE. Live-cell imaging showed that the phagosomes moved bidirectionally along microtubules in RPE cells, with kinesin-1 light chain 1 (KLC1) remaining associated in both directions and during pauses. Lack of KLC1 did not inhibit phagosome speed, but run length was decreased, and phagosome localization and degradation were impaired. In old mice, lack of KLC1 resulted in RPE pathogenesis that was strikingly comparable to aspects of age-related macular degeneration (AMD), with an excessive accumulation of RPE and sub-RPE deposits, as well as oxidative and inflammatory stress responses. These results elucidate mechanisms of POS phagosome transport in relation to degradation, and demonstrate that defective microtubule motor transport in the RPE leads to phenotypes associated with AMD. PMID:26261180

  15. Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis.

    PubMed

    Jiang, Mei; Esteve-Rudd, Julian; Lopes, Vanda S; Diemer, Tanja; Lillo, Concepción; Rump, Agrani; Williams, David S

    2015-08-17

    The degradation of phagosomes, derived from the ingestion of photoreceptor outer segment (POS) disk membranes, is a major role of the retinal pigment epithelium (RPE). Here, POS phagosomes were observed to associate with myosin-7a, and then kinesin-1, as they moved from the apical region of the RPE. Live-cell imaging showed that the phagosomes moved bidirectionally along microtubules in RPE cells, with kinesin-1 light chain 1 (KLC1) remaining associated in both directions and during pauses. Lack of KLC1 did not inhibit phagosome speed, but run length was decreased, and phagosome localization and degradation were impaired. In old mice, lack of KLC1 resulted in RPE pathogenesis that was strikingly comparable to aspects of age-related macular degeneration (AMD), with an excessive accumulation of RPE and sub-RPE deposits, as well as oxidative and inflammatory stress responses. These results elucidate mechanisms of POS phagosome transport in relation to degradation, and demonstrate that defective microtubule motor transport in the RPE leads to phenotypes associated with AMD.

  16. Action spectrum for photochemical retinal pigment epithelium (RPE) disruption in an in vivo monkey model

    NASA Astrophysics Data System (ADS)

    Zhang, Jie; Sabarinathan, Ranjani; Bubel, Tracy; Williams, David R.; Hunter, Jennifer J.

    2016-03-01

    Observations of RPE disruption and autofluorescence (AF) photobleaching at light levels below the ANSI photochemical maximum permissible exposure (MPE) (Morgan et al., 2008) indicates a demand to modify future light safety standards to protect the retina from harm. To establish safe light exposures, we measured the visible light action spectrum for RPE disruption in an in vivo monkey model with fluorescence adaptive optics retinal imaging. Using this high resolution imaging modality can provide insight into the consequences of light on a cellular level and allow for longitudinal monitoring of retinal changes. The threshold retinal radiant exposures (RRE) for RPE disruption were determined for 4 wavelengths (460, 488, 544, and 594 nm). The anaesthetized macaque retina was exposed to a uniform 0.5° × 0.5° field of view (FOV). Imaging within a 2° × 2° FOV was performed before, immediately after and at 2 week intervals for 10 weeks. At each wavelength, multiple RREs were tested with 4 repetitions each to determine the threshold for RPE disruption. For qualitative analysis, RPE disruption is defined as any detectable change from the pre exposure condition in the cell mosaic in the exposed region relative to the corresponding mosaic in the immediately surrounding area. We have tested several metrics to evaluate the RPE images obtained before and after exposure. The measured action spectrum for photochemical RPE disruption has a shallower slope than the current ANSI photochemical MPE for the same conditions and suggests that longer wavelength light is more hazardous than other measurements would suggest.

  17. Energy barriers and cell migration in confluent tissues

    NASA Astrophysics Data System (ADS)

    Bi, Dapeng; Lopez, J. H.; Schwarz, J. M.; Manning, M. Lisa

    2014-03-01

    Biological processes such as embryogensis, tumorigenesis and wound healing require cells to move within a tissue. While the migration of single cells has been extensively studied, it has remained unclear how single cell properties control migration through a confluent tissue. We develop numerical and theoretical models to calculate energy barriers to cell rearrangements, which govern cell motility. In contrast to sheared foams where energy barriers are power-law distributed, energy barriers in tissues are exponentially distributed and depend systematically on the cell's number of neighbors. Using simple extensions of `trap' and `Soft Glassy Rheology' models, we demonstrate that these energy barrier distributions give rise to glassy behavior and use the models to make testable predictions for two-time correlation functions and caging times. We incorporate these ideas into a continuum model that combines glassy rheology with active polarization to better understand collective migration in epithelial sheets.

  18. Modelling Rho GTPase biochemistry to predict collective cell migration

    NASA Astrophysics Data System (ADS)

    Merchant, Brian; Feng, James

    The collective migration of cells, due to individual cell polarization and intercellular contact inhibition of locomotion, features prominently in embryogenesis and metastatic cancers. Existing methods for modelling collectively migrating cells tend to rely either on highly abstracted agent-based models, or on continuum approximations of the group. Both of these frameworks represent intercellular interactions such as contact inhibition of locomotion as hard-coded rules defining model cells. In contrast, we present a vertex-dynamics framework which predicts polarization and contact inhibition of locomotion naturally from an underlying model of Rho GTPase biochemistry and cortical mechanics. We simulate the interaction between many such model cells, and study how modulating Rho GTPases affects migratory characteristics of the group, in the context of long-distance collective migration of neural crest cells during embryogenesis.

  19. Laser-photophoretic migration and fractionation of human blood cells.

    PubMed

    Monjushiro, Hideaki; Tanahashi, Yuko; Watarai, Hitoshi

    2013-05-13

    Laser photophoretic migration behavior of human blood cells in saline solution was investigated under the irradiation of Nd:YAG laser beam (532 nm) in the absence and the presence of the flow in a fused silica capillary. Red blood cells (RBC) were migrated faster than white blood cells (WBC) and blood pellets to the direction of propagation of laser light. The observed photophoretic velocity of RBC was about 11 times faster than those of others. This was understood from the larger photophoretic efficiency of RBC than that of WBC, which was simulated based on the Mie scattering theory. Furthermore, it was found that, during the photophoretic migration, RBCs spontaneously orientated parallel to the migration direction so as to reduce the drag force. Finally, it was demonstrated that RBC and WBC were separated in a micro-channel flow system by the laser photophoresis.

  20. Cell migration in confinement: a micro-channel-based assay.

    PubMed

    Heuzé, Mélina L; Collin, Olivier; Terriac, Emmanuel; Lennon-Duménil, Ana-Maria; Piel, Matthieu

    2011-01-01

    This chapter describes a method to study cells migrating in micro-channels, a confining environment of well-defined geometry. This assay is a complement to more complex 3D migration systems and provides several advantages even if it does not recapitulate the full complexity of 3D migration. Important parameters such as degree of adhesion, degree of confinement, mechanical properties, and geometry can be varied independently of each other. The device is fully compatible with almost any type of light microscopy and the simple geometry makes automated analysis very easy to perform, which allows screening strategy. The chapters is divided into five parts describing the design of different types of migration chambers, the fabrication of a mold by photolithography, the assembly of the chamber, the loading of cells, and finally the imaging on live or fixed cells. PMID:21748692

  1. Selective impairment of a subset of Ran-GTP-binding domains of ran-binding protein 2 (Ranbp2) suffices to recapitulate the degeneration of the retinal pigment epithelium (RPE) triggered by Ranbp2 ablation.

    PubMed

    Patil, Hemangi; Saha, Arjun; Senda, Eugene; Cho, Kyoung-in; Haque, MdEmdadul; Yu, Minzhong; Qiu, Sunny; Yoon, Dosuk; Hao, Ying; Peachey, Neal S; Ferreira, Paulo A

    2014-10-24

    Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2(-/-), with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2(-/-) background. Among the transgenic lines produced, only Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-)-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2(-/-). By contrast, Tg(RBD2/3*-HA) expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2(-/-) and Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-) share proteostatic deregulation of Ran GTPase, serotransferrin, and γ-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting

  2. Mechanics in Mechanosensitivity of Cell Adhesion and its Roles in Cell Migration

    NASA Astrophysics Data System (ADS)

    Zhong, Yuan; He, Shijie; Ji, Baohua

    2012-12-01

    Cells sense and respond to external stimuli and properties of their environment through focal adhesion complexes (FACs) to regulate a broad range of physiological and pathological processes, including cell migration. Currently, the basic principles in mechanics of the mechanosensitivity of cell adhesion and migration have not been fully understood. In this paper, an FEM-based mechano-chemical coupling model is proposed for studying the cell migration behaviors in which the dynamics of stability of FACs and the effect of cell shape on cell traction force distribution are considered. We find that the driving force of cell migration is produced by the competition of stability of cell adhesion between the cell front and cell rear, which consequently controls the speed of cell migration. We show that the rigidity gradient of matrix can bias this competition which allows cell to exhibit a durotaxis behavior, i.e. the larger the gradient, the higher the cell speed.

  3. Commensal bacteria promote migration of mast cells into the intestine.

    PubMed

    Kunii, Junichi; Takahashi, Kyoko; Kasakura, Kazumi; Tsuda, Masato; Nakano, Kou; Hosono, Akira; Kaminogawa, Shuichi

    2011-06-01

    Mast cells differentiate from hematopoietic stem cells in the bone marrow and migrate via the circulation to peripheral tissues, where they play a pivotal role in induction of both innate and adaptive immune responses. In this study, the effect of intestinal commensal bacteria on the migration of mast cells into the intestine was investigated. Histochemical analyses showed that germ-free (GF) mice had lower mast cell densities in the small intestine than normal mice. It was also shown that GF mice had lower mast cell proportion out of lamina propria leukocytes in the small intestine and higher mast cell percentages in the blood than normal mice by flow cytometry. These results indicate that migration of mast cells from the blood to the intestine is promoted by intestinal commensal bacteria. In addition, MyD88⁻/⁻ mice had lower densities of intestinal mast cells than CV mice, suggesting that the promotive effect of commensals is, at least in part, TLR-dependent. The ligands of CXC chemokine receptor 2 (CXCR2), which is critical for homing of mast cells to the intestine, were expressed higher in intestinal tissues and in intestinal epithelial cells (IECs) of normal mice than in those of GF or MyD88⁻/⁻ mice. Collectively, it is suggested that commensals promote migration of mast cells into the intestine through the induction of CXCR2 ligands from IECs in a TLR-dependent manner.

  4. Nuclear stiffening inhibits migration of invasive melanoma cells

    PubMed Central

    Ribeiro, Alexandre J.S.; Khanna, Payal; Sukumar, Aishwarya; Dong, Cheng; Dahl, Kris Noel

    2014-01-01

    During metastasis, melanoma cells must be sufficiently deformable to squeeze through extracellular barriers with small pore sizes. We visualize and quantify deformability of single cells using micropipette aspiration and examine the migration potential of a population of melanoma cells using a flow migration apparatus. We artificially stiffen the nucleus with recombinant overexpression of Δ50 lamin A, which is found in patients with Hutchison Gilford progeria syndrome and in aged individuals. Melanoma cells, both WM35 and Lu1205, both show reduced nuclear deformability and reduced cell invasion with the expression of Δ50 lamin A. These studies suggest that cellular aging including expression of Δ50 lamin A and nuclear stiffening may reduce the potential for metastatic cancer migration. Thus, the pathway of cancer metastasis may be kept in check by mechanical factors in addition to known chemical pathway regulation. PMID:25544862

  5. Mathematical Modeling of Eukaryotic Cell Migration: Insights Beyond Experiments

    PubMed Central

    Danuser, Gaudenz; Allard, Jun; Mogilner, Alex

    2014-01-01

    A migrating cell is a molecular machine made of tens of thousands of short-lived and interacting parts. Understanding migration means understanding the self-organization of these parts into a system of functional units. This task is one of tackling complexity: First, the system integrates numerous chemical and mechanical component processes. Second, these processes are connected in feedback interactions and over a large range of spatial and temporal scales. Third, many processes are stochastic, which leads to heterogeneous migration behaviors. Early on in the research of cell migration it became evident that this complexity exceeds human intuition. Thus, the cell migration community has led the charge to build mathematical models that could integrate the diverse experimental observations and measurements in consistent frameworks, first in conceptual and more recently in molecularly explicit models. The main goal of this review is to sift through a series of important conceptual and explicit mathematical models of cell migration and to evaluate their contribution to the field in their ability to integrate critical experimental data. PMID:23909278

  6. Chemokine-Mediated Migration of Mesencephalic Neural Crest Cells

    PubMed Central

    Rezzoug, Francine; Seelan, Ratnam S.; Bhattacherjee, Vasker; Greene, Robert M.; Pisano, M. Michele

    2011-01-01

    Clefts of the lip and/or palate are among the most prevalent birth defects affecting approximately 7000 newborns in the United States annually. Disruption of the developmentally programmed migration of neural crest cells (NCCs) into the orofacial region is thought to be one of the major causes of orofacial clefting. Signaling of the chemokine SDF-1 (Stromal Derived Factor-1) through its specific receptor, CXCR4, is required for the migration of many stem cell and progenitor cell populations from their respective sites of emergence to the regions where they differentiate into complex cell types, tissues and organs. In the present study, “transwell” assays of chick embryo mesencephalic (cranial) NCC migration and ex ovo whole embryo “bead implantation” assays were utilized to determine whether SDF-1/CXCR4 signaling mediates mesencephalic NCC migration. Results from this study demonstrate that attenuation of SDF-1 signaling, through the use of specific CXCR4 antagonists (AMD3100 and TN14003), disrupts the migration of mesencephalic NCCs into the orofacial region, suggesting a novel role for SDF-1/CXCR4 signaling in the directed migration of mesencephalic NCCs in the early stage embryo. PMID:22015108

  7. Cell collectivity regulation within migrating cell cluster during Kupffer's vesicle formation in zebrafish

    PubMed Central

    Matsui, Takaaki; Ishikawa, Hiroshi; Bessho, Yasumasa

    2015-01-01

    Although cell adhesion is thought to fasten cells tightly, cells that adhere to each other can migrate directionally. This group behavior, called “collective cell migration,” is observed during normal development, wound healing, and cancer invasion. Loss-of-function of cell adhesion molecules in several model systems of collective cell migration results in delay or inhibition of migration of cell groups but does not lead to dissociation of the cell groups, suggesting that mechanisms of cells staying assembled as a single cell cluster, termed as “cell collectivity,” remain largely unknown. During the formation of Kupffer's vesicle (KV, an organ of laterality in zebrafish), KV progenitors form a cluster and migrate together toward the vegetal pole. Importantly, in this model system of collective cell migration, knockdown of cell adhesion molecules or signal components leads to failure of cell collectivity. In this review, we summarize recent findings in cell collectivity regulation during collective migration of KV progenitor cells and describe our current understanding of how cell collectivity is regulated during collective cell migration. PMID:26000276

  8. Phototherapy promotes cell migration in the presence of hydroxyurea.

    PubMed

    Zungu, I L; Mbene, A B; Hawkins Evans, D H; Houreld, N N; Abrahamse, H

    2009-03-01

    Phototherapy has been shown to cause an increase in cell proliferation and migration. This study focused on viability (trypan blue), proliferation [sodium 3'-(1-(phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro)-benzene sulphonic acid hydrate (XTT) and adenosine triphosphate (ATP)] and migration of WS1 cells following irradiation in the presence of hydroxyurea (HU), which is an inhibitor of proliferation. Wounded cells were irradiated on days 1 and 4 with a fluence of 5 J/cm(2) with a helium-neon (He-Ne) laser at 632.8 nm. After a repair time of 24 h, cellular responses were assessed. Wounded irradiated cells without HU showed an increase in cell viability and proliferation, which was confirmed by complete wound closure by day 4. Although wounded irradiated cells treated with 5 mM HU showed incomplete wound closure, these cells showed increased migration compared with that of control cells. This study showed that laser irradiation using an He-Ne laser with a fluence of 5 J/cm(2) stimulates cell viability. The HU results confirmed that laser irradiation promotes cell migration and proliferation. PMID:18214574

  9. Protrusive waves guide 3D cell migration along nanofibers.

    PubMed

    Guetta-Terrier, Charlotte; Monzo, Pascale; Zhu, Jie; Long, Hongyan; Venkatraman, Lakshmi; Zhou, Yue; Wang, PeiPei; Chew, Sing Yian; Mogilner, Alexander; Ladoux, Benoit; Gauthier, Nils C

    2015-11-01

    In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electrospun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Rac1/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions.

  10. Protrusive waves guide 3D cell migration along nanofibers

    PubMed Central

    Guetta-Terrier, Charlotte; Monzo, Pascale; Zhu, Jie; Long, Hongyan; Venkatraman, Lakshmi; Zhou, Yue; Wang, PeiPei; Chew, Sing Yian; Mogilner, Alexander

    2015-01-01

    In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electrospun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Rac1/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions. PMID:26553933

  11. [RPE melanosomes bind A2E fluorophore of lipofuscin granules and products of its photooxidation].

    PubMed

    Sakina, N L; Koromyslova, A D; Dontsov, A E; Ostrovskiĭ, M A

    2013-05-01

    The ability of melanosomes from human, bovine and frog retinal pigment epithelium cells (RPE) to bind A2E fluorophore of RPE lipofuscin granules and products of A2E photooxidation is investigated. RPE melanosomes are found to bind A2E molecules themselves as well as the molecules formed after A2E irradiation by visible light. In our experiments single melanosome was able to bind up to 0.08 fmol A2E. Antioxidant activity of melanosomes is compared to antioxidant activity of their complexes with A2E. It is shown by luminal chemiluminescence quenching in the presence of hydrogen peroxide that in A2E/melanosomes complex the chemiluminescence quenching is not significantly reduced. Comparison of inhibitory activity of melanosomes and their complexes with A2E on UV-induced (light conditions) and Fe(2+)-ascorbate-induced (dark conditions) peroxidation of photoreceptor outer segments (POS) demonstrated that bound A2E does not affect inhibitory ability of melanosomes in both systems. Thus, binding of A2E to RPE melanosomes in concentrations from 0.01 to 0.1 fmol A2E per melanosome does not significantly alter their antioxidant properties. It is supposed that both A2E and hydrophilic products of its photooxidation could be bound by RPE melanosomes and, thus, it lost the ability to exhibit toxic properties. PMID:24459874

  12. Molecular mechanisms underlying progesterone-enhanced breast cancer cell migration

    PubMed Central

    Wang, Hui-Chen; Lee, Wen-Sen

    2016-01-01

    Progesterone (P4) was demonstrated to inhibit migration in vascular smooth muscle cells (VSMCs), but to enhance migration in T47D breast cancer cells. To investigate the mechanism responsible for this switch in P4 action, we examined the signaling pathway responsible for the P4-induced migration enhancement in breast cancer cell lines, T47D and MCF-7. Here, we demonstrated that P4 activated the cSrc/AKT signaling pathway, subsequently inducing RSK1 activation, which in turn increased phosphorylation of p27 at T198 and formation of the p27pT198-RhoA complex in the cytosol, thereby preventing RhoA degradation, and eventually enhanced migration in T47D cells. These findings were confirmed in the P4-treated MCF-7. Comparing the P4-induced molecular events in between breast cancer cells and VSMCs, we found that P4 increased p27 phosphorylation at T198 in breast cancer cells through RSK1 activation, while P4 increased p27 phosphorlation at Ser10 in VSMCs through KIS activation. P27pT198 formed the complex with RhoA and prevented RhoA degradation in T47D cells, whereas p-p27Ser10 formed the complex with RhoA and caused RhoA degradation in VSMCs. The results of this study highlight the molecular mechanism underlying P4-enhanced breast cancer cell migration, and suggest that RSK1 activation is responsible for the P4-induced migration enhancement in breast cancer cells. PMID:27510838

  13. Multidisciplinary approaches to understanding collective cell migration in developmental biology.

    PubMed

    Schumacher, Linus J; Kulesa, Paul M; McLennan, Rebecca; Baker, Ruth E; Maini, Philip K

    2016-06-01

    Mathematical models are becoming increasingly integrated with experimental efforts in the study of biological systems. Collective cell migration in developmental biology is a particularly fruitful application area for the development of theoretical models to predict the behaviour of complex multicellular systems with many interacting parts. In this context, mathematical models provide a tool to assess the consistency of experimental observations with testable mechanistic hypotheses. In this review, we showcase examples from recent years of multidisciplinary investigations of neural crest cell migration. The neural crest model system has been used to study how collective migration of cell populations is shaped by cell-cell interactions, cell-environmental interactions and heterogeneity between cells. The wide range of emergent behaviours exhibited by neural crest cells in different embryonal locations and in different organisms helps us chart out the spectrum of collective cell migration. At the same time, this diversity in migratory characteristics highlights the need to reconcile or unify the array of currently hypothesized mechanisms through the next generation of experimental data and generalized theoretical descriptions. PMID:27278647

  14. Cerium migration during PEM fuel cell accelerated stress testing

    SciTech Connect

    Baker, Andrew M.; Mukundan, Rangachary; Borup, Rodney L.; Spernjak, Dusan; Judge, Elizabeth J.; Advani, Suresh G.; Prasad, Ajay K.

    2016-01-01

    Cerium is a radical scavenger which improves polymer electrolyte membrane (PEM) fuel cell durability. During operation, however, cerium rapidly migrates in the PEM and into the catalyst layers (CLs). In this work, membrane electrode assemblies (MEAs) were subjected to accelerated stress tests (ASTs) under different humidity conditions. Cerium migration was characterized in the MEAs after ASTs using X-ray fluorescence. During fully humidified operation, water flux from cell inlet to outlet generated in-plane cerium gradients. Conversely, cerium profiles were flat during low humidity operation, where in-plane water flux was negligible, however, migration from the PEM into the CLs was enhanced. Humidity cycling resulted in both in-plane cerium gradients due to water flux during the hydration component of the cycle, and significant migration into the CLs. Fluoride and cerium emissions into effluent cell waters were measured during ASTs and correlated, which signifies that ionomer degradation products serve as possible counter-ions for cerium emissions. Fluoride emission rates were also correlated to final PEM cerium contents, which indicates that PEM degradation and cerium migration are coupled. Lastly, it is proposed that cerium migrates from the PEM due to humidification conditions and degradation, and is subsequently stabilized in the CLs by carbon catalyst supports.

  15. How Tissue Mechanical Properties Affect Enteric Neural Crest Cell Migration

    NASA Astrophysics Data System (ADS)

    Chevalier, N. R.; Gazguez, E.; Bidault, L.; Guilbert, T.; Vias, C.; Vian, E.; Watanabe, Y.; Muller, L.; Germain, S.; Bondurand, N.; Dufour, S.; Fleury, V.

    2016-02-01

    Neural crest cells (NCCs) are a population of multipotent cells that migrate extensively during vertebrate development. Alterations to neural crest ontogenesis cause several diseases, including cancers and congenital defects, such as Hirschprung disease, which results from incomplete colonization of the colon by enteric NCCs (ENCCs). We investigated the influence of the stiffness and structure of the environment on ENCC migration in vitro and during colonization of the gastrointestinal tract in chicken and mouse embryos. We showed using tensile stretching and atomic force microscopy (AFM) that the mesenchyme of the gut was initially soft but gradually stiffened during the period of ENCC colonization. Second-harmonic generation (SHG) microscopy revealed that this stiffening was associated with a gradual organization and enrichment of collagen fibers in the developing gut. Ex-vivo 2D cell migration assays showed that ENCCs migrated on substrates with very low levels of stiffness. In 3D collagen gels, the speed of the ENCC migratory front decreased with increasing gel stiffness, whereas no correlation was found between porosity and ENCC migration behavior. Metalloprotease inhibition experiments showed that ENCCs actively degraded collagen in order to progress. These results shed light on the role of the mechanical properties of tissues in ENCC migration during development.

  16. Cerium migration during PEM fuel cell accelerated stress testing

    DOE PAGES

    Baker, Andrew M.; Mukundan, Rangachary; Borup, Rodney L.; Spernjak, Dusan; Judge, Elizabeth J.; Advani, Suresh G.; Prasad, Ajay K.

    2016-01-01

    Cerium is a radical scavenger which improves polymer electrolyte membrane (PEM) fuel cell durability. During operation, however, cerium rapidly migrates in the PEM and into the catalyst layers (CLs). In this work, membrane electrode assemblies (MEAs) were subjected to accelerated stress tests (ASTs) under different humidity conditions. Cerium migration was characterized in the MEAs after ASTs using X-ray fluorescence. During fully humidified operation, water flux from cell inlet to outlet generated in-plane cerium gradients. Conversely, cerium profiles were flat during low humidity operation, where in-plane water flux was negligible, however, migration from the PEM into the CLs was enhanced. Humiditymore » cycling resulted in both in-plane cerium gradients due to water flux during the hydration component of the cycle, and significant migration into the CLs. Fluoride and cerium emissions into effluent cell waters were measured during ASTs and correlated, which signifies that ionomer degradation products serve as possible counter-ions for cerium emissions. Fluoride emission rates were also correlated to final PEM cerium contents, which indicates that PEM degradation and cerium migration are coupled. Lastly, it is proposed that cerium migrates from the PEM due to humidification conditions and degradation, and is subsequently stabilized in the CLs by carbon catalyst supports.« less

  17. How Tissue Mechanical Properties Affect Enteric Neural Crest Cell Migration

    PubMed Central

    Chevalier, N.R.; Gazguez, E.; Bidault, L.; Guilbert, T.; Vias, C.; Vian, E.; Watanabe, Y.; Muller, L.; Germain, S.; Bondurand, N.; Dufour, S.; Fleury, V.

    2016-01-01

    Neural crest cells (NCCs) are a population of multipotent cells that migrate extensively during vertebrate development. Alterations to neural crest ontogenesis cause several diseases, including cancers and congenital defects, such as Hirschprung disease, which results from incomplete colonization of the colon by enteric NCCs (ENCCs). We investigated the influence of the stiffness and structure of the environment on ENCC migration in vitro and during colonization of the gastrointestinal tract in chicken and mouse embryos. We showed using tensile stretching and atomic force microscopy (AFM) that the mesenchyme of the gut was initially soft but gradually stiffened during the period of ENCC colonization. Second-harmonic generation (SHG) microscopy revealed that this stiffening was associated with a gradual organization and enrichment of collagen fibers in the developing gut. Ex-vivo 2D cell migration assays showed that ENCCs migrated on substrates with very low levels of stiffness. In 3D collagen gels, the speed of the ENCC migratory front decreased with increasing gel stiffness, whereas no correlation was found between porosity and ENCC migration behavior. Metalloprotease inhibition experiments showed that ENCCs actively degraded collagen in order to progress. These results shed light on the role of the mechanical properties of tissues in ENCC migration during development. PMID:26887292

  18. Plasma-mediated transfection of RPE

    NASA Astrophysics Data System (ADS)

    Palanker, D.; Chalberg, T.; Vankov, A.; Huie, P.; Molnar, F. E.; Butterwick, A.; Calos, M.; Marmor, M.; Blumenkranz, M. S.

    2006-02-01

    A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

  19. Junctional communication is induced in migrating capillary endothelial cells

    PubMed Central

    1989-01-01

    Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration. PMID:2592412

  20. Cellular Polarization and Contractility in Collective Cell Migration

    NASA Astrophysics Data System (ADS)

    Utuje, Kazage J. Christophe; Notbohm, Jacob; Banerjee, Shiladitya; Gweon, Bomi; Jang, Hwanseok; Park, Yongdoo; Shin, Jennifer; Butler, James P.; Fredberg, Jeffrey J.; Marchetti, M. Cristina

    Collective cell migration drives many biological processes such as metastasis, morphogenesis and wound healing. These coordinated motions are driven by active forces. The physical nature of these forces and the mechanisms by which they generate collective cell migration are still not fully understood. We have developed a minimum physical model of a cell monolayer as an elastic continuum whose deformation field is coupled to two internal degrees of freedom: the concentration of a chemical signal, controlling cell Contractility, and the polarization field controlling the direction of local cell motion. By combining theory with experiments, we show that these two internal variables account for the sloshing waves and the systematic deviations of the direction of cell polarization from that of local cell velocity observed in confined cell monolayers. KJCU and MCM were supported by the Simons Foundation.

  1. Controlled architectural and chemotactic studies of 3D cell migration

    PubMed Central

    Tayalia, Prakriti; Mazur, Eric; Mooney, David

    2010-01-01

    Chemotaxis plays a critical role in tissue development and wound repair, and is widely studied using ex vivo model systems in applications such as immunotherapy. However, typical chemotactic models employ 2D systems that are less physiologically relevant or use end-point assays, that reveal little about the stepwise dynamics of the migration process. To overcome these limitations, we developed a new model system using microfabrication techniques, sustained drug delivery approaches, and theoretical modeling of chemotactic agent diffusion. This model system allows us to study the effects of 3D architecture and chemotactic agent gradient on immune cell migration in real time. We find that dendritic cell migration is characterized by a strong interplay between matrix architecture and chemotactic gradients, and migration is also influenced dramatically by the cell activation state. Our results indicate that Lipopolysaccharide-activated dendritic cells studied in a traditional transwell system actually exhibit anomalous migration behavior. Such a 3D ex vivo system lends itself for analyzing cell migratory behavior in response to single or multiple competitive cues and could prove useful in vaccine development. PMID:21237507

  2. Nanotopography guides and directs cell migration in amoeboid and epithelial cells

    NASA Astrophysics Data System (ADS)

    Lee, Rachel; Das, Satarupa; Hourwitz, Matthew; Sun, Xiaoyu; Parent, Carole; Fourkas, John; Losert, Wolfgang

    Cell migration plays a critical role in development, angiogenesis, immune response, wound healing, and cancer metastasis. In many cases, cells also move in the context of a matrix of collagen fibers, and the alignment of these fibers can both affect the migration phenotype and guide cells. Here we show that both fast and slow migrating cells - amoeboid HL-60 and epithelial MCF10A - are affected in similar ways by micro/nanostructures with dimensions similar to those of collagen fibers. Cell alignment enhances the efficiency of migration by increasing directional persistence.

  3. Live cell imaging analysis of the epigenetic regulation of the human endothelial cell migration at single-cell resolution.

    PubMed

    Zheng, Chunhong; Yu, Zhilong; Zhou, Ying; Tao, Louis; Pang, Yuhong; Chen, Tao; Zhang, Xiannian; Qiu, Haiwei; Zhou, Hongwei; Chen, Zitian; Huang, Yanyi

    2012-09-01

    Epigenetic regulation plays an important role in cell migration. Although many methods have been developed to measure the motility of mammalian cells, accurate quantitative assessments of the migration speed of individual cells remain a major challenge. It is difficult for conventional scratch assays to differentiate proliferation from migration during the so-called wound-healing processes because of the long experimental time required. In addition, it is also challenging to create identical conditions for evaluating cell migration by conventional methods. We developed a microfluidic device with precisely created blanks allowing for robust and reproducible cell migration inside accurately-controlled microenvironments to study the regulatory effect of the epigenetic regulator histone deacetylase 7 (HDAC7) on cell migration. Through analyzing time-lapse imaging of the cells migrating into individual blank regions, we can measure the migration speed parameter for human primary cells within a few hours, eliminating the confounding effect of cell proliferation. We also developed an automatic image analysis and a numeric model-based data fitting to set up an integrated cell migration analysis system at single-cell resolution. Using this system, we measured the motility of primary human umbilical vein endothelial cells (HUVECs) and the migration speed reduction due to the silencing of HDAC7 and various other genes. We showed that the migration behaviour of these human primary cells are clearly regulated by epigenetic mechanisms, demonstrating the great potential of this accurate and robust assay in the fields of quantitatively migration studies and high-throughput screening.

  4. Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos.

    PubMed

    Giger, Florence A; Dumortier, Julien G; David, Nicolas B

    2016-04-29

    Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

  5. Flow and Diffusion in Channel-Guided Cell Migration

    PubMed Central

    Marel, Anna-Kristina; Zorn, Matthias; Klingner, Christoph; Wedlich-Söldner, Roland; Frey, Erwin; Rädler, Joachim O.

    2014-01-01

    Collective migration of mechanically coupled cell layers is a notable feature of wound healing, embryonic development, and cancer progression. In confluent epithelial sheets, the dynamics have been found to be highly heterogeneous, exhibiting spontaneous formation of swirls, long-range correlations, and glass-like dynamic arrest as a function of cell density. In contrast, the flow-like properties of one-sided cell-sheet expansion in confining geometries are not well understood. Here, we studied the short- and long-term flow of Madin-Darby canine kidney (MDCK) cells as they moved through microchannels. Using single-cell tracking and particle image velocimetry (PIV), we found that a defined averaged stationary cell current emerged that exhibited a velocity gradient in the direction of migration and a plug-flow-like profile across the advancing sheet. The observed flow velocity can be decomposed into a constant term of directed cell migration and a diffusion-like contribution that increases with density gradient. The diffusive component is consistent with the cell-density profile and front propagation speed predicted by the Fisher-Kolmogorov equation. To connect diffusion-mediated transport to underlying cellular motility, we studied single-cell trajectories and occurrence of vorticity. We discovered that the directed large-scale cell flow altered fluctuations in cellular motion at short length scales: vorticity maps showed a reduced frequency of swirl formation in channel flow compared with resting sheets of equal cell density. Furthermore, under flow, single-cell trajectories showed persistent long-range, random-walk behavior superimposed on drift, whereas cells in resting tissue did not show significant displacements with respect to neighboring cells. Our work thus suggests that active cell migration manifests itself in an underlying, spatially uniform drift as well as in randomized bursts of short-range correlated motion that lead to a diffusion-mediated transport

  6. The effects of acoustic vibration on fibroblast cell migration.

    PubMed

    Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

    2016-12-01

    Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. PMID:27612824

  7. Leukotrienes induce the migration of Th17 cells.

    PubMed

    Lee, Wonyong; Su Kim, Hyeong; Lee, Gap Ryol

    2015-01-01

    Th17 cell trafficking in response to leukotriene signaling is poorly understood. Here we showed that Th17 cells express high levels of leukotriene B4 receptor 1 (LTB4R1) and cysteinyl leukotriene receptor 1 (CysLTR1). Th17 cells migrated under the guidance of leukotriene B4 and D4. The migration of Th17 cells was more efficient than that of Th1 and Th2 cells, and it was blocked by specific inhibitors of LTB4R1 or CysLTR1. Studies in an animal model of experimental autoimmune encephalomyelitis revealed that treatment with montelukast alleviated disease symptoms and inhibited the recruitment of Th17 cells to the central nervous system. Thus, leukotrienes may act as chemoattractants for Th17 cells. PMID:25512344

  8. Inhibition of REST Suppresses Proliferation and Migration in Glioblastoma Cells.

    PubMed

    Zhang, Dianbao; Li, Ying; Wang, Rui; Li, Yunna; Shi, Ping; Kan, Zhoumi; Pang, Xining

    2016-01-01

    Glioblastoma (GBM) is the most common primary brain tumor, with poor prognosis and a lack of effective therapeutic options. The aberrant expression of transcription factor REST (repressor element 1-silencing transcription factor) had been reported in different kinds of tumors. However, the function of REST and its mechanisms in GBM remain elusive. Here, REST expression was inhibited by siRNA silencing in U-87 and U-251 GBM cells. Then CCK-8 assay showed significantly decreased cell proliferation, and the inhibition of migration was verified by scratch wound healing assay and transwell assay. Using cell cycle analysis and Annexin V/PI straining assay, G1 phase cell cycle arrest was found to be a reason for the suppression of cell proliferation and migration upon REST silencing, while apoptosis was not affected by REST silencing. Further, the detection of REST-downstream genes involved in cytostasis and migration inhibition demonstrated that CCND1 and CCNE1 were reduced; CDK5R1, BBC3, EGR1, SLC25A4, PDCD7, MAPK11, MAPK12, FADD and DAXX were enhanced, among which BBC3 and DAXX were direct targets of REST, as verified by ChIP (chromatin immunoprecipitation) and Western blotting. These data suggested that REST is a master regulator that maintains GBM cells proliferation and migration, partly through regulating cell cycle by repressing downstream genes, which might represent a potential target for GBM therapy. PMID:27153061

  9. Inhibition of REST Suppresses Proliferation and Migration in Glioblastoma Cells

    PubMed Central

    Zhang, Dianbao; Li, Ying; Wang, Rui; Li, Yunna; Shi, Ping; Kan, Zhoumi; Pang, Xining

    2016-01-01

    Glioblastoma (GBM) is the most common primary brain tumor, with poor prognosis and a lack of effective therapeutic options. The aberrant expression of transcription factor REST (repressor element 1-silencing transcription factor) had been reported in different kinds of tumors. However, the function of REST and its mechanisms in GBM remain elusive. Here, REST expression was inhibited by siRNA silencing in U-87 and U-251 GBM cells. Then CCK-8 assay showed significantly decreased cell proliferation, and the inhibition of migration was verified by scratch wound healing assay and transwell assay. Using cell cycle analysis and Annexin V/PI straining assay, G1 phase cell cycle arrest was found to be a reason for the suppression of cell proliferation and migration upon REST silencing, while apoptosis was not affected by REST silencing. Further, the detection of REST-downstream genes involved in cytostasis and migration inhibition demonstrated that CCND1 and CCNE1 were reduced; CDK5R1, BBC3, EGR1, SLC25A4, PDCD7, MAPK11, MAPK12, FADD and DAXX were enhanced, among which BBC3 and DAXX were direct targets of REST, as verified by ChIP (chromatin immunoprecipitation) and Western blotting. These data suggested that REST is a master regulator that maintains GBM cells proliferation and migration, partly through regulating cell cycle by repressing downstream genes, which might represent a potential target for GBM therapy. PMID:27153061

  10. Multidisciplinary approaches to understanding collective cell migration in developmental biology

    PubMed Central

    Schumacher, Linus J.; Kulesa, Paul M.; McLennan, Rebecca; Baker, Ruth E.; Maini, Philip K.

    2016-01-01

    Mathematical models are becoming increasingly integrated with experimental efforts in the study of biological systems. Collective cell migration in developmental biology is a particularly fruitful application area for the development of theoretical models to predict the behaviour of complex multicellular systems with many interacting parts. In this context, mathematical models provide a tool to assess the consistency of experimental observations with testable mechanistic hypotheses. In this review, we showcase examples from recent years of multidisciplinary investigations of neural crest cell migration. The neural crest model system has been used to study how collective migration of cell populations is shaped by cell–cell interactions, cell–environmental interactions and heterogeneity between cells. The wide range of emergent behaviours exhibited by neural crest cells in different embryonal locations and in different organisms helps us chart out the spectrum of collective cell migration. At the same time, this diversity in migratory characteristics highlights the need to reconcile or unify the array of currently hypothesized mechanisms through the next generation of experimental data and generalized theoretical descriptions. PMID:27278647

  11. High Glucose-Mediated Oxidative Stress Impairs Cell Migration

    PubMed Central

    Lamers, Marcelo L.; Almeida, Maíra E. S.; Vicente-Manzanares, Miguel; Horwitz, Alan F.; Santos, Marinilce F.

    2011-01-01

    Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control - OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients. PMID:21826213

  12. Cell migration in paediatric glioma; characterisation and potential therapeutic targeting

    PubMed Central

    Cockle, J V; Picton, S; Levesley, J; Ilett, E; Carcaboso, A M; Short, S; Steel, L P; Melcher, A; Lawler, S E; Brüning-Richardson, A

    2015-01-01

    Background: Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed. Methods: Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays. Results: All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging. Conclusions: Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours. PMID:25628092

  13. Epac Activation Regulates Human Mesenchymal Stem Cells Migration and Adhesion.

    PubMed

    Yu, Jiao-Le; Deng, Ruixia; Chung, Sookja K; Chan, Godfrey Chi-Fung

    2016-04-01

    How to enhance the homing of human mesenchymal stem cells (hMSCs) to the target tissues remains a clinical challenge nowadays. To overcome this barrier, the mechanism responsible for the hMSCs migration and engraftment has to be defined. Currently, the exact mechanism involved in migration and adhesion of hMSCs remains unknown. Exchange protein directly activated by cAMP (Epac), a novel protein discovered in cAMP signaling pathway, may have a potential role in regulating cells adhesion and migration by triggering the downstream Rap family signaling cascades. However, the exact role of Epac in cells homing is elusive. Our study evaluated the role of Epac in the homing of hMSCs. We confirmed that hMSCs expressed functional Epac and its activation enhanced the migration and adhesion of hMSCs significantly. The Epac activation was further found to be contributed directly to the chemotactic responses induced by stromal cell derived factor-1 (SDF-1) which is a known chemokine in regulating hMSCs homing. These findings suggested Epac is connected to the SDF-1 signaling cascades. In conclusion, our study revealed that Epac plays a role in hMSCs homing by promoting adhesion and migration. Appropriate manipulation of Epac may enhance the homing of hMSCs and facilitate their future clinical applications. PMID:26727165

  14. Intermediate filaments in cell migration and invasion: the unusual suspects.

    PubMed

    Leduc, Cécile; Etienne-Manneville, Sandrine

    2015-02-01

    Cell migration is a multistep process which relies on the coordination of cytoskeletal structures in space and time. While the roles of actin and microtubules have been investigated in great details, the lack of inhibitors and visualizing tools and the large number of proteins forming intermediate filaments (IFs) have delayed the characterization of IF functions during migration. However, a large body of evidence has progressively pointed to changes in IF composition as an important parameter in the regulation of cell migratory properties both during development and tumor invasion. More recent in-depth analyses show that IFs are dynamically reorganized to participate, together with microfilaments and microtubules, to the key steps leading to cell migration.

  15. Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

    PubMed

    Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

    2016-07-01

    The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing.

  16. Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

    PubMed

    Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

    2016-07-01

    The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing. PMID:27105673

  17. Collisions of deformable cells lead to collective migration

    SciTech Connect

    Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

    2015-03-17

    Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility – acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignment of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.

  18. Collisions of deformable cells lead to collective migration

    DOE PAGES

    Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

    2015-03-17

    Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility – acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignmentmore » of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.« less

  19. Single-cell Migration Chip for Chemotaxis-based Microfluidic Selection of Heterogeneous Cell Populations

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Allen, Steven G.; Ingram, Patrick N.; Buckanovich, Ronald; Merajver, Sofia D.; Yoon, Euisik

    2015-05-01

    Tumor cell migration toward and intravasation into capillaries is an early and key event in cancer metastasis, yet not all cancer cells are imbued with the same capability to do so. This heterogeneity within a tumor is a fundamental property of cancer. Tools to help us understand what molecular characteristics allow a certain subpopulation of cells to spread from the primary tumor are thus critical for overcoming metastasis. Conventional in vitro migration platforms treat populations in aggregate, which leads to a masking of intrinsic differences among cells. Some migration assays reported recently have single-cell resolution, but these platforms do not provide for selective retrieval of the distinct migrating and non-migrating cell populations for further analysis. Thus, to study the intrinsic differences in cells responsible for chemotactic heterogeneity, we developed a single-cell migration platform so that individual cells’ migration behavior can be studied and the heterogeneous population sorted based upon chemotactic phenotype. Furthermore, after migration, the highly chemotactic and non-chemotactic cells were retrieved and proved viable for later molecular analysis of their differences. Moreover, we modified the migration channel to resemble lymphatic capillaries to better understand how certain cancer cells are able to move through geometrically confining spaces.

  20. Electrolytic cell stack with molten electrolyte migration control

    DOEpatents

    Kunz, H.R.; Guthrie, R.J.; Katz, M.

    1987-03-17

    An electrolytic cell stack includes inactive electrolyte reservoirs at the upper and lower end portions thereof. The reservoirs are separated from the stack of the complete cells by impermeable, electrically conductive separators. Reservoirs at the negative end are initially low in electrolyte and the reservoirs at the positive end are high in electrolyte fill. During stack operation electrolyte migration from the positive to the negative end will be offset by the inactive reservoir capacity. In combination with the inactive reservoirs, a sealing member of high porosity and low electrolyte retention is employed to limit the electrolyte migration rate. 5 figs.

  1. Electrolytic cell stack with molten electrolyte migration control

    DOEpatents

    Kunz, H. Russell; Guthrie, Robin J.; Katz, Murray

    1988-08-02

    An electrolytic cell stack includes inactive electrolyte reservoirs at the upper and lower end portions thereof. The reservoirs are separated from the stack of the complete cells by impermeable, electrically conductive separators. Reservoirs at the negative end are initially low in electrolyte and the reservoirs at the positive end are high in electrolyte fill. During stack operation electrolyte migration from the positive to the negative end will be offset by the inactive reservoir capacity. In combination with the inactive reservoirs, a sealing member of high porosity and low electrolyte retention is employed to limit the electrolyte migration rate.

  2. SIRT1 regulates lamellipodium extension and migration of melanoma cells.

    PubMed

    Kunimoto, Risa; Jimbow, Kowichi; Tanimura, Akihiko; Sato, Masahiro; Horimoto, Kouhei; Hayashi, Takashi; Hisahara, Shin; Sugino, Toshiya; Hirobe, Tomohisa; Yamashita, Toshiharu; Horio, Yoshiyuki

    2014-06-01

    Melanoma is highly metastatic, but the mechanism of melanoma cell migration is still unclear. We found that melanoma cells expressed the nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 in the cytoplasm. Cell membrane extension and migration of melanoma cells were inhibited by SIRT1 inhibitors or SIRT1 knockdown, whereas SIRT1 activators enhanced elongation of protrusion and cellular motility. In B16F1 cells, growth factor stimulation induced lamellipodium extension, a characteristic feature at the leading edge of migrating cells, and SIRT1 was found in the lamellipodium. SIRT1 inhibitor nicotinamide (NAM) or SIRT1 small interfering RNAs suppressed the lamellipodium extension by serum or platelet-derived growth factor (PDGF). The lamellipodium formation by dominant-active Rac1 was also inhibited by NAM, a SIRT1 inhibitor. NAM inhibited the accumulation of phosphorylated Akt at the submembrane by serum or PDGF. Using fluorescence resonance energy transfer, we found that NAM impaired PDGF-dependent increase in the phosphatidylinositol-3,4,5-trisphosphate level at the leading edge. NAM inhibited the abdominal metastasis of transplanted B16F1 melanoma cells in C57BL6/J mice and improved survival. Finally, SIRT1-knockdown B16F1 cells showed significantly reduced metastasis in transplanted mice compared with that in control B16F1 cells. These results indicate that SIRT1 inhibition is a strategy to suppress metastasis of melanoma cells. PMID:24480879

  3. Nuclear envelope rupture and repair during cancer cell migration

    PubMed Central

    Denais, Celine M.; Gilbert, Rachel M.; Isermann, Philipp; McGregor, Alexandra L.; te Lindert, Mariska; Weigelin, Bettina; Davidson, Patricia M.; Friedl, Peter; Wolf, Katarina; Lammerding, Jan

    2016-01-01

    During cancer metastasis, tumor cells penetrate tissues through tight interstitial spaces, requiring extensive deformation of the cell and its nucleus. Here, we investigated tumor cell migration in confining microenvironments in vitro and in vivo. Nuclear deformation caused localized loss of nuclear envelope (NE) integrity, which led to the uncontrolled exchange of nucleo-cytoplasmic content, herniation of chromatin across the NE, and DNA damage. The incidence of NE rupture increased with cell confinement and with depletion of nuclear lamins, NE proteins that structurally support the nucleus. Cells restored NE integrity using components of the endosomal sorting complexes required for transport-III (ESCRT-III) machinery. Our findings indicate that cell migration incurs substantial physical stress on the NE and its content, requiring efficient NE and DNA damage repair for survival. PMID:27013428

  4. The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65.

    PubMed

    Lopes, Vanda S; Gibbs, Daniel; Libby, Richard T; Aleman, Tomas S; Welch, Darcy L; Lillo, Concepción; Jacobson, Samuel G; Radu, Roxana A; Steel, Karen P; Williams, David S

    2011-07-01

    Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B.  In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place.  We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle.  We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells.  This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light.  RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels.  Following a 50-60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity.  Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction.  Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision. PMID:21493626

  5. Molecular analysis of cell surface beta-1,4-galactosyltransferase function during cell migration.

    PubMed Central

    Appeddu, P A; Shur, B D

    1994-01-01

    Despite the identification and characterization of cell surface receptors for the extracellular matrix, it is unknown how their relative expression and cytoskeletal association regulate cell migration. Previous studies have identified beta-1,4-galactosyltransferase (GalTase; EC 2.4.1.38) on the surface of migrating cells, where it mediates cell migration on basal lamina matrices by associating with the cytoskeleton and binding to N-linked oligosaccharides in the E8 domain of laminin. In this study, the function of GalTase during cell migration was examined directly by analyzing the migration rate of stably transfected cell lines in which the relative level of surface GalTase and its ability to associate with the cytoskeleton were altered. We show here that the cytoskeleton contains a limiting, saturable, number of binding sites for surface GalTase. Furthermore, the rate of cell migration was inversely related to the ability of surface GalTase to associate with the cytoskeleton. Elevating surface GalTase in excess of the number of cytoskeleton-binding sites reduced the rate of cell migration, whereas decreasing the amount of surface GalTase available to bind the cytoskeleton increased migration rates. These results show that the rate of cell migration on basal lamina is directly dependent upon the expression of surface GalTase and the ability of this protein to associate with a limiting number of cytoskeleton-binding sites. Images PMID:8134355

  6. Live Imaging of LysoTracker-Labelled Phagolysosomes Tracks Diurnal Phagocytosis of Photoreceptor Outer Segment Fragments in Rat RPE Tissue Ex Vivo.

    PubMed

    Mao, Yingyu; Finnemann, Silvia C

    2016-01-01

    Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 μm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.

  7. Study of dendritic cell migration using micro-fabrication.

    PubMed

    Vargas, Pablo; Chabaud, Mélanie; Thiam, Hawa-Racine; Lankar, Danielle; Piel, Matthieu; Lennon-Dumenil, Ana-Maria

    2016-05-01

    Cell migration is a hallmark of dendritic cells (DCs) function. It is needed for DCs to scan their environment in search for antigens as well as to reach lymphatic organs in order to trigger T lymphocyte's activation. Such interaction leads to tolerance in the case of DCs migrating under homeostatic conditions or to immunity in the case of DCs migrating upon encounter with pathogen-associated molecular patterns. Cell migration is therefore essential for DCs to transfer information from peripheral tissues to lymphoid organs, thereby linking innate to adaptive immunity. This stresses the need to unravel the molecular mechanisms involved. However, the tremendous complexity of the tissue microenvironment as well as the limited spatio-temporal resolution of in vivo imaging techniques has made this task difficult. To bypass this problem, we have developed microfabrication-based experimental tools that are compatible with high-resolution imaging. Here, we will discuss how such devices can be used to study DC migration under controlled conditions that mimic their physiological environment in a robust quantitative manner.

  8. Glucocorticoid receptor beta increases migration of human bladder cancer cells.

    PubMed

    McBeth, Lucien; Nwaneri, Assumpta C; Grabnar, Maria; Demeter, Jonathan; Nestor-Kalinoski, Andrea; Hinds, Terry D

    2016-05-10

    Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p < 0.05) increased expression of GRβ compared to UMUC-3, which also correlated with higher migration rates. Knockdown of GRβ in the T24 cells resulted in a decreased migration rate. Mutational analysis of the 3' untranslated region (UTR) of human GRβ revealed that miR144 might positively regulate expression. Indeed, overexpression of miR144 increased GRβ by 3.8 fold. In addition, miR144 and GRβ were upregulated during migration. We used a peptide nucleic acid conjugated to a cell penetrating-peptide (Sweet-P) to block the binding site for miR144 in the 3'UTR of GRβ. Sweet-P effectively prevented miR144 actions and decreased GRβ expression, as well as the migration of the T24 human bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease. PMID:27036026

  9. A lateral signalling pathway coordinates shape volatility during cell migration

    PubMed Central

    Zhang, Liang; Luga, Valbona; Armitage, Sarah K.; Musiol, Martin; Won, Amy; Yip, Christopher M.; Plotnikov, Sergey V.; Wrana, Jeffrey L.

    2016-01-01

    Cell migration is fundamental for both physiological and pathological processes. Migrating cells usually display high dynamics in morphology, which is orchestrated by an integrative array of signalling pathways. Here we identify a novel pathway, we term lateral signalling, comprised of the planar cell polarity (PCP) protein Pk1 and the RhoGAPs, Arhgap21/23. We show that the Pk1–Arhgap21/23 complex inhibits RhoA, is localized on the non-protrusive lateral membrane cortex and its disruption leads to the disorganization of the actomyosin network and altered focal adhesion dynamics. Pk1-mediated lateral signalling confines protrusive activity and is regulated by Smurf2, an E3 ubiquitin ligase in the PCP pathway. Furthermore, we demonstrate that dynamic interplay between lateral and protrusive signalling generates cyclical fluctuations in cell shape that we quantify here as shape volatility, which strongly correlates with migration speed. These studies uncover a previously unrecognized lateral signalling pathway that coordinates shape volatility during productive cell migration. PMID:27226243

  10. Glucocorticoid receptor beta increases migration of human bladder cancer cells

    PubMed Central

    McBeth, Lucien; Nwaneri, Assumpta C.; Grabnar, Maria; Demeter, Jonathan; Nestor-Kalinoski, Andrea; Hinds, Terry D.

    2016-01-01

    Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p < 0.05) increased expression of GRβ compared to UMUC-3, which also correlated with higher migration rates. Knockdown of GRβ in the T24 cells resulted in a decreased migration rate. Mutational analysis of the 3′ untranslated region (UTR) of human GRβ revealed that miR144 might positively regulate expression. Indeed, overexpression of miR144 increased GRβ by 3.8 fold. In addition, miR144 and GRβ were upregulated during migration. We used a peptide nucleic acid conjugated to a cell penetrating-peptide (Sweet-P) to block the binding site for miR144 in the 3′UTR of GRβ. Sweet-P effectively prevented miR144 actions and decreased GRβ expression, as well as the migration of the T24 human bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease. PMID:27036026

  11. Optimal chemotaxis in intermittent migration of animal cells

    NASA Astrophysics Data System (ADS)

    Romanczuk, P.; Salbreux, G.

    2015-04-01

    Animal cells can sense chemical gradients without moving and are faced with the challenge of migrating towards a target despite noisy information on the target position. Here we discuss optimal search strategies for a chaser that moves by switching between two phases of motion ("run" and "tumble"), reorienting itself towards the target during tumble phases, and performing persistent migration during run phases. We show that the chaser average run time can be adjusted to minimize the target catching time or the spatial dispersion of the chasers. We obtain analytical results for the catching time and for the spatial dispersion in the limits of small and large ratios of run time to tumble time and scaling laws for the optimal run times. Our findings have implications for optimal chemotactic strategies in animal cell migration.

  12. Optimal chemotaxis in intermittent migration of animal cells.

    PubMed

    Romanczuk, P; Salbreux, G

    2015-04-01

    Animal cells can sense chemical gradients without moving and are faced with the challenge of migrating towards a target despite noisy information on the target position. Here we discuss optimal search strategies for a chaser that moves by switching between two phases of motion ("run" and "tumble"), reorienting itself towards the target during tumble phases, and performing persistent migration during run phases. We show that the chaser average run time can be adjusted to minimize the target catching time or the spatial dispersion of the chasers. We obtain analytical results for the catching time and for the spatial dispersion in the limits of small and large ratios of run time to tumble time and scaling laws for the optimal run times. Our findings have implications for optimal chemotactic strategies in animal cell migration.

  13. Short-Lived, Transitory Cell-Cell Interactions Foster Migration-Dependent Aggregation

    PubMed Central

    Pope, Melissa D.; Asthagiri, Anand R.

    2012-01-01

    During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications. PMID:22912835

  14. Knockdown of Legumain Suppresses Cervical Cancer Cell Migration and Invasion.

    PubMed

    Meng, Fei; Liu, Wei

    2016-01-01

    Cervical cancer is the second leading type of cancer in women living in less developed countries. The pathological and molecular mechanisms of cervical cancer are not comprehensively known. Though legumain has been found to be highly expressed in various types of solid tumors, its expression and biological function in cervical cancer remain unknown. In this study, we aimed to investigate legumain expression and functions in cervical cancer. We found that legumain was highly expressed in cervical cancer cells. When knocked down, legumain expression in HeLa and SiHa cells significantly reduced its migration and invasion abilities compared with control cells. Furthermore, legumain silencing suppressed the activation of matrix metalloproteases (MMP2 and MMP3) in cervical cancer cells. This study indicates that legumain might play an important role in cervical cancer cell migration and invasion. Legumain might be a potential therapeutic target for cervical cancer therapy.

  15. Fascin Regulates Nuclear Movement and Deformation in Migrating Cells.

    PubMed

    Jayo, Asier; Malboubi, Majid; Antoku, Susumu; Chang, Wakam; Ortiz-Zapater, Elena; Groen, Christopher; Pfisterer, Karin; Tootle, Tina; Charras, Guillaume; Gundersen, Gregg G; Parsons, Maddy

    2016-08-22

    Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion. PMID:27554857

  16. Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD.

    PubMed

    Mitter, Sayak K; Song, Chunjuan; Qi, Xiaoping; Mao, Haoyu; Rao, Haripriya; Akin, Debra; Lewin, Alfred; Grant, Maria; Dunn, William; Ding, Jindong; Bowes Rickman, Catherine; Boulton, Michael

    2014-01-01

    Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD. PMID:25484094

  17. Quantification of hydrodynamic factors influencing cell lateral migration

    NASA Astrophysics Data System (ADS)

    Nix, Stephanie; Imai, Yohsuke; Ishikawa, Takuji

    2015-11-01

    The study of the migration of blood cells perpendicular to the direction of blood flow, or lateral migration, is motivated by the differing behavior of the various types of blood cells. In vivo, red blood cells are observed to flow in the central region of the blood vessel, particularly in the microcirculation, while other types of cells in the blood, including white blood cells and platelets, are observed to flow disproportionately near the vessel wall. However, the specifics regarding the effect of hydrodynamic and biological factors are still unknown. Thus, in this study, we aim to quantify the effect of hydrodynamic factors on a cell model numerically using the boundary integral method. By using the boundary integral method, we can isolate the effect of a single hydrodynamic factor, such as a wall or given flow distribution, in an otherwise infinite flow. Then, we can use the obtained numerical results to develop a semi-analytical model describing the cell lateral migration dependent on only the flow geometry and the viscosity ratio between the cell and external fluid.

  18. Syndecan-1 Regulates Cell Migration and Fibronectin Fibril Assembly

    PubMed Central

    Stepp, Mary Ann; Daley, William P.; Bernstein, Audrey M.; Pal-Ghosh, Sonali; Tadvalkar, Gauri; Shashurin, Alexey; Palsen, Sarah; Jurjus, Rosalyn A.; Larsen, Melinda

    2011-01-01

    Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1 null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN-fibrillogenesis and migration in sdc1 null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1 null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis. PMID:20580707

  19. Probing cell migration in confined environments by plasma lithography.

    PubMed

    Junkin, Michael; Wong, Pak Kin

    2011-03-01

    Cellular processes are regulated by various mechanical and physical factors in their local microenvironment such as geometric confinements, cell-substrate interactions, and cell-cell contact. Systematic elucidation of these regulatory mechanisms is crucial for fundamental understanding of cell biology and for rational design of biomedical devices and regenerative medicine. Here, we report a generally applicable plasma lithography technique, which performs selective surface functionalization on large substrate areas, for achieving long-term, stable confinements with length scales from 100 nm to 1 cm toward the investigation of cell-microenvironment interactions. In particular, we applied plasma lithography for cellular confinement of neuroblastomas, myoblasts, endothelial cells, and mammary gland epithelial cells, and examined the motion of mouse embryonic fibroblasts in directionality-confined environments for studying the effect of confinements on migratory behavior. In conjunction with live cell imaging, the distance traveled, velocity, and angular motion of individual cells and collective cell migration behaviors were measured in confined environments with dimensions comparable to a cell. A critical length scale that a cell could conceivably occupy and migrate to was also identified by investigating the behaviors of cells using confined environments with subcellular length scales.

  20. A new role for GABA: inhibition of tumor cell migration.

    PubMed

    Ortega, Arturo

    2003-04-01

    GABA, the main inhibitory neurotransmitter in the vertebrate brain, participates outside the CNS in diverse functions such as platelet aggregation and the acrosomal reaction in spermatozoa. A recent study now demonstrates that GABA inhibits the migration of colon carcinoma cells, paving the way to the development of specific pharmacological agents that delay or inhibit invasion and metastasis of various cancer types.

  1. Describing Directional Cell Migration with a Characteristic Directionality Time

    PubMed Central

    Loosley, Alex J.; O’Brien, Xian M.; Reichner, Jonathan S.; Tang, Jay X.

    2015-01-01

    Many cell types can bias their direction of locomotion by coupling to external cues. Characteristics such as how fast a cell migrates and the directedness of its migration path can be quantified to provide metrics that determine which biochemical and biomechanical factors affect directional cell migration, and by how much. To be useful, these metrics must be reproducible from one experimental setting to another. However, most are not reproducible because their numerical values depend on technical parameters like sampling interval and measurement error. To address the need for a reproducible metric, we analytically derive a metric called directionality time, the minimum observation time required to identify motion as directionally biased. We show that the corresponding fit function is applicable to a variety of ergodic, directionally biased motions. A motion is ergodic when the underlying dynamical properties such as speed or directional bias do not change over time. Measuring the directionality of nonergodic motion is less straightforward but we also show how this class of motion can be analyzed. Simulations are used to show the robustness of directionality time measurements and its decoupling from measurement errors. As a practical example, we demonstrate the measurement of directionality time, step-by-step, on noisy, nonergodic trajectories of chemotactic neutrophils. Because of its inherent generality, directionality time ought to be useful for characterizing a broad range of motions including intracellular transport, cell motility, and animal migration. PMID:25992908

  2. Endogenous electric fields as guiding cue for cell migration

    PubMed Central

    Funk, Richard H. W.

    2015-01-01

    This review covers two topics: (1) “membrane potential of low magnitude and related electric fields (bioelectricity)” and (2) “cell migration under the guiding cue of electric fields (EF).”Membrane potentials for this “bioelectricity” arise from the segregation of charges by special molecular machines (pumps, transporters, ion channels) situated within the plasma membrane of each cell type (including eukaryotic non-neural animal cells). The arising patterns of ion gradients direct many cell- and molecular biological processes such as embryogenesis, wound healing, regeneration. Furthermore, EF are important as guiding cues for cell migration and are often overriding chemical or topographic cues. In osteoblasts, for instance, the directional information of EF is captured by charged transporters on the cell membrane and transferred into signaling mechanisms that modulate the cytoskeleton and motor proteins. This results in a persistent directional migration along an EF guiding cue. As an outlook, we discuss questions concerning the fluctuation of EF and the frequencies and mapping of the “electric” interior of the cell. Another exciting topic for further research is the modeling of field concepts for such distant, non-chemical cellular interactions. PMID:26029113

  3. Expression profiling of the RPE in zebrafish smarca4 mutant revealed altered signals that potentially affect RPE and retinal differentiation

    PubMed Central

    Ma, Ping; Collery, Ross; Trowbridge, Sara; Zhong, Wenxuan; Leung, Yuk Fai

    2014-01-01

    Purpose The purpose of this study was to develop a framework for analyzing retinal pigment epithelium (RPE) expression profiles from zebrafish eye mutants. Methods The fish model we used was SWI/SNF-related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (smarca4), a retinal dystrophic mutant with a previously described retinal phenotype and expression profiles. Histological and Affymetrix GeneChip analyses were conducted to characterize the RPE defects and underlying differential expression, respectively. Results Histological analysis revealed that smarca4 RPE was formed, but its differentiation was abnormal. In particular, ultrastructural analysis of smarca4 RPE by transmission electron microscopy demonstrated several defects in melanogenesis. The nature of these defects also suggests that the cytoskeletal dynamics, which are tightly linked with melanogenesis, were impaired in smarca4 RPE. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, the gene expression profiles of microdissected retinas and RPE-attached retinas were measured with Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples by subtracting the retinal expression values from the expression values of the RPE-attached retinas. A factorial analysis was conducted using the expression values of the RPE, retinal, and whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted models to select for three groups of genes: 1) smarca4-regulated RPE genes, 2) smarca4-regulated retinal genes, and 3) smarca4-regulated RPE genes that are not differentially expressed in the retina. Interestingly, the third group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, and paracrine and intracellular signal transduction. Conclusions Our analytical framework provides an experimental approach to identify differentially-regulated genes in the

  4. Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

    PubMed Central

    Zhao, Yuting; Liu, Jianglan; Yang, Changsong; Capraro, Benjamin R.; Baumgart, Tobias; Bradley, Ryan P.; Ramakrishnan, N.; Xu, Xiaowei; Radhakrishnan, Ravi; Svitkina, Tatyana; Guo, Wei

    2013-01-01

    Dynamic shape changes of the plasma membrane are fundamental to many processes ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations towards the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. In cells, the membrane-deformation function of Exo70 is required for protrusion formation and directional cell migration. Exo70 thus represents a membrane-bending protein that may couple actin dynamics and plasma membrane remodeling for morphogenesis. PMID:23948253

  5. IAP proteins: regulators of cell migration and development.

    PubMed

    Kenneth, Niall S; Duckett, Colin S

    2012-12-01

    The cytoprotective properties of vertebrate inhibitor of apoptosis (IAP) proteins have been the subject of much study. These proteins have, however, emerged as key signaling intermediates modulating a variety of cellular functions through their ability to act as E3 ubiquitin ligases. This review will focus on the cell death-independent roles of the IAP proteins, focusing on recent reports indicating that c-IAPs and XIAP are key molecules involved in modulating cell migration and development.

  6. Migration of amoeba cells in an electric field

    NASA Astrophysics Data System (ADS)

    Guido, Isabella; Bodenschatz, Eberhard

    2015-03-01

    Exogenous and endogenous electric fields play a role in cell physiology as a guiding mechanism for the orientation and migration of cells. Electrotaxis of living cells has been observed for several cell types, e.g. neurons, fibroblasts, leukocytes, neural crest cells, cancer cells. Dictyostelium discoideum (Dd), an intensively investigated chemotactic model organism, also exhibits a strong electrotactic behavior moving toward the cathode under the influence of electric fields. Here we report experiments on the effects of DC electric fields on the directional migration of Dd cells. We apply the electric field to cells seeded into microfluidic devices equipped with agar bridges to avoid any harmful effects of the electric field on the cells (ions formation, pH changes, etc.) and a constant flow to prevent the build-up of chemical gradient that elicits chemotaxis. Our results show that the cells linearly increase their speed over time when a constant electric field is applied for a prolonged duration (2 hours). This novel phenomenon cannot be attributed to mechanotaxis as the drag force of the electroosmotic flow is too small to produce shear forces that can reorient cells. It is independent of the cellular developmental stage and to our knowledge, it was not observed in chemotaxis. This work is supported by MaxSynBio project of the Max Planck Society.

  7. Expression of IGFBP-6 in a proliferative vitreoretinopathy rat model and its effects on retinal pigment epithelial cell proliferation and migration

    PubMed Central

    Zhao, Hong-Mei; Sheng, Min-Jie; Yu, Jing

    2014-01-01

    AIM To investigate the expression of insulin-like growth factor binding protein-6 (IGFBP-6) in a proliferative vitreoretinopathy (PVR) model and its effects on proliferation and migration in retinal pigment epithelial (RPE) cells. METHODS A PVR Wistar rat model was established by the intravitreal injection of RPE-J cells combined with platelet-rich plasma (PRP). The expression levels of IGFBP-6 were tested by ELISA. ARPE-19 cell proliferation was evaluated by the MTS method, and cell migration was evaluated by wound healing assays. RESULTS The success rate of the PVR model was 89.3% (25/28). IGFBP-6 was expressed at higher levels in the vitreous, serum and retina of rats experiencing advanced PVR (grade 3) than in the control group (vitreous: 152.80±15.08ng/mL vs 105.44±24.81ng/mL, P>0.05; serum: 93.48±9.27ng/mL vs 80.59±5.20ng/mL, P<0.05; retina: 3.02±0.38ng/mg vs 2.05±0.53ng/mg, P<0.05). In vitro, IGFBP-6 (500ng/mL) inhibited the IGF-II (50ng/mL) induced ARPE-19 cell proliferation (OD value at 24h: from 1.38±0.05 to 1.30±0.02; 48h: from 1.44±0.06 to 1.35±0.05). However, it did not affect basal or VEGF-, TGF-β- and PDGF-induced cell proliferation. IGFBP-6 (500ng/mL) reduced the IGF-II (50ng/mL)-induced would healing rate [24h: from (43.91±3.85)% to (29.76±2.49)%; 48 h: from (66.09±1.67)% to (59.88±3.43)%]. CONCLUSION Concentrations of IGFBP-6 increased in the vitreous, serum, and retinas only in advanced PVR in vivo. IGFBP-6 also inhibited IGF-II-induced cell proliferation in a not dose or time dependent manner and migration. IGFBP-6 participates in the development of PVR and might play a protective role in PVR. PMID:24634859

  8. T-cell Migration, Search Strategies and Mechanisms

    PubMed Central

    Krummel, Matthew F; Bartumeus, Frederic; Gérard, Audrey

    2016-01-01

    T cell migration is essential for T cell responses, allowing for detection of cognate antigen at the surface of an Antigen-Presenting Cell (APC) and for interactions with other cells involved in the immune response. Although appearing random, growing evidence supports that T cell motility patterns are strategic and governed by mechanisms that are optimized for both activation-stage and environment-specific attributes. In this Opinion Article, we will discuss how to understand the combined effects of T cell- intrinsic and -extrinsic forces upon these motility patterns when viewed in highly complex tissues filled with other cells involved in parallel motility. In particular, we will examine how insights from ‘search theory’ describe T cell movement across exploitation-exploration gradients, in the context of activation versus effector function and in the context of lymph nodes versus peripheral tissues. PMID:26852928

  9. Chemokine-guided cell migration and motility in zebrafish development.

    PubMed

    Bussmann, Jeroen; Raz, Erez

    2015-05-12

    Chemokines are vertebrate-specific, structurally related proteins that function primarily in controlling cell movements by activating specific 7-transmembrane receptors. Chemokines play critical roles in a large number of biological processes and are also involved in a range of pathological conditions. For these reasons, chemokines are at the focus of studies in developmental biology and of clinically oriented research aimed at controlling cancer, inflammation, and immunological diseases. The small size of the zebrafish embryos, their rapid external development, and optical properties as well as the large number of eggs and the fast expansion in genetic tools available make this model an extremely useful one for studying the function of chemokines and chemokine receptors in an in vivo setting. Here, we review the findings relevant to the role that chemokines play in the context of directed single-cell migration, primarily in neutrophils and germ cells, and compare it to the collective cell migration of the zebrafish lateral line. We present the current knowledge concerning the formation of the chemokine gradient, its interpretation within the cell, and the molecular mechanisms underlying the cellular response to chemokine signals during directed migration.

  10. Optogenetic approaches to cell migration and beyond

    PubMed Central

    Weitzman, Matthew; Hahn, Klaus M.

    2014-01-01

    Optogenetics, the use of genetically encoded tools to control protein function with light, can generate localized changes in signaling within living cells and animals. For years it has been focused on channel proteins for neurobiology, but has recently expanded to cover many different types of proteins, using a broad array of different protein engineering approaches. These methods have largely been directed at proteins involved in motility, cytoskeletal regulation and gene expression. This review provides a survey of non-channel proteins that have been engineered for optogenetics. Existing molecules are used to illustrate the advantages and disadvantages of the many imaginative new approaches that the reader can use to create light-controlled proteins. PMID:25216352

  11. Anandamide inhibits adhesion and migration of breast cancer cells

    SciTech Connect

    Grimaldi, Claudia; Pisanti, Simona; Laezza, Chiara; Malfitano, Anna Maria; Santoro, Antonietta; Vitale, Mario; Caruso, Maria Gabriella; Notarnicola, Maria; Iacuzzo, Irma; Portella, Giuseppe; Di Marzo, Vincenzo . E-mail: vdimarzo@icmib.na.cnr.it; Bifulco, Maurizio . E-mail: maubiful@unina.it

    2006-02-15

    The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB{sub 1} receptors could induce a non-invasive phenotype in breast mtastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB{sub 1} antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB{sub 1} receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB{sub 1} receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo.

  12. The Origin And Migration Of Primordial Germ Cells In Sturgeons

    PubMed Central

    Saito, Taiju; Pšenička, Martin; Goto, Rie; Adachi, Shinji; Inoue, Kunio; Arai, Katsutoshi; Yamaha, Etsuro

    2014-01-01

    Primordial germ cells (PGCs) arise elsewhere in the embryo and migrate into developing gonadal ridges during embryonic development. In several model animals, formation and migration patterns of PGCs have been studied, and it is known that these patterns vary. Sturgeons (genus Acipenser) have great potential for comparative and evolutionary studies of development. Sturgeons belong to the super class Actinoptergii, and their developmental pattern is similar to that of amphibians, although their phylogenetic position is an out-group to teleost fishes. Here, we reveal an injection technique for sturgeon eggs allowing visualization of germplasm and PGCs. Using this technique, we demonstrate that the PGCs are generated at the vegetal pole of the egg and they migrate on the yolky cell mass toward the gonadal ridge. We also provide evidence showing that PGCs are specified by inheritance of maternally supplied germplasm. Furthermore, we demonstrate that the migratory mechanism is well-conserved between sturgeon and other remotely related teleosts, such as goldfish, by a single PGCs transplantation (SPT) assay. The mode of PGCs specification in sturgeon is similar to that of anurans, but the migration pattern resembles that of teleosts. PMID:24505272

  13. Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

    PubMed Central

    Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

    2015-01-01

    The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

  14. Actein Inhibits Cell Proliferation and Migration in Human Osteosarcoma

    PubMed Central

    Chen, Zhi; Wu, Jingdong; Guo, Qinghao

    2016-01-01

    Background Osteosarcoma is one of the most common malignant bone cancers worldwide. Although the traditional chemotherapies have made some progression in the past decades, the mortality of osteosarcoma in children and adolescent is very high. Herein, the role of actein in osteosarcoma was explored. Material/Methods Cell viability assay was performed in osteosarcoma cell lines 143B and U2OS. Colony formation analysis was included when cells were treated with different doses of actin. Cell cycle assay was conducted to further examine the role of actein. Cell apoptotic rate and the relative activities of caspase-3, caspase-8, and caspase-9 were detected in 143B and U2OS osteosarcoma cells. Moreover, transwell assays were used to explore the effects of actein on cell metastasis. Results Actein significantly inhibited osteosarcoma cell viability in a time- and dose-dependent manner. Actein also dramatically suppressed the colony formation ability in osteosarcoma143B and U2OS cells. It was revealed that osteosarcoma cells were arrested in G0/G1 phase in the cell cycle progression and induced to apoptosis by administration of actein. The activities of pro-apoptotic factors such as caspase-3 and caspase-9 were significantly increased by actein. Furthermore, administration of actein decreased cell migrated and invasive abilities in both 143B and U2OS cell lines. Conclusions Actein inhibits tumor growth by inducing cell apoptosis in osteosarcoma. The inhibitive roles of actein in cell proliferation, migration and invasion suggest that actein may serve as a potential therapeutic agent in the treatment of osteosarcoma. PMID:27173526

  15. Migration of Drosophila intestinal stem cells across organ boundaries

    PubMed Central

    Takashima, Shigeo; Paul, Manash; Aghajanian, Patrick; Younossi-Hartenstein, Amelia; Hartenstein, Volker

    2013-01-01

    All components of the Drosophila intestinal tract, including the endodermal midgut and ectodermal hindgut/Malpighian tubules, maintain populations of dividing stem cells. In the midgut and hindgut, these stem cells originate from within larger populations of intestinal progenitors that proliferate during the larval stage and form the adult intestine during metamorphosis. The origin of stem cells found in the excretory Malpighian tubules (‘renal stem cells’) has not been established. In this paper, we investigate the migration patterns of intestinal progenitors that take place during metamorphosis. Our data demonstrate that a subset of adult midgut progenitors (AMPs) move posteriorly to form the adult ureters and, consecutively, the renal stem cells. Inhibiting cell migration by AMP-directed expression of a dominant-negative form of Rac1 protein results in the absence of stem cells in the Malpighian tubules. As the majority of the hindgut progenitor cells migrate posteriorly and differentiate into hindgut enterocytes, a group of the progenitor cells, unexpectedly, invades anteriorly into the midgut territory. Consequently, these progenitor cells differentiate into midgut enterocytes. The midgut determinant GATAe is required for the differentiation of midgut enterocytes derived from hindgut progenitors. Wingless signaling acts to balance the proportion of hindgut progenitors that differentiate as midgut versus hindgut enterocytes. Our findings indicate that a stable boundary between midgut and hindgut/Malpighian tubules is not established during early embryonic development; instead, pluripotent progenitor populations cross in between these organs in both directions, and are able to adopt the fate of the organ in which they come to reside. PMID:23571215

  16. Nifedipine Promotes the Proliferation and Migration of Breast Cancer Cells

    PubMed Central

    Guo, Dong-Qing; Zhang, Hao; Tan, Sheng-Jiang; Gu, Yu-Chun

    2014-01-01

    Nifedipine is widely used as a calcium channel blocker (CCB) to treat angina and hypertension,but it is controversial with respect the risk of stimulation of cancers. In this study, we demonstrated that nifedipine promoted the proliferation and migration of breast cancer cells both invivo and invitro. However, verapamil, another calcium channel blocker, didn’t exert the similar effects. Nifedipine and high concentration KCl failed to alter the [Ca2+]i in MDA-MB-231 cells, suggesting that such nifedipine effect was not related with calcium channel. Moreover, nifedipine decreased miRNA-524-5p, resulting in the up-regulation of brain protein I3 (BRI3). Erk pathway was consequently activated and led to the proliferation and migration of breast cancer cells. Silencing BRI3 reversed the promoting effect of nifedipine on the breast cancer. In a summary, nifedipine stimulated the proliferation and migration of breast cancer cells via the axis of miRNA-524-5p-BRI3–Erk pathway independently of its calcium channel-blocking activity. Our findings highlight that nifedipine but not verapamil is conducive for breast cancer growth and metastasis, urging that the caution should be taken in clinic to prescribe nifedipine to women who suffering both hypertension and breast cancer, and hypertension with a tendency in breast cancers. PMID:25436889

  17. SENP1 regulates cell migration and invasion in neuroblastoma.

    PubMed

    Xiang-Ming, Yan; Zhi-Qiang, Xu; Ting, Zhang; Jian, Wang; Jian, Pan; Li-Qun, Yuan; Ming-Cui, Fu; Hong-Liang, Xia; Xu, Cao; Yun, Zhou

    2016-05-01

    Neuroblastoma (NB) is an embryonic solid tumor derived from precursor cells of the sympathetic nervous system, and accounts for 11% of childhood cancers and around 15% of cancer deaths in children. SUMOylation and deSUMOylation are dynamic mechanisms regulating a spectrum of protein activities. The SUMO proteases (SENP) remove SUMO conjugate from proteins, and their expression is deregulated in diverse cancers. However, nothing is known about the role of SENPs in NBL. In the present study, we found that SENP1 expression was significantly high in metastatic NB tissues compared with primary NB tissues. Overexpression of SENP1 promoted NB cells migration and invasion. Inhibition of SENP1 could significantly suppress NB cell migration and invasion. Moreover, we found that SENP1 could regulate the expression of CDH1, MMP9, and MMP2. In summary, the data presented here indicate a significant role of SENP1 in the regulation of cell migration and invasion in NB and suppress SENP1 expression as promising candidates for novel treatment strategies of NB.

  18. Rpe65 as a modifier gene for inherited retinal degeneration

    PubMed Central

    Samardzija, M.; Wenzel, A.; Naash, M.; Remé, C. E.; Grimm, C.

    2009-01-01

    Light accelerates progression of retinal degeneration in many animal models of retinitis pigmentosa (RP). A sequence variant in the Rpe65 gene (Rpe65450Leu or Rpe65450Met) can act as a modulator of light-damage susceptibility in mice by influencing the kinetics of rhodopsin regeneration and thus by modulating the photon absorption. Depending on exposure duration and light intensity applied, white fluorescent light induces photoreceptor apoptosis and retinal degeneration in wild-type mice by the activation of one of two known molecular pathways. These pathways depend, respectively, on activation of the transcription factor c-Fos/AP-1 and on phototransduction activity. Here we tested Rpe65 as a genetic modifier for inherited retinal degeneration and analysed which degenerative pathway is activated in a transgenic mouse model of autosomal dominant RP. We show that retinal degeneration was reduced in mice expressing the Rpe65450Met variant and that these mice retained more visual pigment rhodopsin than did transgenic mice expressing the Rpe65450Leu variant. In addition, lack of phototransduction slowed retinal degeneration whereas ablation of c-Fos had no effect. We conclude that sequence variations in the Rpe65 gene can act as genetic modifiers in inherited retinal degeneration, presumably by regulating the daily rate of photon absorption through the modulation of rhodopsin regeneration kinetics. Increased absorption of photons and/or light sensitivity appear to accelerate retinal degeneration via an apoptotic cascade which involves phototransduction but not c-Fos. PMID:16519667

  19. Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures — a new donor for cell therapy

    PubMed Central

    Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

    2016-01-01

    Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases. PMID:27009841

  20. Capturing relevant extracellular matrices for investigating cell migration

    PubMed Central

    Keely, Patricia; Nain, Amrinder

    2015-01-01

    Much progress in understanding cell migration has been determined by using classic two-dimensional (2D) tissue culture platforms. However, increasingly, it is appreciated that certain properties of cell migration in vivo are not represented by strictly 2D assays. There is much interest in creating relevant three-dimensional (3D) culture environments and engineered platforms to better represent features of the extracellular matrix and stromal microenvironment that are not captured in 2D platforms. Important to this goal is a solid understanding of the features of the extracellular matrix—composition, stiffness, topography, and alignment—in different tissues and disease states and the development of means to capture these features PMID:26918156

  1. Capsaicin modulates proliferation, migration, and activation of hepatic stellate cells.

    PubMed

    Bitencourt, Shanna; Mesquita, Fernanda; Basso, Bruno; Schmid, Júlia; Ferreira, Gabriela; Rizzo, Lucas; Bauer, Moises; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis; Mannaerts, Inge; van Grunsven, Leo Adrianus; Oliveira, Jarbas

    2014-03-01

    Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro. PMID:23955514

  2. Epithelial bridges maintain tissue integrity during collective cell migration

    NASA Astrophysics Data System (ADS)

    Vedula, Sri Ram Krishna; Hirata, Hiroaki; Nai, Mui Hoon; Brugués, Agustí; Toyama, Yusuke; Trepat, Xavier; Lim, Chwee Teck; Ladoux, Benoit

    2014-01-01

    The ability of skin to act as a barrier is primarily determined by the efficiency of skin cells to maintain and restore its continuity and integrity. In fact, during wound healing keratinocytes migrate collectively to maintain their cohesion despite heterogeneities in the extracellular matrix. Here, we show that monolayers of human keratinocytes migrating along functionalized micropatterned surfaces comprising alternating strips of extracellular matrix (fibronectin) and non-adherent polymer form suspended multicellular bridges over the non-adherent areas. The bridges are held together by intercellular adhesion and are subjected to considerable tension, as indicated by the presence of prominent actin bundles. We also show that a model based on force propagation through an elastic material reproduces the main features of bridge maintenance and tension distribution. Our findings suggest that multicellular bridges maintain tissue integrity during wound healing when cell-substrate interactions are weak and may prove helpful in the design of artificial scaffolds for skin regeneration.

  3. Pancreatic Tumor Cell Secreted CCN1/Cyr61 Promotes Endothelial cell migration and Aberrant Neovascularization

    PubMed Central

    Maity, Gargi; Mehta, Smita; Haque, Inamul; Dhar, Kakali; Sarkar, Sandipto; Banerjee, Sushanta K.; Banerjee, Snigdha

    2014-01-01

    The complex signaling networks between cancer cells and adjacent endothelial cells make it challenging to unravel how cancer cells send extracellular messages to promote aberrant vascularization or tumor angiogenesis. Here, in vitro and in vivo models show that pancreatic cancer cell generated unique microenvironments can underlie endothelial cell migration and tumor angiogenesis. Mechanistically, we find that pancreatic cancer cell secreted CCN1/Cyr61 matricellular protein rewires the microenvironment to promote endothelial cell migration and tumor angiogenesis. This event can be overcome by Sonic Hedgehog (SHh) antibody treatment. Collectively, these studies identify a novel CCN1 signaling program in pancreatic cancer cells which activates SHh through autocrine-paracrine circuits to promote endothelial cell migration and tumor angiogenesis and suggests that CCN1 signaling of pancreatic cancer cells is vital for the regulation of tumor angiogenesis. Thus CCN1 signaling could be an ideal target for tumor vascular disruption in pancreatic cancer. PMID:24833309

  4. Iodine Affects Differentiation and Migration Process in Trophoblastic Cells.

    PubMed

    Olivo-Vidal, Zendy Evelyn; Rodríguez, Roció Coutiño; Arroyo-Helguera, Omar

    2016-02-01

    Iodine deficiency is associated with oxidative stress increase and preeclampsia during gestation, suggesting that iodine concentration plays an important role in the normal placenta physiology. The question raised is to analyze the effect of iodine deficiency on oxidative stress, viability, differentiation, and migration process and changes in the expression of differentiation and migration markers. Iodine deprivation was done using potassium perchlorate (KCLO4) to block sodium iodide symporter (NIS) transporter and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS to inhibit pendrine (PEN) transport for 3-48 h. Then trophoblast cells were treated with low iodine doses of 5-500 μM and high iodine doses of 100-5000 μM. Oxidative stress, viability, and human chorionic gonadotropin (hGC) were measured by colorimetric methods. Migration throphoblast cells were evaluated by both wound healing and Boyden chamber assays. Changes in mRNA expression were analyzed by real-time RT-PCR. Iodine deprivation induces a significant increase of reactive oxygen species (ROS), viability, and migration process vs control cells. We found a significant overregulation in the mRNA's peroxisome proliferator-activated receptor (PPAR-gamma), Snail, and matrix metalloproteinase-9 (MMP-9) mRNA's in cells deprived of iodine, as well as a down glial cell missing-1 (GCM-1) regulation, hGC, pregnancy-associated plasma protein-A (PAPP-A), and E-cadherin mRNA expression. The expression of hypoxic induction factor alpha (HIFα) mRNA does not change with iodine deprivation. In cells deprived of iodine, supplementing low iodine doses (5-500 μM) does not induce any significant changes in viability. However, ROS and migration process were decreased, although we found an increased human chorionic gonadotropin (hCG) secretion as a differentiation marker. In addition, we found that PPAR-gamma, Snail, and MPP-9 mRNAs expression are downregulated with low iodine doses, in contrast with GCM-1, PAPP

  5. Directional Collective Cell Migration Emerges as a Property of Cell Interactions

    PubMed Central

    Woods, Mae L.; Carmona-Fontaine, Carlos; Barnes, Chris P.; Couzin, Iain D.; Mayor, Roberto; Page, Karen M.

    2014-01-01

    Collective cell migration is a fundamental process, occurring during embryogenesis and cancer metastasis. Neural crest cells exhibit such coordinated migration, where aberrant motion can lead to fatality or dysfunction of the embryo. Migration involves at least two complementary mechanisms: contact inhibition of locomotion (a repulsive interaction corresponding to a directional change of migration upon contact with a reciprocating cell), and co-attraction (a mutual chemoattraction mechanism). Here, we develop and employ a parameterized discrete element model of neural crest cells, to investigate how these mechanisms contribute to long-range directional migration during development. Motion is characterized using a coherence parameter and the time taken to reach, collectively, a target location. The simulated cell group is shown to switch from a diffusive to a persistent state as the response-rate to co-attraction is increased. Furthermore, the model predicts that when co-attraction is inhibited, neural crest cells can migrate into restrictive regions. Indeed, inhibition of co-attraction in vivo and in vitro leads to cell invasion into restrictive areas, confirming the prediction of the model. This suggests that the interplay between the complementary mechanisms may contribute to guidance of the neural crest. We conclude that directional migration is a system property and does not require action of external chemoattractants. PMID:25181349

  6. Novel interactions between erythroblast macrophage protein and cell migration.

    PubMed

    Javan, Gulnaz T; Can, Ismail; Yeboah, Fred; Lee, Youngil; Soni, Shivani

    2016-09-01

    Erythroblast macrophage protein is a novel protein known to mediate attachment of erythroid cells to macrophages to form erythroblastic islands in bone marrow during erythropoiesis. Emp-null macrophages are small with round morphologies, and lack cytoplasmic projections which imply immature structure. The role of Emp in macrophage development and function is not fully elucidated. Macrophages perform varied functions (e.g. homeostasis, erythropoiesis), and are implicated in numerous pathophysiological conditions such as cellular malignancy. The objective of the current study is to investigate the interaction of Emp with cytoskeletal- and cell migration-associated proteins involved in macrophage functions. A short hairpin RNA lentiviral system was use to down-regulate the expression of Emp in macrophage cells. A cell migration assay revealed that the relocation of macrophages was significantly inhibited when Emp expression was decreased. To further analyze changes in gene expression related to cell motility, PCR array was performed by down-regulating Emp expression. The results indicated that expression of mitogen-activated protein kinase 1 and thymoma viral proto-oncogene 1 were significantly higher when Emp was down-regulated. The results implicate Emp in abnormal cell motility, thus, warrants to assess its role in cancer where tumor cell motility is required for invasion and metastasis. PMID:27519940

  7. Novel interactions between erythroblast macrophage protein and cell migration.

    PubMed

    Javan, Gulnaz T; Can, Ismail; Yeboah, Fred; Lee, Youngil; Soni, Shivani

    2016-09-01

    Erythroblast macrophage protein is a novel protein known to mediate attachment of erythroid cells to macrophages to form erythroblastic islands in bone marrow during erythropoiesis. Emp-null macrophages are small with round morphologies, and lack cytoplasmic projections which imply immature structure. The role of Emp in macrophage development and function is not fully elucidated. Macrophages perform varied functions (e.g. homeostasis, erythropoiesis), and are implicated in numerous pathophysiological conditions such as cellular malignancy. The objective of the current study is to investigate the interaction of Emp with cytoskeletal- and cell migration-associated proteins involved in macrophage functions. A short hairpin RNA lentiviral system was use to down-regulate the expression of Emp in macrophage cells. A cell migration assay revealed that the relocation of macrophages was significantly inhibited when Emp expression was decreased. To further analyze changes in gene expression related to cell motility, PCR array was performed by down-regulating Emp expression. The results indicated that expression of mitogen-activated protein kinase 1 and thymoma viral proto-oncogene 1 were significantly higher when Emp was down-regulated. The results implicate Emp in abnormal cell motility, thus, warrants to assess its role in cancer where tumor cell motility is required for invasion and metastasis.

  8. Lysophosphatidic acid induces cell migration through the selective activation of Akt1

    PubMed Central

    Kim, Eun Kyoung; Yun, Sung Ji; Do, Kee Hun; Kim, Min Sung; Cho, Mong; Suh, Dong-Soo; Kim, Chi Dae; Kim, Jae Ho; Birnbaum, Morris J.

    2008-01-01

    Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration. PMID:18779657

  9. Serotonin induces pulmonary artery smooth muscle cell migration

    PubMed Central

    Day, Regina M.; Agyeman, Abena S.; Segel, Michael J.; Chévere, Rubén D.; Angelosanto, Jill M.; Suzuki, Yuichiro J.; Fanburg, Barry L.

    2007-01-01

    The chronic phase of pulmonary arterial hypertension (PAH) is associated with vascular remodeling, especially thickening of the smooth muscle layer of large pulmonary arteries and muscularization of small pulmonary vessels, which normally have no associated smooth muscle. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMC), and may be important for in vivo pulmonary vascular remodeling. Here, we show that 5-HT stimulates migration of pulmonary artery PASMC. Treatment with 5-HT for 16 h increased migration of PASMC up to four-fold as monitored in a modified Boyden chamber assay. Increased migratory responses were associated with cellular morphological changes and reorganization of the actin cytoskeleton. 5-HT-induced alterations in morphology were previously shown in our laboratory to require cAMP [Lee SL, Fanburg BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Phys 1992;150(2):396–405], and the 5-HT4 receptor was pharmacologically determined to be the primary activator of cAMP in bovine PASMC [Becker BN, Gettys TW, Middleton JP, Olsen CL, Albers FJ, Lee SL, et al. 8-Hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992;42(5):817–25]. We examined the role of the 5-HT4 receptor and cAMP in 5-HT-induced bovine PASMC migration. PASMC express 5-HT4 receptor mRNA, and a 5-HT4 receptor antagonist and a cAMP antagonist completely blocked 5-HT-induced cellular migration. Consistent with our previous report that a cAMP-dependent Cl− channel is required for 5-HT-induced morphological changes in PASMC, phenylanthranilic acid, a Cl− channel blocker, inhibited actin cytoskeletal reorganization and migration produced by 5-HT. We conclude that 5-HT stimulates PASMC migration and

  10. Cu Migration in Polycrystalline CdTe Solar Cells

    SciTech Connect

    Guo, Da; Akis, Richard; Brinkman, Daniel; Sankin, Igor; Fang, Tian; Vasileska, Dragica; Ringhofer, Christian

    2014-03-12

    An impurity reaction-diffusion model is applied to Cu defects and related intrinsic defects in polycrystalline CdTe for a better understanding of Cu’s role in the cell level reliability of CdTe PV devices. The simulation yields transient Cu distributions in polycrystalline CdTe during solar cell processing and stressing. Preliminary results for Cu migration using available diffusivity and solubility data show that Cu accumulates near the back contact, a phenomena that is commonly observed in devices after back-contact processing or stress conditions.

  11. Functional screening with a live cell imaging-based random cell migration assay.

    PubMed

    van Roosmalen, Wies; Le Dévédec, Sylvia E; Zovko, Sandra; de Bont, Hans; van de Water, Bob

    2011-01-01

    Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrinsic cell signaling, a number of different tools and approaches have been developed. In order to unravel the key pathways and players involved in the regulation of (cancer) cell migration, a systematical mapping of the players/pathways is required. For this purpose, we developed a cell migration assay based on automatic high-throughput microscopy screen. This approach allows for screening of hundreds of genes, e.g., those encoding various kinases and phosphatases but can also be used for screening of drugs libraries. Moreover, we have developed an automatic analysis pipeline comprising of (a) automatic data acquisition (movie) and (b) automatic analysis of the acquired movies of the migrating cells. Here, we describe various facets of this approach. Since cell migration is essential in progression of cancer metastasis, we describe two examples of experiments performed on highly motile (metastatic) cancer cells.

  12. Cell traction in collective cell migration and morphogenesis: The chase and run mechanism

    PubMed Central

    Szabó, András; Mayor, Roberto

    2015-01-01

    Directional collective cell migration plays an important role in development, physiology, and disease. An increasing number of studies revealed key aspects of how cells coordinate their movement through distances surpassing several cell diameters. While physical modeling and measurements of forces during collective cell movements helped to reveal key mechanisms, most of these studies focus on tightly connected epithelial cultures. Less is known about collective migration of mesenchymal cells. A typical example of such behavior is the migration of the neural crest cells, which migrate large distances as a group. A recent study revealed that this persistent migration is aided by the interaction between the neural crest and the neighboring placode cells, whereby neural crest chase the placodes via chemotaxis, but upon contact both populations undergo contact inhibition of locomotion and a rapid reorganization of cellular traction. The resulting asymmetric traction field of the placodes forces them to run away from the chasers. We argue that this chase and run interaction may not be specific only to the neural crest system, but could serve as the underlying mechanism for several morphogenetic processes involving collective cell migration. PMID:26267782

  13. Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces.

    PubMed

    Jeon, Hojeong; Koo, Sangmo; Reese, Willie Mae; Loskill, Peter; Grigoropoulos, Costas P; Healy, Kevin E

    2015-09-01

    Although adhesive interactions between cells and nanostructured interfaces have been studied extensively, there is a paucity of data on how nanostructured interfaces repel cells by directing cell migration and cell-colony organization. Here, by using multiphoton ablation lithography to pattern surfaces with nanoscale craters of various aspect ratios and pitches, we show that the surfaces altered the cells' focal-adhesion size and distribution, thus affecting cell morphology, migration and ultimately localization. We also show that nanocrater pitch can disrupt the formation of mature focal adhesions to favour the migration of cells towards higher-pitched regions, which present increased planar area for the formation of stable focal adhesions. Moreover, by designing surfaces with variable pitch but constant nanocrater dimensions, we were able to create circular and striped cellular patterns. Our surface-patterning approach, which does not involve chemical treatments and can be applied to various materials, represents a simple method to control cell behaviour on surfaces. PMID:26213899

  14. Mechanobiology of cell migration in the context of dynamic two-way cell-matrix interactions.

    PubMed

    Kurniawan, Nicholas A; Chaudhuri, Parthiv Kant; Lim, Chwee Teck

    2016-05-24

    Migration of cells is integral in various physiological processes in all facets of life. These range from embryonic development, morphogenesis, and wound healing, to disease pathology such as cancer metastasis. While cell migratory behavior has been traditionally studied using simple assays on culture dishes, in recent years it has been increasingly realized that the physical, mechanical, and chemical aspects of the matrix are key determinants of the migration mechanism. In this paper, we will describe the mechanobiological changes that accompany the dynamic cell-matrix interactions during cell migration. Furthermore, we will review what is to date known about how these changes feed back to the dynamics and biomechanical properties of the cell and the matrix. Elucidating the role of these intimate cell-matrix interactions will provide not only a better multi-scale understanding of cell motility in its physiological context, but also a more holistic perspective for designing approaches to regulate cell behavior.

  15. Human Mesenchymal Stem Cell Morphology and Migration on Microtextured Titanium

    PubMed Central

    Banik, Brittany L.; Riley, Thomas R.; Platt, Christina J.; Brown, Justin L.

    2016-01-01

    The implant used in spinal fusion procedures is an essential component to achieving successful arthrodesis. At the cellular level, the implant impacts healing and fusion through a series of steps: first, mesenchymal stem cells (MSCs) need to adhere and proliferate to cover the implant; second, the MSCs must differentiate into osteoblasts; third, the osteoid matrix produced by the osteoblasts needs to generate new bone tissue, thoroughly integrating the implant with the vertebrate above and below. Previous research has demonstrated that microtextured titanium is advantageous over smooth titanium and PEEK implants for both promoting osteogenic differentiation and integrating with host bone tissue; however, no investigation to date has examined the early morphology and migration of MSCs on these surfaces. This study details cell spreading and morphology changes over 24 h, rate and directionality of migration 6–18 h post-seeding, differentiation markers at 10 days, and the long-term morphology of MSCs at 7 days, on microtextured, acid-etched titanium (endoskeleton), smooth titanium, and smooth PEEK surfaces. The results demonstrate that in all metrics, the two titanium surfaces outperformed the PEEK surface. Furthermore, the rough acid-etched titanium surface presented the most favorable overall results, demonstrating the random migration needed to efficiently cover a surface in addition to morphologies consistent with osteoblasts and preosteoblasts. PMID:27243001

  16. Exploring the control circuit of cell migration by mathematical modeling.

    PubMed

    Satulovsky, Javier; Lui, Roger; Wang, Yu-li

    2008-05-01

    We have developed a top-down, rule-based mathematical model to explore the basic principles that coordinate mechanochemical events during animal cell migration, particularly the local-stimulation-global-inhibition model suggested originally for chemotaxis. Cells were modeled as a shape machine that protrudes or retracts in response to a combination of local protrusion and global retraction signals. Using an optimization algorithm to identify parameters that generate specific shapes and migration patterns, we show that the mechanism of local stimulation global inhibition can readily account for the behavior of Dictyostelium under a large collection of conditions. Within this collection, some parameters showed strong correlation, indicating that a normal phenotype may be maintained by complementation among functional modules. In addition, comparison of parameters for control and nocodazole-treated Dictyostelium identified the most prominent effect of microtubules as regulating the rates of retraction and protrusion signal decay, and the extent of global inhibition. Other changes in parameters can lead to profound transformations from amoeboid cells into cells mimicking keratocytes, neurons, or fibroblasts. Thus, a simple circuit of local stimulation-global inhibition can account for a wide range of cell behaviors. A similar top-down approach may be applied to other complex problems and combined with molecular manipulations to define specific protein functions.

  17. The effects of laser immunotherapy on cancer cell migration

    NASA Astrophysics Data System (ADS)

    Bahavar, Cody F.; Zhou, Feifan; Hasanjee, Aamr M.; Layton, Elivia; Lam, Anh; Chen, Wei R.; Vaughan, Melville B.

    2016-03-01

    Laser immunotherapy (LIT) uses laser irradiation and immunological stimulation to target all types of metastases and creates a long-term tumor resistance. Glycated chitosan (GC) is the immunological stimulant used in LIT. Interestingly, GC can act as a surfactant for single-walled carbon nanotubes (SWNTs) to immunologically modify SWNTs. SWNT-GC retains the optical properties of SWNTs and the immunological functions of GC to help increase the selectivity of the laser and create a more optimal immune response. One essential aspect of understanding this immune response is knowing how laser irradiation affects cancer cells' ability to metastasize. In this experiment, a cell migration assay was performed. A 2mm circular elastomer plugs were placed at the bottom of multi-well dishes. Pre-cancerous keratinocytes, different tumor cells, and fibroblasts were then plated separately in treated wells. Once the cells reached 100% confluence, they were irradiated by either a 980nm or 805nm wavelength laser. The goal was to determine the effects of laser irradiation and immunological stimulation on cancer cell migration in vitro, paying the way to understand the mechanism of LIT in treating metastatic tumors in cancer patients.

  18. Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq.

    PubMed

    Whitmore, S Scott; Wagner, Alex H; DeLuca, Adam P; Drack, Arlene V; Stone, Edwin M; Tucker, Budd A; Zeng, Shemin; Braun, Terry A; Mullins, Robert F; Scheetz, Todd E

    2014-12-01

    Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with

  19. Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq

    PubMed Central

    Whitmore, S. Scott; Wagner, Alex H.; DeLuca, Adam P.; Drack, Arlene V.; Stone, Edwin M.; Tucker, Budd A.; Zeng, Shemin; Braun, Terry A.; Mullins, Robert F.; Scheetz, Todd E.

    2014-01-01

    Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell-types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with

  20. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    SciTech Connect

    Zhang, Fenxi; Hong, Yan; Liang, Wenmei; Ren, Tongming; Jing, Suhua; Lin, Juntang

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  1. Vimentin contributes to human mammary epithelial cell migration.

    PubMed

    Gilles, C; Polette, M; Zahm, J M; Tournier, J M; Volders, L; Foidart, J M; Birembaut, P

    1999-12-01

    Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound's edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound's edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound's edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.

  2. Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines.

    PubMed

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Chen, Wei-Ching; Fang, Jung-Hua; Weng, Tzu-Yang; Chen, Yi-Ling

    2015-01-01

    Gastric cancer metastasis remains a major cause of cancer-related deaths. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. Argininosuccinate synthetase 1 (ASS1) expression is increased in gastric cancer. We detected the protein expression of ASS1 in human gastric cancer cell lines (AGS, NCI-N87, and MKN45) and in murine gastric cancer cell lines (3I and 3IB2). We used vector-mediated short hairpin RNA (shRNA) expression to silence ASS1 expression in the MKN45 and 3IB2 cell lines, and analyzed the effects of this protein on cell migration and metastasis. We demonstrated that ASS1 silencing suppressed cell migration in the MKN45 and 3IB2 cell lines. ASS1 knockdown significantly reduced liver metastasis in mice after the intrasplenic implantation of 3IB2 cancer cell clones. To determine whether arginine restriction may represent a therapeutic approach to treat gastric cancer, the sensitivity of tumor cells to arginine depletion was determined in gastric cancer cells. Arginine depletion significantly inhibited cell migration in the gastric cancer cell line. The silencing of ASS1 expression in MKN45 and 3IB2 gastric cancer cells markedly decreased STAT3 protein expression. In conclusion, our results indicate that the ASS1 protein is required for cell migration in gastric cancer cell lines. PMID:25928182

  3. Anabolic androgens affect the competitive interactions in cell migration and adhesion between normal mouse urothelial cells and urothelial carcinoma cells.

    PubMed

    Huang, Chi-Ping; Hsieh, Teng-Fu; Chen, Chi-Cheng; Hung, Xiao-Fan; Yu, Ai-Lin; Chang, Chawnshang; Shyr, Chih-Rong

    2014-09-26

    The urothelium is constantly rebuilt by normal urothelial cells to regenerate damaged tissues caused by stimuli in urine. However, the urothelial carcinoma cells expand the territory by aberrant growth of tumor cells, which migrate and occupy the damaged tissues to spread outside and disrupt the normal cells and organized tissues and form a tumor. Therefore, the interaction between normal urothelial cells and urothelial carcinoma cells affect the initiation and progression of urothelial tumors if normal urothelial cells fail to migrate and adhere to the damages sites to regenerate the tissues. Here, comparing normal murine urothelial cells with murine urothelial carcinoma cells (MBT-2), we found that normal cells had less migration ability than carcinoma cells. And in our co-culture system we found that carcinoma cells had propensity migrating toward normal urothelial cells and carcinoma cells had more advantages to adhere than normal cells. To reverse this condition, we used anabolic androgen, dihyrotestosterone (DHT) to treat normal cells and found that DHT treatment increased the migration ability of normal urothelial cells toward carcinoma cells and the adhesion capacity in competition with carcinoma cells. This study provides the base of a novel therapeutic approach by using anabolic hormone-enforced normal urothelial cells to regenerate the damage urothelium and defend against the occupancy of carcinoma cells to thwart cancer development and recurrence.

  4. Collective migration and cell jamming in asthma, cancer and development.

    PubMed

    Park, Jin-Ah; Atia, Lior; Mitchel, Jennifer A; Fredberg, Jeffrey J; Butler, James P

    2016-09-15

    Collective cellular migration within the epithelial layer impacts upon development, wound healing and cancer invasion, but remains poorly understood. Prevailing conceptual frameworks tend to focus on the isolated role of each particular underlying factor - taken one at a time or at most a few at a time - and thus might not be tailored to describe a cellular collective that embodies a wide palette of physical and molecular interactions that are both strong and complex. To bridge this gap, we shift the spotlight to the emerging concept of cell jamming, which points to only a small set of parameters that govern when a cellular collective might jam and rigidify like a solid, or instead unjam and flow like a fluid. As gateways to cellular migration, the unjamming transition (UJT) and the epithelial-to-mesenchymal transition (EMT) share certain superficial similarities, but their congruence - or lack thereof - remains unclear. In this Commentary, we discuss aspects of cell jamming, its established role in human epithelial cell layers derived from the airways of non-asthmatic and asthmatic donors, and its speculative but emerging roles in development and cancer cell invasion. PMID:27550520

  5. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

    PubMed Central

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S.; Riahi, Reza; Wong, Pak Kin

    2016-01-01

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters. PMID:26936382

  6. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

    PubMed

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S; Riahi, Reza; Wong, Pak Kin

    2016-03-03

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

  7. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

    NASA Astrophysics Data System (ADS)

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S.; Riahi, Reza; Wong, Pak Kin

    2016-03-01

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

  8. The migrations of Drosophila muscle founders and primordial germ cells are interdependent.

    PubMed

    Stepanik, Vincent; Dunipace, Leslie; Bae, Young-Kyung; Macabenta, Frank; Sun, Jingjing; Trisnadi, Nathanie; Stathopoulos, Angelike

    2016-09-01

    Caudal visceral mesoderm (CVM) cells migrate from posterior to anterior of the Drosophila embryo as two bilateral streams of cells to support the specification of longitudinal muscles along the midgut. To accomplish this long-distance migration, CVM cells receive input from their environment, but little is known about how this collective cell migration is regulated. In a screen we found that wunen mutants exhibit CVM cell migration defects. Wunens are lipid phosphate phosphatases known to regulate the directional migration of primordial germ cells (PGCs). PGC and CVM cell types interact while PGCs are en route to the somatic gonadal mesoderm, and previous studies have shown that CVM impacts PGC migration. In turn, we found here that CVM cells exhibit an affinity for PGCs, localizing to the position of PGCs whether mislocalized or trapped in the endoderm. In the absence of PGCs, CVM cells exhibit subtle changes, including more cohesive movement of the migrating collective, and an increased number of longitudinal muscles is found at anterior sections of the larval midgut. These data demonstrate that PGC and CVM cell migrations are interdependent and suggest that distinct migrating cell types can coordinately influence each other to promote effective cell migration during development. PMID:27578182

  9. Immuno-histochemical analysis of rod and cone reaction to RPE65 deficiency in the inferior and superior canine retina.

    PubMed

    Klein, Daniela; Mendes-Madeira, Alexandra; Schlegel, Patrice; Rolling, Fabienne; Lorenz, Birgit; Haverkamp, Silke; Stieger, Knut

    2014-01-01

    Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.

  10. Immuno-Histochemical Analysis of Rod and Cone Reaction to RPE65 Deficiency in the Inferior and Superior Canine Retina

    PubMed Central

    Klein, Daniela; Mendes-Madeira, Alexandra; Schlegel, Patrice; Rolling, Fabienne; Lorenz, Birgit; Haverkamp, Silke; Stieger, Knut

    2014-01-01

    Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model. PMID:24466015

  11. To The Abercrombie Meeting and back again: a journey into the world of cell migration.

    PubMed

    Makowska, Katarzyna Anna

    2013-01-01

    The 7th Abercrombie Meeting took place in Oxford this past summer. It was organized by The Royal Microscopical Society with the support of The British Society for Cell Biology. Michael Abercrombie was a pioneer in the field of investigating cell behavior using time-lapse microscopy. The meeting was focused on "multi-dimensional cell migration in development and disease" and it brought together many of the world's leading researchers in the area, providing an opportunity to discuss the very latest advances and possible future developments in the field. The meeting sessions included Invasive Migration, Invasive Adhesions in Migrating Cells, Signaling in Migration, Immune Cell Migration, Migrations during Morphogenesis and Migration and Disease. As with all Abercrombie meetings, the conference delegates were treated to a staggering array of live cell imaging, in vivo imaging and images generated by the latest developments in microscopy.

  12. Hsc70 regulates cell surface ASIC2 expression and vascular smooth muscle cell migration.

    PubMed

    Grifoni, Samira C; McKey, Susan E; Drummond, Heather A

    2008-05-01

    Recent studies suggest members of the degenerin (DEG)/epithelial Na(+) channel (ENaC)/acid-sensing ion channel (ASIC) protein family play an important role in vascular smooth muscle cell (VSMC) migration. In a previous investigation, we found suppression of a certain DEG/ENaC/ASIC member, ASIC2, increased VSMC chemotactic migration, raising the possibility that ASIC2 may play an inhibitory role. Because ASIC2 protein was retained in the cytoplasm, we reasoned increasing surface expression of ASIC2 might unmask the inhibitory role of ASIC2 in VSMC migration so we could test the hypothesis that ASIC2 inhibits VSMC migration. Therefore, we used the chemical chaperone glycerol to enhance ASIC2 expression. Glycerol 1) increased cytoplasm ASIC2 expression, 2) permitted detection of ASIC2 at the cell surface, and 3) inhibited platelet-derived growth factor (PDGF)-bb mediated VSMC migration. Furthermore, ASIC2 silencing completely abolished the inhibitory effect of glycerol on migration, suggesting upregulation of ASIC2 is responsible for glycerol-induced inhibition of VSMC migration. Because other investigators have shown that glycerol regulates ENaC/ASIC via interactions with a certain heat shock protein, heat shock protein 70 (Hsc70), we wanted to determine the importance of Hsc70 on ASIC2 expression in VSMCs. We found that Hsc70 silencing increases ASIC2 cell surface expression and inhibits VSMC migration, which is abolished by cosilencing ASIC2. These data demonstrate that Hsc70 inhibits ASIC2 expression, and, when the inhibitory effect of Hsc70 is removed, ASIC2 expression increases, resulting in reduced VSMC migration. Because VSMC migration contributes to vasculogenesis and remodeling following vascular injury, our findings raise the possibility that ASIC2-Hsc70 interactions may play a role in these processes. PMID:18310515

  13. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

    SciTech Connect

    Huang, Xionggao; Wei, Yantao; Ma, Haizhi; Zhang, Shaochong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  14. Time-lapse microscopy and image processing for stem cell research: modeling cell migration

    NASA Astrophysics Data System (ADS)

    Gustavsson, Tomas; Althoff, Karin; Degerman, Johan; Olsson, Torsten; Thoreson, Ann-Catrin; Thorlin, Thorleif; Eriksson, Peter

    2003-05-01

    This paper presents hardware and software procedures for automated cell tracking and migration modeling. A time-lapse microscopy system equipped with a computer controllable motorized stage was developed. The performance of this stage was improved by incorporating software algorithms for stage motion displacement compensation and auto focus. The microscope is suitable for in-vitro stem cell studies and allows for multiple cell culture image sequence acquisition. This enables comparative studies concerning rate of cell splits, average cell motion velocity, cell motion as a function of cell sample density and many more. Several cell segmentation procedures are described as well as a cell tracking algorithm. Statistical methods for describing cell migration patterns are presented. In particular, the Hidden Markov Model (HMM) was investigated. Results indicate that if the cell motion can be described as a non-stationary stochastic process, then the HMM can adequately model aspects of its dynamic behavior.

  15. Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells.

    PubMed

    Reffay, M; Parrini, M C; Cochet-Escartin, O; Ladoux, B; Buguin, A; Coscoy, S; Amblard, F; Camonis, J; Silberzan, P

    2014-03-01

    The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

  16. Tre1, a G Protein-Coupled Receptor, Directs Transepithelial Migration of Drosophila Germ Cells

    PubMed Central

    2003-01-01

    In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target. PMID:14691551

  17. Glioma migration: clues from the biology of neural progenitor cells and embryonic CNS cell migration.

    PubMed

    Dirks, P B

    2001-06-01

    Neural stem cells have recently come to the forefront in neurobiology because of the possibilities for CNS repair by transplantation. Further understanding of the biology of these cells is critical for making their use in CNS repair possible. It is likely that these discoveries will also have spin-offs for neuro-oncology as primary brain tumors may arise from a CNS progenitor cell. An understanding of the normal migratory ability of these cells is also likely to have a very important impact on the knowledge of brain tumor invasion.

  18. Notch1-Dll4 signalling and mechanical force regulate leader cell formation during collective cell migration.

    PubMed

    Riahi, Reza; Sun, Jian; Wang, Shue; Long, Min; Zhang, Donna D; Wong, Pak Kin

    2015-03-13

    At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct 'leader' phenotype with characteristic morphology and motility. However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here we use single-cell gene expression analysis and computational modelling to show that the leader cell identity is dynamically regulated by Dll4 signalling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

  19. Migration of Langerhans cells and gammadelta dendritic cells from UV-B-irradiated sheep skin.

    PubMed

    Dandie, G W; Clydesdale, G J; Radcliff, F J; Muller, H K

    2001-02-01

    Depletion of dendritic cells from UV-B-irradiated sheep skin was investigated by monitoring migration of these cells towards regional lymph nodes. By creating and cannulating pseudoafferent lymphatic vessels draining a defined region of skin, migrating cells were collected and enumerated throughout the response to UV-B irradiation. In the present study, the effects of exposing sheep flank skin to UV-B radiation clearly demonstrated a dose-dependent increase in the migration of Langerhans cells (LC) from the UV-B-exposed area to the draining lymph node. The range of UV-B doses assessed in this study included 2.7 kJ/m2, a suberythemal dose; 8 kJ/m2, 1 minimal erythemal dose (MED); 20.1 kJ/m2; 40.2 kJ/m2; and 80.4 kJ/m2, 10 MED. The LC were the cells most sensitive to UV-B treatment, with exposure to 8 kJ/m2 or greater reproducibly causing a significant increase in migration. Migration of gammadelta+ dendritic cells (gammadelta+ DC) from irradiated skin was also triggered by exposure to UV-B radiation, but dose dependency was not evident within the range of UV-B doses examined. This, in conjunction with the lack of any consistent correlation between either the timing or magnitude of migration peaks of these two cell types, suggests that different mechanisms govern the egress of LC and gammadelta+ DC from the skin. It is concluded that the depression of normal immune function in the skin after exposure to erythemal doses of UV-B radiation is associated with changes in the migration patterns of epidermal dendritic cells to local lymph nodes. PMID:11168622

  20. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    NASA Astrophysics Data System (ADS)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

  1. Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines

    PubMed Central

    Serfozo, Peter; Schlarman, Maggie S; Pierret, Chris; Maria, Bernard L; Kirk, Mark D

    2006-01-01

    Background Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF). Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium). RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed. PMID:16436212

  2. Gene Therapy Rescues Cone Structure and Function in the 3-Month-Old rd12 Mouse: A Model for Midcourse RPE65 Leber Congenital Amaurosis

    PubMed Central

    Li, Xia; Li, Wensheng; Dai, Xufeng; Kong, Fansheng; Zheng, Qinxiang; Zhou, Xiangtian; Lü, Fan; Chang, Bo; Rohrer, Bärbel; Hauswirth, William. W.; Qu, Jia; Pang, Ji-jing

    2011-01-01

    Purpose. RPE65 function is necessary in the retinal pigment epithelium (RPE) to generate chromophore for all opsins. Its absence results in vision loss and rapid cone degeneration. Recent Leber congenital amaurosis type 2 (LCA with RPE65 mutations) phase I clinical trials demonstrated restoration of vision on RPE65 gene transfer into RPE cells overlying cones. In the rd12 mouse, a naturally occurring model of RPE65-LCA early cone degeneration was observed; however, some peripheral M-cones remained. A prior study showed that AAV-mediated RPE65 expression can prevent early cone degeneration. The present study was conducted to test whether the remaining cones in older rd12 mice can be rescued. Methods. Subretinal treatment with the scAAV5-smCBA-hRPE65 vector was initiated at postnatal day (P)14 and P90. After 2 months, electroretinograms were recorded, and cone morphology was analyzed by using cone-specific peanut agglutinin and cone opsin–specific antibodies. Results. Cone degeneration started centrally and spread ventrally, with cells losing cone-opsin staining before that for the PNA-lectin–positive cone sheath. Gene therapy starting at P14 resulted in almost wild-type M- and S-cone function and morphology. Delaying gene-replacement rescued the remaining M-cones, and most important, more M-cone opsin–positive cells were identified than were present at the onset of gene therapy, suggesting that opsin expression could be reinitiated in cells with cone sheaths. Conclusions. The results support and extend those of the previous study that gene therapy can stop early cone degeneration, and, more important, they provide proof that delayed treatment can restore the function and morphology of the remaining cones. These results have important implications for the ongoing LCA2 clinical trials. PMID:21169527

  3. Role of medullary progenitor cells in epithelial cell migration and proliferation

    PubMed Central

    Chen, Dong; Chen, Zhiyong; Zhang, Yuning; Park, Chanyoung; Al-Omari, Ahmed

    2014-01-01

    This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive >50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D Matrigel. Moreover, most of the MPCs express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair. PMID:24808539

  4. Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration

    PubMed Central

    Salomon, Carlos; Yee, Sarah; Scholz-Romero, Katherin; Kobayashi, Miharu; Vaswani, Kanchan; Kvaskoff, David; Illanes, Sebastian E.; Mitchell, Murray D.; Rice, Gregory E.

    2014-01-01

    Background: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration. Methods: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37°C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected, and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software). Results: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 × 108 ± 2.5 × 108 particles/106 cells) compared to JEG-3 (2.86 × 108 ± 0.78 × 108 particles/106 cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (−exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, vs. control 25.09 ± 0.58 h, p < 0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes. Conclusion: The data obtained in this study are consistent with the hypothesis that the release, content, and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls. PMID:25157233

  5. Cadherin-11 mediates contact inhibition of locomotion during Xenopus neural crest cell migration.

    PubMed

    Becker, Sarah F S; Mayor, Roberto; Kashef, Jubin

    2013-01-01

    Collective cell migration is an essential feature both in embryonic development and cancer progression. The molecular mechanisms of these coordinated directional cell movements still need to be elucidated. The migration of cranial neural crest (CNC) cells during embryogenesis is an excellent model for collective cell migration in vivo. These highly motile and multipotent cells migrate directionally on defined routes throughout the embryo. Interestingly, local cell-cell interactions seem to be the key force for directionality. CNC cells can change their migration direction by a repulsive cell response called contact inhibition of locomotion (CIL). Cell protrusions collapse upon homotypic cell-cell contact and internal repolarization leads to formation of new protrusions toward cell-free regions. Wnt/PCP signaling was shown to mediate activation of small RhoGTPase RhoA and inhibition of cell protrusions at the contact side. However, the mechanism how a cell recognizes the contact is poorly understood. Here, we demonstrate that Xenopus cadherin-11 (Xcad-11) mediated cell-cell adhesion is necessary in CIL for directional and collective migration of CNC cells. Reduction of Xcad-11 adhesive function resulted in higher invasiveness of CNC due to loss of CIL. Additionally, transplantation analyses revealed that CNC migratory behaviour in vivo is non-directional and incomplete when Xcad-11 adhesive function is impaired. Blocking Wnt/PCP signaling led to similar results underlining the importance of Xcad-11 in the mechanism of CIL and directional migration of CNC.

  6. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient

    PubMed Central

    Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A.

    2014-01-01

    Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

  7. Iodine Migration and its Effect on Hysteresis in Perovskite Solar Cells.

    PubMed

    Li, Cheng; Tscheuschner, Steffen; Paulus, Fabian; Hopkinson, Paul E; Kießling, Johannes; Köhler, Anna; Vaynzof, Yana; Huettner, Sven

    2016-03-23

    The migration and accumulation of iodide ions create a modulation of the respective interfacial barriers causing the hysteresis in solar cells based on methylammonium lead iodide perovskites. Iodide ions are identified as the migrating species by measuring temperature dependent current-transients and photoelectron spectroscopy. The involved changes in the built-in potential due to ion migration are directly measured by electroabsorption spectroscopy.

  8. Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

    PubMed Central

    2014-01-01

    Background Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues. Results Here, we use an assay that allows to biochemically purify extending protrusions of cells migrating in response to three prototypical receptors: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes. Conclusions The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration. PMID:24987309

  9. Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies.

    PubMed

    Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

    2016-01-01

    Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

  10. Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

    PubMed Central

    Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

    2016-01-01

    Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

  11. Polydatin induces bone marrow stromal cells migration by activation of ERK1/2.

    PubMed

    Chen, ZhenQiu; Wei, QiuShi; Hong, GuoJu; Chen, Da; Liang, Jiang; He, Wei; Chen, Mei Hui

    2016-08-01

    Bone marrow stromal cells (BMSCs) have proven to be useful for the treatment of numerous human diseases. However, the reparative ability of BMSCs is limited by their poor migration. Polydatin, widely used in traditional Chinese remedies, has proven to exert protective effects to BMSCs. However, little is known about its role in BMSCs migration. In this study, we studied the effects of polydatin on rat BMSCs migration using the scratch wound healing and transwell migration assays. Our results showed polydatin could promote BMSCs migration. Further experiments showed activation of ERK 1/2, but not JNK, was required for polydatin-induced BMSCs migration, suggesting that polydatin may promote BMSCs migration via the ERK 1/2 signaling pathways. Taken together, our results indicate that polydatin might be beneficial for stem cell replacement therapy by improving BMSCs migration.

  12. Contact inhibition of locomotion probabilities drive solitary versus collective cell migration

    PubMed Central

    Desai, Ravi A.; Gopal, Smitha B.; Chen, Sophia; Chen, Christopher S.

    2013-01-01

    Contact inhibition of locomotion (CIL) is the process whereby cells collide, cease migrating in the direction of the collision, and repolarize their migration machinery away from the collision. Quantitative analysis of CIL has remained elusive because cell-to-cell collisions are infrequent in traditional cell culture. Moreover, whereas CIL predicts mutual cell repulsion and ‘scattering’ of cells, the same cells in vivo are observed to undergo CIL at some developmental times and collective cell migration at others. It remains unclear whether CIL is simply absent during collective cell migration, or if the two processes coexist and are perhaps even related. Here, we used micropatterned stripes of extracellular matrix to restrict cell migration to linear paths such that cells polarized in one of two directions and collisions between cells occurred frequently and consistently, permitting quantitative and unbiased analysis of CIL. Observing repolarization events in different contexts, including head-to-head collision, head-to-tail collision, collision with an inert barrier, or no collision, and describing polarization as a two-state transition indicated that CIL occurs probabilistically, and most strongly upon head-to-head collisions. In addition to strong CIL, we also observed ‘trains’ of cells moving collectively with high persistence that appeared to emerge from single cells. To reconcile these seemingly conflicting observations of CIL and collective cell migration, we constructed an agent-based model to simulate our experiments. Our model quantitatively predicted the emergence of collective migration, and demonstrated the sensitivity of such emergence to the probability of CIL. Thus CIL and collective migration can coexist, and in fact a shift in CIL probabilities may underlie transitions between solitary cell migration and collective cell migration. Taken together, our data demonstrate the emergence of persistently polarized, collective cell movement

  13. Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

    PubMed Central

    Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

    2012-01-01

    Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

  14. RPE and Choroid Mechanisms Underlying Ocular Growth and Myopia.

    PubMed

    Zhang, Yan; Wildsoet, Christine F

    2015-01-01

    Myopia is the most common type of refractive errors and one of the world's leading causes of blindness. Visual manipulations in animal models have provided convincing evidence for the role of environmental factors in myopia development. These models along with in vitro studies have provided important insights into underlying mechanisms. The key locations of the retinal pigment epithelium (RPE) and choroid make them plausible conduits for relaying growth regulatory signals originating in the retina to the sclera, which ultimately determines eye size and shape. Identifying the key signal molecules and their targets may lead to the development of new myopia control treatments. This section summarizes findings implicating the RPE and choroid in myopia development. For RPE and/or choroid, changes in morphology, activity of ion channels/transporters, as well as in gene and protein expression, have been linked to altered eye growth. Both tissues thus represent potential targets for novel therapies for myopia.

  15. RPE and Choroid Mechanisms Underlying Ocular Growth and Myopia

    PubMed Central

    Zhang, Yan; Wildsoet, Christine F.

    2016-01-01

    Myopia is the most common type of refractive errors and one of the world’s leading causes of blindness. Visual manipulations in animal models have provided convincing evidence for the role of environmental factors in myopia development. These models along with in vitro studies have provided important insights into underlying mechanisms. The key locations of the retinal pigment epithelium (RPE) and choroid make them plausible conduits for relaying growth regulatory signals originating in the retina to the sclera, which ultimately determines eye size and shape. Identifying the key signal molecules and their targets may lead to the development of new myopia control treatments. This section summarizes findings implicating the RPE and choroid in myopia development. For RPE and/or choroid, changes in morphology, activity of ion channels/transporters, as well as in gene and protein expression, have been linked to altered eye growth. Both tissues thus represent potential targets for novel therapies for myopia. PMID:26310157

  16. Membrane-Binding and Enzymatic Properties of RPE65

    PubMed Central

    Kiser, Philip D.; Palczewski, Krzysztof

    2010-01-01

    Regeneration of visual pigments is essential for sustained visual function. Although the requirement for non-photochemical regeneration of the visual chromophore, 11-cis-retinal, was recognized early on, it was only recently that the trans to cis retinoid isomerase activity required for this process was assigned to a specific protein, a microsomal membrane enzyme called RPE65. In this review, we outline progress that has been made in the functional characterization of RPE65. We then discuss general concepts related to protein-membrane interactions and the mechanism of the retinoid isomerization reaction and describe some of the important biochemical and structural features of RPE65 with respect to its membrane-binding and enzymatic properties. PMID:20304090

  17. miR-1 Inhibits Cell Growth, Migration, and Invasion by Targeting VEGFA in Osteosarcoma Cells

    PubMed Central

    Niu, Junjie; Guo, Qiaoge; Niu, Dongju; Liu, Bo

    2016-01-01

    microRNAs (miRNAs) are small noncoding RNAs and have been shown to play a crucial role in the osteosarcoma (OS) tumorigenesis and progression. VEGFA is a key regulator of angiogenesis and plays an important role in regulation of tumor metastasis. The objective of this study was to determine whether VEGFA was involved in miR-1-mediated suppression of proliferation, migration, and invasion of OS cells. The expression levels of miR-1 were significantly lower in OS tumor tissues than those in adjacent normal tissues and in SAOS-2 and U2OS cell lines compared to a normal osteoblast (NHOst) cell line. VEGFA was upregulated in OS tumor tissues and SAOS-2 and U2OS cell lines. The results of CCK-8 assay and transwell assay showed that miR-1 acted as a tumor suppressor by inhibiting cell proliferation, migration, and invasion in U2OS cells. Dual luciferase reporter assay demonstrated that VEGFA was a direct and functional target gene of miR-1. miR-1 directly inhibits the protein expression of VEGFA via its 3′-UTR. Knockdown of VEGFA by siRNA inhibited proliferation, migration, and invasion of U2OS cells. Our study suggested the potential inhibitory function of miR-1 in OS cell proliferation, migration, and invasion via inhibiting VEGFA. PMID:27777493

  18. Cell crawling mediates collective cell migration to close undamaged epithelial gaps

    PubMed Central

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C.; Sheetz, Michael P.; Trepat, Xavier; Ladoux, Benoît

    2012-01-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  19. Cell crawling mediates collective cell migration to close undamaged epithelial gaps.

    PubMed

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C; Sheetz, Michael P; Trepat, Xavier; Ladoux, Benoît

    2012-07-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  20. A Macroscopic Mathematical Model for Cell Migration Assays Using a Real-Time Cell Analysis

    PubMed Central

    Angelini, Claudia; Carfora, Maria Francesca; Carriero, Maria Vincenza; Natalini, Roberto

    2016-01-01

    Experiments of cell migration and chemotaxis assays have been classically performed in the so-called Boyden Chambers. A recent technology, xCELLigence Real Time Cell Analysis, is now allowing to monitor the cell migration in real time. This technology measures impedance changes caused by the gradual increase of electrode surface occupation by cells during the course of time and provide a Cell Index which is proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number. In this paper we propose a macroscopic mathematical model, based on advection-reaction-diffusion partial differential equations, describing the cell migration assay using the real-time technology. We carried out numerical simulations to compare simulated model dynamics with data of observed biological experiments on three different cell lines and in two experimental settings: absence of chemotactic signals (basal migration) and presence of a chemoattractant. Overall we conclude that our minimal mathematical model is able to describe the phenomenon in the real time scale and numerical results show a good agreement with the experimental evidences. PMID:27680883

  1. Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling

    SciTech Connect

    Seomun, Young; Joo, Choun-Ki

    2008-07-18

    Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins {alpha}2 and {beta}1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin {beta}1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

  2. Analysis of Cell Migration within a Three-dimensional Collagen Matrix

    PubMed Central

    Rommerswinkel, Nadine; Niggemann, Bernd; Keil, Silvia; Zänker, Kurt S.; Dittmar, Thomas

    2014-01-01

    The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment. PMID:25350138

  3. Analysis of cell migration within a three-dimensional collagen matrix.

    PubMed

    Rommerswinkel, Nadine; Niggemann, Bernd; Keil, Silvia; Zänker, Kurt S; Dittmar, Thomas

    2014-10-05

    The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

  4. Loss of lysophosphatidic acid receptor-3 enhances cell migration in rat lung tumor cells

    SciTech Connect

    Hayashi, Mai; Okabe, Kyoko; Yamawaki, Yasuna; Teranishi, Miki; Honoki, Kanya; Mori, Toshio; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2011-02-18

    Research highlights: {yields} Loss of the Lpar3 expression due to aberrant DNA methylation occurred in rat lung tumor cells. {yields} The Lpar3 inhibited cell migration of rat lung tumor cells. {yields} The Lpar3 may act as a negative regulator of rat lung tumor cells. -- Abstract: Lysophosphatidic acid (LPA) indicates several biological effects, such as cell proliferation, differentiation and migration. LPA interacts with G protein-coupled transmembrane LPA receptors. In our previous report, we detected that loss of the LPA receptor-1 (Lpar1) expression is due to its aberrant DNA methylation in rat tumor cell lines. In this study, to assess an involvement of the other LPA receptor, Lpar3, in the pathogenesis of rat lung tumor cells, we measured the expression levels of the Lpar3 gene and its DNA methylation status by reverse transcription (RT)-polymerase chain reaction (PCR) and bisulfite sequencing analyses, respectively. RLCNR lung adenocarcinoma cells showed reduced expression of the Lpar3, compared with normal lung tissues. In the 5' upstream region of the Lpar3, normal lung tissues were unmethylated. By contrast, RLCNR cells were highly methylated, correlating with reduced expressions of the Lpar3. Based on these results, we generated the Lpar3-expressing RLCNR-a3 cells and measured the cell migration ability. Interestingly, the cell migration of RLCNR-a3 cells was significantly lower than that of RLCNR cells. This study suggests that loss of the Lpar3 due to aberrant DNA methylation may be involved in the progression of rat lung tumor cells.

  5. Endothelial Cell Morphology and Migration are Altered by Changes in Gravitational Fields

    NASA Technical Reports Server (NTRS)

    Melhado, Caroline; Sanford, Gary; Harris-Hooker, Sandra

    1997-01-01

    Endothelial cell migration is important to vascular wall regeneration following injury or stress. However, the mechanism(s) governing this response is not well understood. The microgravity environment of space may complicate the response of these cells to injury. To date, there are no reports in this area. We examined how bovine aortic (BAEC) and pulmonary (BPEC) endothelial cells respond to denudation injury under hypergravity (HGrav) and simulated microgravity (MGrav), using image analysis. In 10% FBS, the migration of confluent BAEC and BPEC into the denuded area was not affected by HGrav or MGrav. However, in low FBS (0.5%), signficantly retarded migration under MGrav, and increased migration under HGrav was found. MGrav also decreased the migration of postconfluent BPEC while HGrav showed no difference. Both MGrav and HGrav strongly decreased the migration of postconfluent BAEC. Also, both cell lines showed significant morphological changes by scanning electron microscopy. These studies indicate that endothelial cell function is affected by changes in gravity.

  6. Manipulation of neutrophil-like HL-60 cells for the study of directed cell migration.

    PubMed

    Millius, Arthur; Weiner, Orion D

    2010-01-01

    Many cells undergo directed cell migration in response to external cues in a process known as chemotaxis. This ability is essential for many single-celled organisms to hunt and mate, the development of multicellular organisms, and the functioning of the immune system. Because of their relative ease of manipulation and their robust chemotactic abilities, the neutrophil-like cell line (HL-60) has been a powerful system to analyze directed cell migration. In this chapter, we describe the maintenance and transient transfection of HL-60 cells and explain how to analyze their behavior with two standard chemotactic assays (micropipette and EZ-TAXIS). Finally, we demonstrate how to fix and stain the actin cytoskeleton of polarized cells for fluorescent microscopy imaging.

  7. Integrin-mediated cell migration is blocked by inhibitors of human neuraminidase.

    PubMed

    Jia, Feng; Howlader, Md Amran; Cairo, Christopher W

    2016-09-01

    Integrins are critical receptors in cell migration and adhesion. A number of mechanisms are known to regulate the function of integrins, including phosphorylation, conformational change, and cytoskeletal anchoring. We investigated whether native neuraminidase (Neu, or sialidase) enzymes which modify glycolipids could play a role in regulating integrin-mediated cell migration. Using a scratch assay, we found that exogenously added Neu3 and Neu4 activity altered rates of cell migration. We observed that Neu4 increased the rate of migration in two cell lines (HeLa, A549); while Neu3 only increased migration in HeLa cells. A bacterial neuraminidase was able to increase the rate of migration in HeLa, but not in A549 cells. Treatment of cells with complex gangliosides (GM1, GD1a, GD1b, and GT1b) resulted in decreased cell migration rates, while LacCer was able to increase rates of migration in both lines. Importantly, our results show that treatment of cells with inhibitors of native Neu enzymes had a dramatic effect on the rates of cell migration. The most potent compound tested targeted the human Neu4 isoenzyme, and was able to substantially reduce the rate of cell migration. We found that the lateral mobility of integrins was reduced by treatment of cells with Neu3, suggesting that Neu3 enzyme activity resulted in changes to integrin-co-receptor or integrin-cytoskeleton interactions. Finally, our results support the hypothesis that inhibitors of human Neu can be used to investigate mechanisms of cell migration and for the development of anti-adhesive therapies.

  8. Cadherin-2 Is Required Cell Autonomously for Collective Migration of Facial Branchiomotor Neurons

    PubMed Central

    Rebman, Jane K.; Kirchoff, Kathryn E.

    2016-01-01

    Collective migration depends on cell-cell interactions between neighbors that contribute to their overall directionality, yet the mechanisms that control the coordinated migration of neurons remains to be elucidated. During hindbrain development, facial branchiomotor neurons (FBMNs) undergo a stereotypic tangential caudal migration from their place of birth in rhombomere (r)4 to their final location in r6/7. FBMNs engage in collective cell migration that depends on neuron-to-neuron interactions to facilitate caudal directionality. Here, we demonstrate that Cadherin-2-mediated neuron-to-neuron adhesion is necessary for directional and collective migration of FBMNs. We generated stable transgenic zebrafish expressing dominant-negative Cadherin-2 (Cdh2ΔEC) driven by the islet1 promoter. Cell-autonomous inactivation of Cadherin-2 function led to non-directional migration of FBMNs and a defect in caudal tangential migration. Additionally, mosaic analysis revealed that Cdh2ΔEC-expressing FBMNs are not influenced to migrate caudally by neighboring wild-type FBMNs due to a defect in collective cell migration. Taken together, our data suggest that Cadherin-2 plays an essential cell-autonomous role in mediating the collective migration of FBMNs. PMID:27716840

  9. ERP44 inhibits human lung cancer cell migration mainly via IP3R2.

    PubMed

    Huang, Xue; Jin, Meng; Chen, Ying-Xiao; Wang, Jun; Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

    2016-06-01

    Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway.

  10. ERP44 inhibits human lung cancer cell migration mainly via IP3R2

    PubMed Central

    Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

    2016-01-01

    Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway. PMID:27347718

  11. Insulin-producing cells from embryonic stem cells rescues hyperglycemia via intra-spleen migration.

    PubMed

    Ren, Meng; Shang, Changzhen; Zhong, Xiaomei; Guo, Ruomi; Lao, Guojuan; Wang, Xiaoyi; Cheng, Hua; Min, Jun; Yan, Li; Shen, Jun

    2014-12-23

    Implantation of embryonic stem cells (ESC)-derived insulin-producing cells has been extensively investigated for treatment of diabetes in animal models. However, the in vivo behavior and migration of transplanted cells in diabetic models remains unclear. Here we investigated the location and migration of insulin-producing cells labeled with superparamagnetic iron oxide (SPIO) using a dynamic MRI tracking method. SPIO labeled cells showed hypointense signal under the kidney subcapsules of diabetic mice on MRI, and faded gradually over the visiting time. However, new hypointense signal appeared in the spleen 1 week after transplantation, and became obvious with the time prolongation. Further histological examination proved the immigrated cells were insulin and C-peptide positive cells which were evenly distributed throughout the spleen. These intra-spleen insulin-producing cells maintained their protective effects against hyperglycemia in vivo, and these effects were reversed upon spleen removal. Transplantation of insulin-producing cells through spleen acquired an earlier blood glucose control as compared with that through kidney subcapsules. In summary, our data demonstrate that insulin-producing cells transplanted through kidney subcapsules were not located in situ but migrated into spleen, and rescues hyperglycemia in diabetic models. MRI may provide a novel tracking method for preclinical cell transplantation therapy of diabetes continuously and non-invasively.

  12. From Cell Differentiation to Cell Collectives: Bacillus subtilis Uses Division of Labor to Migrate

    PubMed Central

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-01-01

    The organization of cells, emerging from cell–cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called “van Gogh bundles”) of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity. PMID:25894589

  13. Insulin-producing cells from embryonic stem cells rescues hyperglycemia via intra-spleen migration

    PubMed Central

    Ren, Meng; Shang, Changzhen; Zhong, Xiaomei; Guo, Ruomi; Lao, Guojuan; Wang, Xiaoyi; Cheng, Hua; Min, Jun; Yan, Li; Shen, Jun

    2014-01-01

    Implantation of embryonic stem cells (ESC)-derived insulin-producing cells has been extensively investigated for treatment of diabetes in animal models. However, the in vivo behavior and migration of transplanted cells in diabetic models remains unclear. Here we investigated the location and migration of insulin-producing cells labeled with superparamagnetic iron oxide (SPIO) using a dynamic MRI tracking method. SPIO labeled cells showed hypointense signal under the kidney subcapsules of diabetic mice on MRI, and faded gradually over the visiting time. However, new hypointense signal appeared in the spleen 1 week after transplantation, and became obvious with the time prolongation. Further histological examination proved the immigrated cells were insulin and C-peptide positive cells which were evenly distributed throughout the spleen. These intra-spleen insulin-producing cells maintained their protective effects against hyperglycemia in vivo, and these effects were reversed upon spleen removal. Transplantation of insulin-producing cells through spleen acquired an earlier blood glucose control as compared with that through kidney subcapsules. In summary, our data demonstrate that insulin-producing cells transplanted through kidney subcapsules were not located in situ but migrated into spleen, and rescues hyperglycemia in diabetic models. MRI may provide a novel tracking method for preclinical cell transplantation therapy of diabetes continuously and non-invasively. PMID:25533571

  14. Silencing of VAMP3 inhibits cell migration and integrin-mediated adhesion

    SciTech Connect

    Luftman, Kevin; Hasan, Nazarul; Day, Paul; Hardee, Deborah; Hu Chuan

    2009-02-27

    Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of {beta}1 integrin at the cell surface but had no effect on total cellular {beta}1 integrin, indicating that VAMP3 is required for trafficking of {beta}1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.

  15. Cinnamtannin B-1 Promotes Migration of Mesenchymal Stem Cells and Accelerates Wound Healing in Mice.

    PubMed

    Fujita, Kosuke; Kuge, Katsunori; Ozawa, Noriyasu; Sahara, Shunya; Zaiki, Kaori; Nakaoji, Koichi; Hamada, Kazuhiko; Takenaka, Yukiko; Tanahashi, Takao; Tamai, Katsuto; Kaneda, Yasufumi; Maeda, Akito

    2015-01-01

    Substances that enhance the migration of mesenchymal stem cells to damaged sites have the potential to improve the effectiveness of tissue repair. We previously found that ethanol extracts of Mallotus philippinensis bark promoted migration of mesenchymal stem cells and improved wound healing in a mouse model. We also demonstrated that bark extracts contain cinnamtannin B-1, a flavonoid with in vitro migratory activity against mesenchymal stem cells. However, the in vivo effects of cinnamtannin B-1 on the migration of mesenchymal stem cells and underlying mechanism of this action remain unknown. Therefore, we examined the effects of cinnamtannin B-1 on in vivo migration of mesenchymal stem cells and wound healing in mice. In addition, we characterized cinnamtannin B-1-induced migration of mesenchymal stem cells pharmacologically and structurally. The mobilization of endogenous mesenchymal stem cells into the blood circulation was enhanced in cinnamtannin B-1-treated mice as shown by flow cytometric analysis of peripheral blood cells. Whole animal imaging analysis using luciferase-expressing mesenchymal stem cells as a tracer revealed that cinnamtannin B-1 increased the homing of mesenchymal stem cells to wounds and accelerated healing in a diabetic mouse model. Additionally, the cinnamtannin B-1-induced migration of mesenchymal stem cells was pharmacologically susceptible to inhibitors of phosphatidylinositol 3-kinase, phospholipase C, lipoxygenase, and purines. Furthermore, biflavonoids with similar structural features to cinnamtannin B-1 also augmented the migration of mesenchymal stem cells by similar pharmacological mechanisms. These results demonstrate that cinnamtannin B-1 promoted mesenchymal stem cell migration in vivo and improved wound healing in mice. Furthermore, the results reveal that cinnamtannin B-1-induced migration of mesenchymal stem cells may be mediated by specific signaling pathways, and the flavonoid skeleton may be relevant to its effects on

  16. Cinnamtannin B-1 Promotes Migration of Mesenchymal Stem Cells and Accelerates Wound Healing in Mice.

    PubMed

    Fujita, Kosuke; Kuge, Katsunori; Ozawa, Noriyasu; Sahara, Shunya; Zaiki, Kaori; Nakaoji, Koichi; Hamada, Kazuhiko; Takenaka, Yukiko; Tanahashi, Takao; Tamai, Katsuto; Kaneda, Yasufumi; Maeda, Akito

    2015-01-01

    Substances that enhance the migration of mesenchymal stem cells to damaged sites have the potential to improve the effectiveness of tissue repair. We previously found that ethanol extracts of Mallotus philippinensis bark promoted migration of mesenchymal stem cells and improved wound healing in a mouse model. We also demonstrated that bark extracts contain cinnamtannin B-1, a flavonoid with in vitro migratory activity against mesenchymal stem cells. However, the in vivo effects of cinnamtannin B-1 on the migration of mesenchymal stem cells and underlying mechanism of this action remain unknown. Therefore, we examined the effects of cinnamtannin B-1 on in vivo migration of mesenchymal stem cells and wound healing in mice. In addition, we characterized cinnamtannin B-1-induced migration of mesenchymal stem cells pharmacologically and structurally. The mobilization of endogenous mesenchymal stem cells into the blood circulation was enhanced in cinnamtannin B-1-treated mice as shown by flow cytometric analysis of peripheral blood cells. Whole animal imaging analysis using luciferase-expressing mesenchymal stem cells as a tracer revealed that cinnamtannin B-1 increased the homing of mesenchymal stem cells to wounds and accelerated healing in a diabetic mouse model. Additionally, the cinnamtannin B-1-induced migration of mesenchymal stem cells was pharmacologically susceptible to inhibitors of phosphatidylinositol 3-kinase, phospholipase C, lipoxygenase, and purines. Furthermore, biflavonoids with similar structural features to cinnamtannin B-1 also augmented the migration of mesenchymal stem cells by similar pharmacological mechanisms. These results demonstrate that cinnamtannin B-1 promoted mesenchymal stem cell migration in vivo and improved wound healing in mice. Furthermore, the results reveal that cinnamtannin B-1-induced migration of mesenchymal stem cells may be mediated by specific signaling pathways, and the flavonoid skeleton may be relevant to its effects on

  17. Microbial desalination cell with capacitive adsorption for ion migration control.

    PubMed

    Forrestal, Casey; Xu, Pei; Jenkins, Peter E; Ren, Zhiyong

    2012-09-01

    A new microbial desalination cell with capacitive adsorption capability (cMDC) was developed to solve the ion migration problem facing current MDC systems. Traditional MDCs remove salts by transferring ions to the anode and cathode chambers, which may prohibit wastewater beneficial reuse due to increased salinity. The cMDC uses adsorptive activated carbon cloth (ACC) as the electrodes and utilizes the formed capacitive double layers for electrochemical ion adsorption. The cMDC removed an average of 69.4% of the salt from the desalination chamber through electrode adsorption during one batch cycle, and it did not add salts to the anode or cathode chamber. It was estimated that 61-82.2mg of total dissolved solids (TDS) was adsorbed to 1g of ACC electrode. The cMDC provides a new approach for salt management, organic removal, and energy production. Further studies will be conducted to optimize reactor configuration and achieve in situ electrode regeneration.

  18. Inhibition of corneal epithelial cell migration by cadmium and mercury

    SciTech Connect

    Ubels, J.L.; Osgood, T.B. Medical Coll. of Wisconsin, Milwaukee )

    1991-02-01

    In a previous comparative study of corneal healing in fish, the authors observed that corneal epithelial healing occurs very rapidly in vivo in the marine teleost Myoxocephalus octodecimspinosus (longhorn sculpin) with a 6-mm diameter wound on the mammalian cornea. This rapid healing which permits prompt restoration of the epithelial barrier is apparently an adaptation to the large ionic and osmotic gradients between the environment and the intraocular fluids of the fish. These observations suggested that epithelial healing in the sculpin cornea might be useful model in aquatic biomedical toxicology if an in vitro method for measurement of healing rates could be developed. In this report the authors demonstrate that sculpin eyes maintained in short-term organ culture have a rapid corneal epithelial healing response and that this model can be used to demonstrate the toxic effects of heavy metals on epithelial cell migration.

  19. Downregulation of SENP1 inhibits cell proliferation, migration and promotes apoptosis in human glioma cells

    PubMed Central

    ZHANG, QIU-SHENG; ZHANG, MENG; HUANG, XIAN-JIAN; LIU, XIAO-JIA; LI, WEI-PING

    2016-01-01

    Small ubiquitin-related modifier protein (SUMO) is an evolutionarily conserved protein in a broad range of eukaryotic organisms. De-SUMOylation, the reverse reaction of SUMOylation, is regulated by a family of SUMO-specific proteases (SENPs). SENP1 is a member of the de-SUMOylation protease family involved in the de-SUMOylation of a variety of SUMOylated proteins. The present study demonstrates that small hairpin RNA (shRNA)-mediated downregulation of SENP1 inhibits cell proliferation and migration, and promotes apoptosis in human glioma cells. Firstly, LN-299 cells were transfected with a plasmid expressing SENP1 shRNA (pGenesil-1-SENP1). The messenger RNA and protein expression of SENP1 was detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Cell proliferation in vitro was assessed using a methyl thiazolyl tetrazolium assay. Flow cytometry (FCM) was used to detect the apoptosis of LN-299 cells. The effect of the downregulation of SENP1 on cell migration was detected by a Transwell migration system. The present results showed that, compared with the control shRNA group, the expression of SENP1 was significantly reduced in the SENP1 shRNA group. The proliferation was markedly inhibited in the SENP1 shRNA group. FCM findings revealed that apoptosis increased significantly in the SENP1 shRNA group. In addition, it was found that downregulation of SENP1 evidently suppressed tumor cell migration. Downregulation of SENP1 expression inhibited the proliferation and migration and promoted apoptosis in LN-299 cells. These results indirectly demonstrate that SENP1 is likely to play a critical role in human glioma cells. PMID:27347128

  20. Impaired SIRT1 promotes the migration of vascular smooth muscle cell-derived foam cells.

    PubMed

    Zhang, Ming-Jie; Zhou, Yi; Chen, Lei; Wang, Xu; Pi, Yan; Long, Chun-Yan; Sun, Meng-Jiao; Chen, Xue; Gao, Chang-Yue; Li, Jing-Cheng; Zhang, Li-Li

    2016-07-01

    The formation of fat-laden foam cells, contributing to the fatty streaks of the plaques of atheroma, is the critical early process in atherosclerosis. The previous study demonstrated that vascular smooth muscle cells (VSMCs) contain a much larger burden of the excess cholesterol in comparison with monocyte-derived macrophages in human coronary atherosclerosis, as the main origin of foam cells. It is noteworthy that VSMC-derived foam cells are deposited in subintima but not media, where VSMCs normally deposit in. Therefore, migration from media to intima is an indispensable step for a VSMC to accrue neutral lipids and form foam cell. Whether this migration occurs paralleled with or prior to the formation of foam cell is still unclear. Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxidized low-density lipoprotein (oxLDL). In conclusion, we provide evidence that oxLDL induces the VSMC-derived foam cells formation with increased migration ability and MMP-9 expression, which were partly attributed to the impaired SIRT1 and enhanced nuclear factor-kappa B (NF-κB) activity. As activation of transient receptor potential vanilloid type 1 (TRPV1) has been reported to have anti-atherosclerotic effects, we investigated its role in oxLDL-treated VSMC migration. It is found that activating TRPV1 by capsaicin inhibits VSMC foam cell formation and the accompanied migration through rescuing the SIRT1 and suppressing NF-κB signaling. The present study provides evidence that SIRT1 may be a promising intervention target of atherosclerosis, and raises the prospect of TRPV1 in prevention and treatment of atherosclerosis.

  1. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration

    PubMed Central

    Ma, Liang; Li, Ying; Peng, Junya; Wu, Danni; Zhao, Xiaoxin; Cui, Yitong; Chen, Lilian; Yan, Xiaojun; Du, Yanan; Yu, Li

    2015-01-01

    Cells communicate with each other through secreting and releasing proteins and vesicles. Many cells can migrate. In this study, we report the discovery of migracytosis, a cell migration-dependent mechanism for releasing cellular contents, and migrasomes, the vesicular structures that mediate migracytosis. As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, we named them “migrasomes”. We also found that cytosolic contents can be transported into migrasomes and released from the cell through migrasomes. We named this migration-dependent release mechanism “migracytosis”. PMID:25342562

  2. The acetylenic tricyclic bis(cyano enone), TBE-31, targets microtubule dynamics and cell polarity in migrating cells.

    PubMed

    Chan, Eddie; Saito, Akira; Honda, Tadashi; Di Guglielmo, Gianni M

    2016-04-01

    Cell migration is dependent on the microtubule network for structural support as well as for the proper delivery and positioning of polarity proteins at the leading edge of migrating cells. Identification of drugs that target cytoskeletal-dependent cell migration and protein transport in polarized migrating cells is important in understanding the cell biology of normal and tumor cells and can lead to new therapeutic targets in disease processes. Here, we show that the tricyclic compound TBE-31 directly binds to tubulin and interferes with microtubule dynamics, as assessed by end binding 1 (EB1) live cell imaging. Interestingly, this interference is independent of in vitro tubulin polymerization. Using immunofluorescence microscopy, we also observed that TBE-31 interferes with the polarity of migratory cells. The polarity proteins Rac1, IQGAP and Tiam1 were localized at the leading edge of DMSO-treated migrating cell, but were observed to be in multiple protrusions around the cell periphery of TBE-31-treated cells. Finally, we observed that TBE-31 inhibits the migration of Rat2 fibroblasts with an IC50 of 0.75 μM. Taken together, our results suggest that the inhibition of cell migration by TBE-31 may result from the improper maintenance of cell polarity of migrating cells.

  3. The acetylenic tricyclic bis(cyano enone), TBE-31, targets microtubule dynamics and cell polarity in migrating cells.

    PubMed

    Chan, Eddie; Saito, Akira; Honda, Tadashi; Di Guglielmo, Gianni M

    2016-04-01

    Cell migration is dependent on the microtubule network for structural support as well as for the proper delivery and positioning of polarity proteins at the leading edge of migrating cells. Identification of drugs that target cytoskeletal-dependent cell migration and protein transport in polarized migrating cells is important in understanding the cell biology of normal and tumor cells and can lead to new therapeutic targets in disease processes. Here, we show that the tricyclic compound TBE-31 directly binds to tubulin and interferes with microtubule dynamics, as assessed by end binding 1 (EB1) live cell imaging. Interestingly, this interference is independent of in vitro tubulin polymerization. Using immunofluorescence microscopy, we also observed that TBE-31 interferes with the polarity of migratory cells. The polarity proteins Rac1, IQGAP and Tiam1 were localized at the leading edge of DMSO-treated migrating cell, but were observed to be in multiple protrusions around the cell periphery of TBE-31-treated cells. Finally, we observed that TBE-31 inhibits the migration of Rat2 fibroblasts with an IC50 of 0.75 μM. Taken together, our results suggest that the inhibition of cell migration by TBE-31 may result from the improper maintenance of cell polarity of migrating cells. PMID:26775215

  4. Tyrosinase-Cre-Mediated Deletion of the Autophagy Gene Atg7 Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice.

    PubMed

    Sukseree, Supawadee; Chen, Ying-Ting; Laggner, Maria; Gruber, Florian; Petit, Valérie; Nagelreiter, Ionela-Mariana; Mlitz, Veronika; Rossiter, Heidemarie; Pollreisz, Andreas; Schmidt-Erfurth, Ursula; Larue, Lionel; Tschachler, Erwin; Eckhart, Leopold

    2016-01-01

    Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7f/f Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene Atg7. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of Atg7f/f Tyr-Cre mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to Sqstm1, genes involved in retinal regeneration, i.e. Lrat, Rdh5, Rgr, and Rpe65, were expressed at higher mRNA levels. Sequencing of the Rpe65 gene showed that Atg7f/f and Atg7f/f Tyr-Cre mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of Atg7f/f Tyr-Cre mice is, at least partly, mediated by upregulation of Rpe65 transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by Atg7-dependent autophagy in Atg7f/f mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms. PMID:27537685

  5. Tyrosinase-Cre-Mediated Deletion of the Autophagy Gene Atg7 Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice

    PubMed Central

    Sukseree, Supawadee; Chen, Ying-Ting; Laggner, Maria; Gruber, Florian; Petit, Valérie; Nagelreiter, Ionela-Mariana; Mlitz, Veronika; Rossiter, Heidemarie; Pollreisz, Andreas; Schmidt-Erfurth, Ursula; Larue, Lionel; Tschachler, Erwin

    2016-01-01

    Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7f/f Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene Atg7. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of Atg7f/f Tyr-Cre mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to Sqstm1, genes involved in retinal regeneration, i.e. Lrat, Rdh5, Rgr, and Rpe65, were expressed at higher mRNA levels. Sequencing of the Rpe65 gene showed that Atg7f/f and Atg7f/f Tyr-Cre mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of Atg7f/f Tyr-Cre mice is, at least partly, mediated by upregulation of Rpe65 transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by Atg7-dependent autophagy in Atg7f/f mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms. PMID:27537685

  6. A simple non-perturbing cell migration assay insensitive to proliferation effects

    PubMed Central

    Glenn, Honor L.; Messner, Jacob; Meldrum, Deirdre R.

    2016-01-01

    Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells. PMID:27535324

  7. A simple non-perturbing cell migration assay insensitive to proliferation effects.

    PubMed

    Glenn, Honor L; Messner, Jacob; Meldrum, Deirdre R

    2016-01-01

    Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells. PMID:27535324

  8. Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells.

    PubMed

    Liu, Yan-Jun; Le Berre, Maël; Lautenschlaeger, Franziska; Maiuri, Paolo; Callan-Jones, Andrew; Heuzé, Mélina; Takaki, Tohru; Voituriez, Raphaël; Piel, Matthieu

    2015-02-12

    The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration--one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility.

  9. Multi-function microsystem for cells migration analysis and evaluation of photodynamic therapy procedure in coculture

    PubMed Central

    Jastrzebska (Jedrych), Elzbieta; Grabowska-Jadach, Ilona; Chudy, Michal; Dybko, Artur; Brzozka, Zbigniew

    2012-01-01

    Cell migration is an important physiological process, which is involved in cancer metastasis. Therefore, the investigation of cell migration may lead to the development of novel therapeutic approaches. In this study, we have successfully developed a microsystem for culture of two cell types (non-malignant and carcinoma) and for analysis of cell migration dependence on distance between them. Finally, we studied quantitatively the influence of photodynamic therapy (PDT) procedures on the viability of pairs of non-malignant (MRC5 or Balb/3T3) and carcinoma (A549) cells coculture. The proposed geometry of the microsystem allowed for separate introduction of two cell lines and analysis of cells migration dependence on distance between the cells. We found that a length of connecting microchannel has an influence on cell migration and viability of non-malignant cells after PDT procedure. Summarizing, the developed microsystem can constitute a new tool for carrying out experiments, which offers a few functions: cell migration analysis, carcinoma and non-malignant cells coculture, and evaluation of PDT procedure in the various steps of cell migration. PMID:24339849

  10. Microfluidic gradient device for studying mesothelial cell migration and the effect of chronic carbon nanotube exposure

    NASA Astrophysics Data System (ADS)

    Zhang, Hanyuan; Lohcharoenkal, Warangkana; Sun, Jianbo; Li, Xiang; Wang, Liying; Wu, Nianqiang; Rojanasakul, Yon; Liu, Yuxin

    2015-07-01

    Cell migration is one of the crucial steps in many physiological and pathological processes, including cancer development. Our recent studies have shown that carbon nanotubes (CNTs), similarly to asbestos, can induce accelerated cell growth and invasiveness that contribute to their mesothelioma pathogenicity. Malignant mesothelioma is a very aggressive tumor that develops from cells of the mesothelium, and is most commonly caused by exposure to asbestos. CNTs have a similar structure and mode of exposure to asbestos. This has raised a concern regarding the potential carcinogenicity of CNTs, especially in the pleural area which is a key target for asbestos-related diseases. In this paper, a static microfluidic gradient device was applied to study the migration of human pleural mesothelial cells which had been through a long-term exposure (4 months) to subcytotoxic concentration (0.02 µg cm-2) of single-walled CNTs (SWCNTs). Multiple migration signatures of these cells were investigated using the microfluidic gradient device for the first time. During the migration study, we observed that cell morphologies changed from flattened shapes to spindle shapes prior to their migration after their sensing of the chemical gradient. The migration of chronically SWCNT-exposed mesothelial cells was evaluated under different fetal bovine serum (FBS) concentration gradients, and the migration speeds and number of migrating cells were extracted and compared. The results showed that chronically SWCNT-exposed mesothelial cells are more sensitive to the gradient compared to non-SWCNT-exposed cells. The method described here allows simultaneous detection of cell morphology and migration under chemical gradient conditions, and also allows for real-time monitoring of cell motility that resembles in vivo cell migration. This platform would be much needed for supporting the development of more physiologically relevant cell models for better assessment and characterization of the

  11. Progesterone promotes cell migration, invasion and cofilin activation in human astrocytoma cells.

    PubMed

    Piña-Medina, Ana Gabriela; Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Cerbón, Marco; Camacho-Arroyo, Ignacio

    2016-01-01

    Astrocytomas are the most common and aggressive primary brain tumors in humans. Invasiveness of these tumors has been attributed in part to deregulation of cell motility-dependent cytoskeletal dynamics that involves actin-binding proteins such as cofilin. Progesterone (P4) has been found to induce migration and invasion of cells derived from breast cancer and endothelium. However, the role of P4 in migration and invasion of astrocytoma cells as well as its effects on astrocytomas cytoskeleton remodeling is not known. In this work we evaluated these aspects in D54 and U251 cells derived from human astrocytomas from the highest degree of malignancy (grade IV, glioblastoma). Our results showed that in scratch-wound assays P4 increased the number of D54 and U251 cells migrating from 3 to 48 h. Both RU486, a P4 receptor (PR) antagonist, and an oligonucleotide antisense against PR significantly blocked P4 effects. Transwell assays showed that P4 significantly increased the number of invasive cells at 24h. As in the case of migration, this effect was blocked by RU486. Finally, by Western blotting, an increase in the cofilin/p-cofilin ratio at 15 and 30 min and a decrease at 30 and 60 min in U251 and D54 cells, respectively, was observed after P4, P4+RU486 and RU486 treatments. These data suggest that P4 increases human astrocytoma cells migration and invasion through its intracellular receptor, and that cofilin activation by P4 is independent of PR action. PMID:26639431

  12. P-cadherin-mediated Rho GTPase regulation during collective cell migration

    PubMed Central

    Plutoni, Cédric; Bazellières, Elsa; Gauthier-Rouvière, Cécile

    2016-01-01

    ABSTRACT This commentary addresses the role of P-cadherin in collective cell migration (CCM), a cooperative and coordinated migration mode, used by cells during normal and pathological migration processes. We discuss how cadherin-mediated cell-cell junctions (CCJs) play a critical role in CCM through their ability to regulate Rho GTPase-dependent pathways and how this leads to the generation and orientation of mechanical forces. We will also highlight the key function of P-cadherin (a poor prognostic marker in several tumors) in promoting collective cell movement in epithelial and mesenchymal cells. PMID:27152729

  13. ASB2α regulates migration of immature dendritic cells.

    PubMed

    Lamsoul, Isabelle; Métais, Arnaud; Gouot, Emmanuelle; Heuzé, Mélina L; Lennon-Duménil, Ana-Maria; Moog-Lutz, Christel; Lutz, Pierre G

    2013-07-25

    The actin-binding protein filamins (FLNs) are major organizers of the actin cytoskeleton. They control the elasticity and stiffness of the actin network and provide connections with the extracellular microenvironment by anchoring transmembrane receptors to the actin filaments. Although numerous studies have revealed the importance of FLN levels, relatively little is known about the regulation of its stability in physiological relevant settings. Here, we show that the ASB2α cullin 5-ring E3 ubiquitin ligase is highly expressed in immature dendritic cells (DCs) and is down-regulated after DC maturation. We further demonstrate that FLNs are substrates of ASB2α in immature DCs and therefore are not stably expressed in these cells, whereas they exhibit high levels of expression in mature DCs. Using ASB2 conditional knockout mice, we show that ASB2α is a critical regulator of cell spreading and podosome rosette formation in immature DCs. Furthermore, we show that ASB2(-/-) immature DCs exhibit reduced matrix-degrading function leading to defective migration. Altogether, our results point to ASB2α and FLNs as newcomers in DC biology. PMID:23632887

  14. ASB2α regulates migration of immature dendritic cells.

    PubMed

    Lamsoul, Isabelle; Métais, Arnaud; Gouot, Emmanuelle; Heuzé, Mélina L; Lennon-Duménil, Ana-Maria; Moog-Lutz, Christel; Lutz, Pierre G

    2013-07-25

    The actin-binding protein filamins (FLNs) are major organizers of the actin cytoskeleton. They control the elasticity and stiffness of the actin network and provide connections with the extracellular microenvironment by anchoring transmembrane receptors to the actin filaments. Although numerous studies have revealed the importance of FLN levels, relatively little is known about the regulation of its stability in physiological relevant settings. Here, we show that the ASB2α cullin 5-ring E3 ubiquitin ligase is highly expressed in immature dendritic cells (DCs) and is down-regulated after DC maturation. We further demonstrate that FLNs are substrates of ASB2α in immature DCs and therefore are not stably expressed in these cells, whereas they exhibit high levels of expression in mature DCs. Using ASB2 conditional knockout mice, we show that ASB2α is a critical regulator of cell spreading and podosome rosette formation in immature DCs. Furthermore, we show that ASB2(-/-) immature DCs exhibit reduced matrix-degrading function leading to defective migration. Altogether, our results point to ASB2α and FLNs as newcomers in DC biology.

  15. Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma.

    PubMed

    Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant'Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

    2016-01-01

    Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

  16. Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

    PubMed Central

    Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

    2016-01-01

    Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

  17. Nerve growth factor and its low-affinity receptor promote Schwann cell migration.

    PubMed Central

    Anton, E S; Weskamp, G; Reichardt, L F; Matthew, W D

    1994-01-01

    Migrating Schwann cells in developing or regenerating peripheral nerves are known to express dramatically increased levels of nerve growth factor (NGF) and the low-affinity NGF receptor (LNGFR). Schwann cells do not express detectable pp140trk, the NGF-activated receptor tyrosine kinase which is essential for neuronal responses to NGF. The temporal correlation observed in Schwann cells between migration and the enhanced expression of NGF and LNGFR suggests that NGF and LNGFR may promote Schwann cell migration. To test this possibility, we examined the effects of NGF on Schwann cell migration on cryostat sections of biologically relevant NGF-poor and NGF-rich substrates--normal or denervated peripheral (sciatic) nerve, untreated or pretreated with NGF. Results show that Schwann cells migrate more rapidly on denervated than on normal sciatic nerve. Antibodies to NGF or to LNGFR strongly, but incompletely, inhibit enhanced migration on denervated nerves. Pretreatment of denervated nerve sections with NGF increases further the rate of Schwann cell migration. The same antibodies to NGF or to LNGFR abolish this response. These results suggest that one function of the elevated levels of NGF known to be present in embryonic and regenerating peripheral nerves is to promote the migration of Schwann cells. In contrast to neurons, where pp140trk appears to be the functionally critical NGF receptor, NGF responses in Schwann cells depend on LNGFR. Images PMID:8146193

  18. X-ray microprobe analysis of the retina and RPE in sheep with ovine ceroid-lipofuscinosis

    SciTech Connect

    Samuelson, D.A.; Armstrong, D.; Jolly, R. )

    1990-11-01

    Ovine ceroid-lipofuscinosis (OCL) is one animal model for the human condition, and because autofluorescent lipopigments are prominent in the brain and eye, it may also prove useful as a model for aging. For example, a progressive decline in electrical recording from brain and retina are observed in both aging and OCL. Samples of retinal and retinal pigment epithelial (RPE) tissues were obtained from a young control. 2 animals with OCL and a normal aged sheep. Specimens were cryo-fractured and examined by scanning electron microscopy/x-ray microanalysis. Measurements made of 6 individual cells in the ganglion layer of OCL specimens, the remainder of the retina, and RPE showed age-related changes in zinc, iron, and copper which were associated with lipopigment accumulation in the RPE. There was marked decrease in phosphate, sulfur, and manganese levels, as photoreceptor cells and their outer segments are lost in the disease process. This is the first report of metal analysis in the retina and RPE in a disease entity, and as a function of normal aging.

  19. ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells.

    PubMed

    Ding, You-Quan; Li, Xuan-Yang; Xia, Guan-Nan; Ren, Hong-Yi; Zhou, Xin-Fu; Su, Bing-Yin; Qi, Jian-Guo

    2016-10-01

    Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since "the yin and yang of neurotrophin action" has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of "the yin and yang of neurotrophin action" and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration.

  20. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

    PubMed

    Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

    2015-11-01

    Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.

  1. Coexpressing shRNA with fluorescence tags for quantification of cell migration studies.

    PubMed

    Koo, Christine Xing'er; Fang, Wanru; Salto-Tellez, Manuel; Leong, David Tai

    2012-07-01

    Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.

  2. Established and Novel Methods of Interrogating Two-Dimensional Cell Migration

    PubMed Central

    Ashby, William J.; Zijlstra, Andries

    2014-01-01

    The regulation of cell motility is central to living systems. Consequently, cell migration assays are some of the most frequently used in vitro assays. This article provides a comprehensive, detailed review of in vitro cell migration assays both currently in use and possible with existing technology. Emphasis is given to two-dimensional migration assays using densely organized cells such as the scratch assay. Assays are compared and categorized in an outline format according to their primary biological readout and physical parameters. The individual benefits of the various methods and quantification strategies are also discussed. This review provides an in-depth, structured overview of in vitro cell migration assays as a means of enabling the reader to make informed decisions among the growing number of options available for their specific cell migration application. PMID:23038152

  3. Cell-surface proteoglycan in sea urchin primary mesenchyme cell migration

    SciTech Connect

    Lane, M.C.

    1989-01-01

    Early in the development of the sea urchin embryo, the primary mesenchyme cells (PMC) migrate along the basal lamina of the blastocoel. Migration is inhibited in L. pictus embryos cultured in sulfate-free seawater and in S. purpuratus embryos exposed to exogenous {beta}-D-xylosides. An in vitro assay was developed to test the migratory capacity of normal PMC on normal and treated blastocoelic matrix. Sulfate deprivation and exposure to exogenous xyloside render PMC nonmotile on either matrix. Materials removed from the surface of normal PMC by treatment with 1 M urea restored migratory ability to defective cells, whereas a similar preparation isolated from the surface of epithelial cells at the same stage did not. Migration also resumed when cells were removed from the xyloside or returned to normal seawater. The urea extract was partially purified and characterized by radiolabeling, gel electrophoresis, fluorography, ion exchange chromatography, and western blotting. The PMC synthesize a large chondroitin sulfate/dermatan sulfate proteoglycan that is present in an active fraction isolated by chromatography. Chondroitinase ABC digestion of live cells blocked migration reversibly, further supporting the identification of the chondroitin sulfate/dermatan sulfate proteoglycan as the active component in the urea extract. Much of the incorporated sulfate was distributed along the filopodia in {sup 35}SO{sub 4}-labelled PMC by autoradiography. The morphology of normal and treated S. purpuratus PMC was examined by scanning electron microscopy, and differences in spreading, particularly of the extensive filopodia present on the cells, was observed. A model for the role of the chondroitin sulfate/dermatan sulfate proteoglycan in cell detachment during migration is proposed.

  4. Nonpolarized signaling reveals two distinct modes of 3D cell migration.

    PubMed

    Petrie, Ryan J; Gavara, Núria; Chadwick, Richard S; Yamada, Kenneth M

    2012-04-30

    We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix.

  5. Nonpolarized signaling reveals two distinct modes of 3D cell migration

    PubMed Central

    Gavara, Núria; Chadwick, Richard S.

    2012-01-01

    We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix. PMID:22547408

  6. Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth

    SciTech Connect

    Wang, Jiying; Rao, Qing; Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang

    2009-09-04

    Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

  7. Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth.

    PubMed

    Wang, Jiying; Rao, Qing; Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang

    2009-09-01

    Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

  8. Microfluidic Assay To Study the Combinatorial Impact of Substrate Properties on Mesenchymal Stem Cell Migration.

    PubMed

    Menon, Nishanth V; Chuah, Yon Jin; Phey, Samantha; Zhang, Ying; Wu, Yingnan; Chan, Vincent; Kang, Yuejun

    2015-08-12

    As an alternative to complex and costly in vivo models, microfluidic in vitro models are being widely used to study various physiological phenomena. It is of particular interest to study cell migration in a controlled microenvironment because of its vital role in a large number of physiological processes, such as wound healing, disease progression, and tissue regeneration. Cell migration has been shown to be affected by variations in the biochemical and physical properties of the extracellular matrix (ECM). To study the combinatorial impact of the ECM physical properties on cell migration, we have developed a microfluidic assay to induce migration of human bone marrow derived mesenchymal stem cells (hBMSCs) on polydimethylsiloxane (PDMS) substrates with varying combinatorial properties (hydrophobicity, stiffness, and roughness). The results show that although the initial cell adhesion and viability appear similar on all PDMS samples, the cell spreading and migration are enhanced on PDMS samples exhibiting intermediate levels of hydrophobicity, stiffness, and roughness. This study suggests that there is a particular range of substrate properties for optimal cell spreading and migration. The influence of substrate properties on hBMSC migration can help understand the physical cues that affect cell migration, which may facilitate the development of optimized engineered scaffolds with desired properties for tissue regeneration applications. PMID:26186177

  9. Random migration precedes stable target cell interactions of tumor-infiltrating T cells.

    PubMed

    Mrass, Paulus; Takano, Hajime; Ng, Lai Guan; Daxini, Sachin; Lasaro, Marcio O; Iparraguirre, Amaya; Cavanagh, Lois L; von Andrian, Ulrich H; Ertl, Hildegund C J; Haydon, Philip G; Weninger, Wolfgang

    2006-11-27

    The tumor microenvironment is composed of an intricate mixture of tumor and host-derived cells that engage in a continuous interplay. T cells are particularly important in this context as they may recognize tumor-associated antigens and induce tumor regression. However, the precise identity of cells targeted by tumor-infiltrating T lymphocytes (TILs) as well as the kinetics and anatomy of TIL-target cell interactions within tumors are incompletely understood. Furthermore, the spatiotemporal conditions of TIL locomotion through the tumor stroma, as a prerequisite for establishing contact with target cells, have not been analyzed. These shortcomings limit the rational design of immunotherapeutic strategies that aim to overcome tumor-immune evasion. We have used two-photon microscopy to determine, in a dynamic manner, the requirements leading to tumor regression by TILs. Key observations were that TILs migrated randomly throughout the tumor microenvironment and that, in the absence of cognate antigen, they were incapable of sustaining active migration. Furthermore, TILs in regressing tumors formed long-lasting (>or=30 min), cognate antigen-dependent contacts with tumor cells. Finally, TILs physically interacted with macrophages, suggesting tumor antigen cross-presentation by these cells. Our results demonstrate that recognition of cognate antigen within tumors is a critical determinant of optimal TIL migration and target cell interactions, and argue against TIL guidance by long-range chemokine gradients.

  10. Transfection microarrays for high-throughput phenotypic screening of genes involved in cell migration.

    PubMed

    Onuki-Nagasaki, Reiko; Nagasaki, Akira; Hakamada, Kazumi; Uyeda, Taro Q P; Fujita, Satoshi; Miyake, Masato; Miyake, Jun

    2010-01-01

    Cell migration is important in several biological phenomena, such as cancer metastasis. Therefore, the identification of genes involved in cell migration might facilitate the discovery of antimetastatic drugs. However, screening of genes by the current methods can be complicated by factors related to cell stimulation, for example, abolition of contact inhibition and the release inflammatory cytokines from wounded cells during examinations of wound healing in vitro. To overcome these problems and identify genes involved in cell migration, in this chapter we describe the use of transfection microarrays for high-throughput phenotypic screening. PMID:20387151

  11. A novel role for Lh3 dependent ECM modifications during neural crest cell migration in zebrafish.

    PubMed

    Banerjee, Santanu; Isaacman-Beck, Jesse; Schneider, Valerie A; Granato, Michael

    2013-01-01

    During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3-Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.

  12. Common mechanisms linking connexin43 to neural progenitor cell migration and glioma invasion.

    PubMed

    Naus, Christian C; Aftab, Qurratulain; Sin, Wun Chey

    2016-02-01

    Cell migration is critical for cell differentiation, tissue formation and organ development. Several mechanisms come to play in the process of cell migration, orchestrating changes in cell polarity, adhesion, process extension and motility. Recent findings have shown that gap junctions, and specifically connexin43 (Cx43), can play a significant role in these processes, impacting adhesion and cytoskeletal rearrangements. Thus Cx43 within a cell regulates its motility and migration via intracellular signaling. Furthermore, Cx43 in the host cells can impact the degree of cellular migration through that tissue. Similarities in these connexin-based processes account for both neural progenitor migration in the developing brain, and for glioma cell invasion in the mature brain. In both cases, Cx43 in the tissue ("soil") in which cells ("seeds") exist facilitates their migration and, for glioma cells, tissue invasion. Cx43 mediates these effects through channel- and non-channel-dependent mechanisms which have similarities in both paradigms of cell migration. This provides insight into developmental processes and pathological situations, as well as possible therapeutic approaches regarding specific functional domains of gap junction proteins.

  13. Lipid raft association restricts CD44-ezrin interaction and promotion of breast cancer cell migration.

    PubMed

    Donatello, Simona; Babina, Irina S; Hazelwood, Lee D; Hill, Arnold D K; Nabi, Ivan R; Hopkins, Ann M

    2012-12-01

    Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration.

  14. Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces

    PubMed Central

    Jeon, Hojeong; Koo, Sangmo; Reese, Willie Mae; Loskill, Peter; Grigoropoulos, Costas P.; Healy, Kevin E.

    2015-01-01

    Although adhesive interactions between cells and nanostructured interfaces have been studied extensively1–6, there is a paucity of data on how nanostructured interfaces repel cells by directing cell migration and cell-colony organization. Here, by using multiphoton ablation lithography7 to pattern surfaces with nanoscale craters of various aspect ratios and pitches, we show that the surfaces altered the cells’ focal-adhesion size and distribution, thus affecting cell morphology, migration and ultimately localization. We also show that nanocrater pitch can disrupt the formation of mature focal adhesions to favour the migration of cells toward higher-pitched regions, which present increased planar area for the formation of stable focal adhesions. Moreover, by designing surfaces with variable pitch but constant nanocrater dimensions, we were able to create circular and striped cellular patterns. Our surface-patterning approach, which does not involve chemical treatments and can be applied to various materials, represents a simple method to control cell behaviour on surfaces. PMID:26213899

  15. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front.

    PubMed

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M; Meyer, Tobias; Heo, Won Do

    2016-09-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration. PMID:27555588

  16. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front

    PubMed Central

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M.; Meyer, Tobias; Heo, Won Do

    2016-01-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration. PMID:27555588

  17. Expression of complete keratin filaments in mouse L cells augments cell migration and invasion.

    PubMed Central

    Chu, Y W; Runyan, R B; Oshima, R G; Hendrix, M J

    1993-01-01

    Intermediate filament proteins have been used to diagnose the origin of specific cells. Classically, vimentin is found in mesenchymal cells, and keratins are present in epithelial cells. However, recent evidence suggests that the coexpression of these phenotype-specific proteins augments tumor cell motility, and hence, metastasis. In the present study, we used the mouse L-cell model to determine if a direct correlation exists between the expression of additional keratins in these cells, which normally express only vimentin, and their migratory ability. Mouse L cells were transfected with human keratins 8, 18, and both 8 and 18. The results indicate that the cells expressing complete keratin filaments have a higher migratory and invasive ability (through extracellular matrix-coated filters) compared with the parental and control-transfected clones. Furthermore, there is an enrichment of keratin-positive cells from a heterogeneous population of L clones selected over serial migrations. This migratory activity was directly correlated with the spreading ability of the cells on Matrigel matrix, in which the keratin-positive transfectants maintain a round morphology for a longer duration, compared with the other L-cell populations. Collectively, these data suggest that keratins may play an important role(s) in migration, through a special interaction with the extracellular environment, thereby influencing cell shape. Images Fig. 1 Fig. 4 PMID:7683431

  18. Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation

    SciTech Connect

    Hogan, Niamh M.; Joyce, Myles R.; Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy; Kerin, Michael J.; Dwyer, Roisin M.

    2013-06-14

    Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the

  19. The newt (Cynops pyrrhogaster) RPE65 promoter: molecular cloning, characterization and functional analysis.

    PubMed

    Casco-Robles, Martin Miguel; Miura, Tomoya; Chiba, Chikafumi

    2015-06-01

    The adult newt has the ability to regenerate the neural retina following injury, a process achieved primarily by the retinal pigment epithelium (RPE). To deliver exogenous genes to the RPE for genetic manipulation of regenerative events, we isolated the newt RPE65 promoter region by genome walking. First, we cloned the 2.8 kb RPE65 promoter from the newt, Cynops pyrrhogaster. Sequence analysis revealed several conserved regulatory elements described previously in mouse and human RPE65 promoters. Second, having previously established an I-SceI-mediated transgenic protocol for the newt, we used it here to examine the -657 bp proximal promoter of RPE65. The promoter assay used with F0 transgenic newts confirmed transgene expression of mCherry fluorescent protein in the RPE. Using bioinformatic tools and the TRANSFAC database, we identified a 340 bp CpG island located between -635 and -296 bp in the promoter; this region contains response elements for the microphthalmia-associated transcription factor known as MITF (CACGTG, CATGTG), and E-boxes (CANNTG). Sex-determining region box 9 (or SOX9) response element previously reported in the regulation of RPE genes (including RPE65) was also identified in the newt RPE65 promoter. Third, we identified DNA motif boxes in the newt RPE65 promoter that are conserved among other vertebrates. The newt RPE65 promoter is an invaluable tool for site-specific delivery of exogenous genes or genetic manipulation systems for the study of retinal regeneration in this animal.

  20. Directional Migration of MDA-MB-231 Cells Under Oxygen Concentration Gradients.

    PubMed

    Yahara, D; Yoshida, T; Enokida, Y; Takahashi, E

    2016-01-01

    To elucidate the initial mechanism of hematogenous metastasis of cancer cells, we hypothesized that cancer cells migrate toward regions with higher oxygen concentration such as intratumor micro vessels along the oxygen concentration gradient. To produce gradients of oxygen concentration in vitro, we devised the gap cover glass (GCG). After placing a GCG onto cultured MDA-MB-231 cells (a metastatic breast cancer cell line), the migration of individual cells under the GCG was tracked up to 12 h at 3 % oxygen in the micro incubator. We quantified the migration of individual cells using forward migration index (FMI). The cell migration perpendicular to the oxygen gradients was random in the direction whereas FMIs of the cell located at 300, 500, 700, and 1500 μm from the oxygen inlet were positive (p < 0.05) indicating a unidirectional migration toward the oxygen inlet. Present results are consistent with our hypothesis that MDA-MB-231 cells migrate toward regions with higher oxygen concentration. PMID:27526134

  1. Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo

    PubMed Central

    Law, Ah-Lai; Vehlow, Anne; Kotini, Maria; Dodgson, Lauren; Soong, Daniel; Theveneau, Eric; Bodo, Cristian; Taylor, Eleanor; Navarro, Christel; Perera, Upamali; Michael, Magdalene; Dunn, Graham A.; Bennett, Daimark; Mayor, Roberto

    2013-01-01

    Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd’s Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo. PMID:24247431

  2. Hypoxia promotes Rab5 activation, leading to tumor cell migration, invasion and metastasis.

    PubMed

    Silva, Patricio; Mendoza, Pablo; Rivas, Solange; Díaz, Jorge; Moraga, Carolina; Quest, Andrew F G; Torres, Vicente A

    2016-05-17

    Hypoxia, a common condition of the tumor microenvironment, is associated with poor patient prognosis, tumor cell migration, invasion and metastasis. Recent evidence suggests that hypoxia alters endosome dynamics in tumor cells, leading to augmented cell proliferation and migration and this is particularly relevant, because endosomal components have been shown to be deregulated in cancer. The early endosome protein Rab5 is a small GTPase that promotes integrin trafficking, focal adhesion turnover, Rac1 activation, tumor cell migration and invasion. However, the role of Rab5 and downstream events in hypoxia remain unknown. Here, we identify Rab5 as a critical player in hypoxia-driven tumor cell migration, invasion and metastasis. Exposure of A549 human lung carcinoma, ZR-75, MDA-MB-231 and MCF-7 human breast cancer and B16-F10 mouse melanoma cells to hypoxia increased Rab5 activation, followed by its re-localization to the leading edge and association with focal adhesions. Importantly, Rab5 was required for hypoxia-driven cell migration, FAK phosphorylation and Rac1 activation, as shown by shRNA-targeting and transfection assays with Rab5 mutants. Intriguingly, the effect of hypoxia on both Rab5 activity and migration was substantially higher in metastatic B16-F10 cells than in poorly invasive B16-F0 cells. Furthermore, exogenous expression of Rab5 in B16-F0 cells predisposed to hypoxia-induced migration, whereas expression of the inactive mutant Rab5/S34N prevented the migration of B16-F10 cells induced by hypoxia. Finally, using an in vivo syngenic C57BL/6 mouse model, Rab5 expression was shown to be required for hypoxia-induced metastasis. In summary, these findings identify Rab5 as a key mediator of hypoxia-induced tumor cell migration, invasion and metastasis.

  3. Hypoxia promotes Rab5 activation, leading to tumor cell migration, invasion and metastasis

    PubMed Central

    Silva, Patricio; Mendoza, Pablo; Rivas, Solange; Díaz, Jorge; Moraga, Carolina; Quest, Andrew F.G.; Torres, Vicente A.

    2016-01-01

    Hypoxia, a common condition of the tumor microenvironment, is associated with poor patient prognosis, tumor cell migration, invasion and metastasis. Recent evidence suggests that hypoxia alters endosome dynamics in tumor cells, leading to augmented cell proliferation and migration and this is particularly relevant, because endosomal components have been shown to be deregulated in cancer. The early endosome protein Rab5 is a small GTPase that promotes integrin trafficking, focal adhesion turnover, Rac1 activation, tumor cell migration and invasion. However, the role of Rab5 and downstream events in hypoxia remain unknown. Here, we identify Rab5 as a critical player in hypoxia-driven tumor cell migration, invasion and metastasis. Exposure of A549 human lung carcinoma, ZR-75, MDA-MB-231 and MCF-7 human breast cancer and B16-F10 mouse melanoma cells to hypoxia increased Rab5 activation, followed by its re-localization to the leading edge and association with focal adhesions. Importantly, Rab5 was required for hypoxia-driven cell migration, FAK phosphorylation and Rac1 activation, as shown by shRNA-targeting and transfection assays with Rab5 mutants. Intriguingly, the effect of hypoxia on both Rab5 activity and migration was substantially higher in metastatic B16-F10 cells than in poorly invasive B16-F0 cells. Furthermore, exogenous expression of Rab5 in B16-F0 cells predisposed to hypoxia-induced migration, whereas expression of the inactive mutant Rab5/S34N prevented the migration of B16-F10 cells induced by hypoxia. Finally, using an in vivo syngenic C57BL/6 mouse model, Rab5 expression was shown to be required for hypoxia-induced metastasis. In summary, these findings identify Rab5 as a key mediator of hypoxia-induced tumor cell migration, invasion and metastasis. PMID:27121131

  4. Hypoxia promotes Rab5 activation, leading to tumor cell migration, invasion and metastasis.

    PubMed

    Silva, Patricio; Mendoza, Pablo; Rivas, Solange; Díaz, Jorge; Moraga, Carolina; Quest, Andrew F G; Torres, Vicente A

    2016-05-17

    Hypoxia, a common condition of the tumor microenvironment, is associated with poor patient prognosis, tumor cell migration, invasion and metastasis. Recent evidence suggests that hypoxia alters endosome dynamics in tumor cells, leading to augmented cell proliferation and migration and this is particularly relevant, because endosomal components have been shown to be deregulated in cancer. The early endosome protein Rab5 is a small GTPase that promotes integrin trafficking, focal adhesion turnover, Rac1 activation, tumor cell migration and invasion. However, the role of Rab5 and downstream events in hypoxia remain unknown. Here, we identify Rab5 as a critical player in hypoxia-driven tumor cell migration, invasion and metastasis. Exposure of A549 human lung carcinoma, ZR-75, MDA-MB-231 and MCF-7 human breast cancer and B16-F10 mouse melanoma cells to hypoxia increased Rab5 activation, followed by its re-localization to the leading edge and association with focal adhesions. Importantly, Rab5 was required for hypoxia-driven cell migration, FAK phosphorylation and Rac1 activation, as shown by shRNA-targeting and transfection assays with Rab5 mutants. Intriguingly, the effect of hypoxia on both Rab5 activity and migration was substantially higher in metastatic B16-F10 cells than in poorly invasive B16-F0 cells. Furthermore, exogenous expression of Rab5 in B16-F0 cells predisposed to hypoxia-induced migration, whereas expression of the inactive mutant Rab5/S34N prevented the migration of B16-F10 cells induced by hypoxia. Finally, using an in vivo syngenic C57BL/6 mouse model, Rab5 expression was shown to be required for hypoxia-induced metastasis. In summary, these findings identify Rab5 as a key mediator of hypoxia-induced tumor cell migration, invasion and metastasis. PMID:27121131

  5. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    SciTech Connect

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  6. Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis.

    PubMed

    Hazan, R B; Phillips, G R; Qiao, R F; Norton, L; Aaronson, S A

    2000-02-21

    E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein

  7. Immature human dendritic cells enhance their migration through KCa3.1 channel activation.

    PubMed

    Crottès, David; Félix, Romain; Meley, Daniel; Chadet, Stéphanie; Herr, Florence; Audiger, Cindy; Soriani, Olivier; Vandier, Christophe; Roger, Sébastien; Angoulvant, Denis; Velge-Roussel, Florence

    2016-04-01

    Migration capacity is essential for dendritic cells (DCs) to present antigen to T cells for the induction of immune response. The DC migration is supposed to be a calcium-dependent process, while not fully understood. Here, we report a role of the KCa3.1/IK1/SK4 channels in the migration capacity of both immature (iDC) and mature (mDC) human CD14(+)-derived DCs. KCa3.1 channels were shown to control the membrane potential of human DC and the Ca(2+) entry, which is directly related to migration capacities. The expression of migration marker such as CCR5 and CCR7 was modified in both types of DCs by TRAM-34 (100nM). But, only the migration of iDC was decreased by use of both TRAM-34 and KCa3.1 siRNA. Confocal analyses showed a close localization of CCR5 with KCa3.1 in the steady state of iDC. Finally, the implication of KCa3.1 seems to be limited to the migration capacities as T cell activation of DCs appeared unchanged. Altogether, these results demonstrated that KCa3.1 channels have a pro-migratory effect on iDC migration. Our findings suggest that KCa3.1 in human iDC play a major role in their migration and constitute an attractive target for the cell therapy optimization.

  8. A Quorum-Sensing Factor in Vegetative Dictyostelium Discoideum Cells Revealed by Quantitative Migration Analysis

    PubMed Central

    Golé, Laurent; Rivière, Charlotte; Hayakawa, Yoshinori; Rieu, Jean-Paul

    2011-01-01

    Background Many cells communicate through the production of diffusible signaling molecules that accumulate and once a critical concentration has been reached, can activate or repress a number of target genes in a process termed quorum sensing (QS). In the social amoeba Dictyostelium discoideum, QS plays an important role during development. However little is known about its effect on cell migration especially in the growth phase. Methods and Findings To investigate the role of cell density on cell migration in the growth phase, we use multisite timelapse microscopy and automated cell tracking. This analysis reveals a high heterogeneity within a given cell population, and the necessity to use large data sets to draw reliable conclusions on cell motion. In average, motion is persistent for short periods of time (), but normal diffusive behavior is recovered over longer time periods. The persistence times are positively correlated with the migrated distances. Interestingly, the migrated distance decreases as well with cell density. The adaptation of cell migration to cell density highlights the role of a secreted quorum sensing factor (QSF) on cell migration. Using a simple model describing the balance between the rate of QSF generation and the rate of QSF dilution, we were able to gather all experimental results into a single master curve, showing a sharp cell transition between high and low motile behaviors with increasing QSF. Conclusion This study unambiguously demonstrates the central role played by QSF on amoeboid motion in the growth phase. PMID:22073217

  9. Substrate stiffness modulates lung cancer cell migration but not epithelial to mesenchymal transition.

    PubMed

    Shukla, V C; Higuita-Castro, N; Nana-Sinkam, P; Ghadiali, S N

    2016-05-01

    Biomechanical properties of the tumor microenvironment, including matrix/substrate stiffness, play a significant role in tumor evolution and metastasis. Epithelial to Mesenchymal Transition (EMT) is a fundamental biological process that is associated with increased cancer cell migration and invasion. The goal of this study was to investigate (1) how substrate stiffness modulates the migration behaviors of lung adenocarcinoma cells (A549) and (2) if stiffness-induced changes in cell migration correlate with biochemical markers of EMT. Collagen-coated polydimethylsiloxane (PDMS) substrates and an Ibidi migration assay were used to investigate how substrate stiffness alters the migration patterns of A549 cells. RT-PCR, western blotting and immunofluorescence were used to investigate how substrate stiffness alters biochemical markers of EMT, that is, E-cadherin and N-cadherin, and the phosphorylation of focal adhesion proteins. Increases in substrate stiffness led to slower, more directional migration but did not alter the biochemical markers of EMT. Interestingly, growth factor (i.e., Transforming Growth Factor-β) stimulation resulted in similar levels of EMT regardless of substrate stiffness. We also observed decreased levels of phosphorylated focal adhesion kinase (FAK) and paxillin on stiffer substrates which correlated with slower cell migration. These results indicate that substrate stiffness modulates lung cancer cell migration via focal adhesion signaling as opposed to EMT signaling. PMID:26779779

  10. Correlation between cell migration and reactive oxygen species under electric field stimulation.

    PubMed

    Wu, Shang-Ying; Hou, Hsien-San; Sun, Yung-Shin; Cheng, Ji-Yen; Lo, Kai-Yin

    2015-09-01

    Cell migration is an essential process involved in the development and maintenance of multicellular organisms. Electric fields (EFs) are one of the many physical and chemical factors known to affect cell migration, a phenomenon termed electrotaxis or galvanotaxis. In this paper, a microfluidics chip was developed to study the migration of cells under different electrical and chemical stimuli. This chip is capable of providing four different strengths of EFs in combination with two different chemicals via one simple set of agar salt bridges and Ag/AgCl electrodes. NIH 3T3 fibroblasts were seeded inside this chip to study their migration and reactive oxygen species (ROS) production in response to different EF strengths and the presence of β-lapachone. We found that both the EF and β-lapachone level increased the cell migration rate and the production of ROS in an EF-strength-dependent manner. A strong linear correlation between the cell migration rate and the amount of intracellular ROS suggests that ROS are an intermediate product by which EF and β-lapachone enhance cell migration. Moreover, an anti-oxidant, α-tocopherol, was found to quench the production of ROS, resulting in a decrease in the migration rate. PMID:26487906

  11. Long-distance cell migration during larval development in the appendicularian, Oikopleura dioica.

    PubMed

    Kishi, Kanae; Onuma, Takeshi A; Nishida, Hiroki

    2014-11-15

    The appendicularian, Oikopleura dioica, is a planktonic chordate. Its simple and transparent body, invariant cell lineages and short life cycle of 5 days make it a promising model organism for studies of chordate development. Here we describe the cell migration that occurs during development of the O. dioica larva. Using time-lapse imaging facilitated by florescent labeling of cells, three cell populations exhibiting long-distance migration were identified and characterized. These included (i) a multinucleated oral gland precursor that migrates anteriorly within the trunk region and eventually separates into the left and right sides, (ii) endodermal strand cells that are collectively retracted from the tail into the trunk in a tractor movement, and (iii) two subchordal cell precursors that individually migrate out from the trunk to the tip of the tail. The migration of subchordal cell precursors starts when all of the endodermal strand cells enter the trunk, and follows the same path but in a direction opposite to that of the latter. Labeling of these cells with a photoconvertible fluorescent protein, Kaede, demonstrated that the endodermal strand cells and subchordal cell precursors have distinct origins and eventual fates. Surgical removal of the trunk from the tail demonstrated that the endodermal strand cells do not require the trunk for migration, and that the subchordal cell precursors would be attracted by the distal part of the tail. This well-defined, invariant and traceable long-distance cell migration provides a unique experimental system for exploring the mechanisms of versatile cell migration in this simple organism with a chordate body plan. PMID:25224225

  12. RLIM interacts with Smurf2 and promotes TGF-{beta} induced U2OS cell migration

    SciTech Connect

    Huang, Yongsheng; Yang, Yang; Gao, Rui; Yang, Xianmei; Yan, Xiaohua; Wang, Chenji; Jiang, Sirui; Yu, Long

    2011-10-14

    Highlights: {yields} RLIM directly binds to Smurf2. {yields} RLIM enhances TGF-{beta} responsiveness in U2OS cells. {yields} RLIM promotes TGF-{beta} driven migration of osteosarcoma U2OS cells. -- Abstract: TGF-{beta} (transforming growth factor-{beta}), a pleiotropic cytokine that regulates diverse cellular processes, has been suggested to play critical roles in cell proliferation, migration, and carcinogenesis. Here we found a novel E3 ubiquitin ligase RLIM which can directly bind to Smurf2, enhancing TGF-{beta} responsiveness in osteosarcoma U2OS cells. We constructed a U2OS cell line stably over-expressing RLIM and demonstrated that RLIM promoted TGF-{beta}-driven migration of U2OS cells as tested by wound healing assay. Our results indicated that RLIM is an important positive regulator in TGF-{beta} signaling pathway and cell migration.

  13. Cell migration or cytokinesis and proliferation? – Revisiting the “go or grow” hypothesis in cancer cells in vitro

    SciTech Connect

    Garay, Tamás; Juhász, Éva; Molnár, Eszter; Eisenbauer, Maria; Czirók, András; Dekan, Barbara; László, Viktória; Hoda, Mir Alireza; Döme, Balázs; Tímár, József; Klepetko, Walter; Berger, Walter; Hegedűs, Balázs

    2013-12-10

    The mortality of patients with solid tumors is mostly due to metastasis that relies on the interplay between migration and proliferation. The “go or grow” hypothesis postulates that migration and proliferation spatiotemporally excludes each other. We evaluated this hypothesis on 35 cell lines (12 mesothelioma, 13 melanoma and 10 lung cancer) on both the individual cell and population levels. Following three-day-long videomicroscopy, migration, proliferation and cytokinesis-length were quantified. We found a significantly higher migration in mesothelioma cells compared to melanoma and lung cancer while tumor types did not differ in mean proliferation or duration of cytokinesis. Strikingly, we found in melanoma and lung cancer a significant positive correlation between mean proliferation and migration. Furthermore, non-dividing melanoma and lung cancer cells displayed slower migration. In contrast, in mesothelioma there were no such correlations. Interestingly, negative correlation was found between cytokinesis-length and migration in melanoma. FAK activation was higher in melanoma cells with high motility. We demonstrate that the cancer cells studied do not defer proliferation for migration. Of note, tumor cells from various organ systems may differently regulate migration and proliferation. Furthermore, our data is in line with the observation of pathologists that highly proliferative tumors are often highly invasive. - Highlights: • We investigated the “go or grow” hypothesis in human cancer cells in vitro. • Proliferation and migration positively correlate in melanoma and lung cancer cells. • Duration of cytokinesis and migration shows inverse correlation. • Increased FAK activation is present in highly motile melanoma cells.

  14. Migration of co-cultured endothelial cells and osteoblasts in composite hydroxyapatite/polylactic acid scaffolds.

    PubMed

    Shah, Amita R; Shah, Sarita R; Oh, Sunho; Ong, Joo L; Wenke, Joseph C; Agrawal, C Mauli

    2011-10-01

    Regeneration of bone in large segmental bone defects requires regeneration of both cortical bone and trabecular bone. A scaffold design consisting of a hydroxyapatite (HA) ring surrounding a polylactic acid (PLA) core simulates the structure of bone and provides an environment for indirect and direct co-culture conditions. In this experiment, human umbilical vein endothelial cells (EC) and normal human primary osteoblasts (OB) were co-cultured to evaluate cell migration and interactions within this biphasic composite scaffold. Both cell types were able to migrate between the different material phases of the scaffold. It was also observed that OB migration increased when they were co-cultured with ECs, whereas EC migration decreased in co-culture. The results show that co-culture of ECs and OBs in this composite biphasic scaffold allows for migration of cells throughout the scaffold and that pre-seeding a scaffold with ECs can increase OB infiltration into desired areas of the scaffold.

  15. WNK1 kinase balances T cell adhesion versus migration in vivo.

    PubMed

    Köchl, Robert; Thelen, Flavian; Vanes, Lesley; Brazão, Tiago F; Fountain, Kathryn; Xie, Jian; Huang, Chou-Long; Lyck, Ruth; Stein, Jens V; Tybulewicz, Victor L J

    2016-09-01

    Adhesion and migration of T cells are controlled by chemokines and by adhesion molecules, especially integrins, and have critical roles in the normal physiological function of T lymphocytes. Using an RNA-mediated interference screen, we identified the WNK1 kinase as a regulator of both integrin-mediated adhesion and T cell migration. We found that WNK1 is a negative regulator of integrin-mediated adhesion, whereas it acts as a positive regulator of migration via the kinases OXSR1 and STK39 and the ion co-transporter SLC12A2. WNK1-deficient T cells home less efficiently to lymphoid organs and migrate more slowly through them. Our results reveal that a pathway previously known only to regulate salt homeostasis in the kidney functions to balance T cell adhesion and migration.

  16. Dynamic tensile forces drive collective cell migration through three-dimensional extracellular matrices

    PubMed Central

    Gjorevski, Nikolce; S. Piotrowski, Alexandra; Varner, Victor D.; Nelson, Celeste M.

    2015-01-01

    Collective cell migration drives tissue remodeling during development, wound repair, and metastatic invasion. The physical mechanisms by which cells move cohesively through dense three-dimensional (3D) extracellular matrix (ECM) remain incompletely understood. Here, we show directly that migration of multicellular cohorts through collagenous matrices occurs via a dynamic pulling mechanism, the nature of which had only been inferred previously in 3D. Tensile forces increase at the invasive front of cohorts, serving a physical, propelling role as well as a regulatory one by conditioning the cells and matrix for further extension. These forces elicit mechanosensitive signaling within the leading edge and align the ECM, creating microtracks conducive to further migration. Moreover, cell movements are highly correlated and in phase with ECM deformations. Migrating cohorts use spatially localized, long-range forces and consequent matrix alignment to navigate through the ECM. These results suggest biophysical forces are critical for 3D collective migration. PMID:26165921

  17. WNK1 kinase balances T cell adhesion versus migration in vivo.

    PubMed

    Köchl, Robert; Thelen, Flavian; Vanes, Lesley; Brazão, Tiago F; Fountain, Kathryn; Xie, Jian; Huang, Chou-Long; Lyck, Ruth; Stein, Jens V; Tybulewicz, Victor L J

    2016-09-01

    Adhesion and migration of T cells are controlled by chemokines and by adhesion molecules, especially integrins, and have critical roles in the normal physiological function of T lymphocytes. Using an RNA-mediated interference screen, we identified the WNK1 kinase as a regulator of both integrin-mediated adhesion and T cell migration. We found that WNK1 is a negative regulator of integrin-mediated adhesion, whereas it acts as a positive regulator of migration via the kinases OXSR1 and STK39 and the ion co-transporter SLC12A2. WNK1-deficient T cells home less efficiently to lymphoid organs and migrate more slowly through them. Our results reveal that a pathway previously known only to regulate salt homeostasis in the kidney functions to balance T cell adhesion and migration. PMID:27400149

  18. Envisioning migration: Mathematics in both experimental analysis and modeling of cell behavior

    PubMed Central

    Zhang, Elizabeth R.; Wu, Lani F.; Altschuler, Steven J.

    2013-01-01

    The complex nature of cell migration highlights the power and challenges of applying mathematics to biological studies. Mathematics may be used to create model equations that recapitulate migration, which can predict phenomena not easily uncovered by experiments or intuition alone. Alternatively, mathematics may be applied to interpreting complex data sets with better resolution—potentially empowering scientists to discern subtle patterns amid the noise and heterogeneity typical of migrating cells. Iteration between these two methods is necessary in order to reveal connections within the cell migration signaling network, as well as to understand the behavior that arises from those connections. Here, we review recent quantitative analysis and mathematical modeling approaches to the cell migration problem. PMID:23660413

  19. An indirect electric field-induced control in directional migration of rat mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Park, Hyoun-Hyang; Jo, Sungkwon; Hoon Seo, Cheong; Jeong, Je Hoon; Yoo, Yeong-Eun; Lee, Dae Hoon

    2014-12-01

    We present the efficacy of an indirect E-field on the directional migration of rat mesenchymal stem cell (MSC). To avoid current flow through culture media and cell, E-fields were generated without exposing electrodes directly to the cell media. MSC migration was observed during wound closure in presence of indirect E-field. MSC migration depended on the E-field strength and occurs predominantly in the anodal direction. Indirect E-field therapy proved as tentative tool for controlled cell movement and healing.

  20. Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis.

    PubMed

    Ohgo, Shiro; Hasegawa, Seiji; Hasebe, Yuichi; Mizutani, Hiroshi; Nakata, Satoru; Akamatsu, Hirohiko

    2015-07-01

    During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.

  1. Matricellular protein Cyr61 bridges lysophosphatidic acid and integrin pathways leading to cell migration.

    PubMed

    Wu, Daniel Dongwei; Zhang, Fuqiang; Hao, Feng; Chun, Jerold; Xu, Xuemin; Cui, Mei-Zhen

    2014-02-28

    Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA1) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA1 is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6β1 and ανβ3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.

  2. Reduced Metabolic Capacity in Aged Primary Retinal Pigment Epithelium (RPE) is Correlated with Increased Susceptibility to Oxidative Stress.

    PubMed

    Rohrer, Bärbel; Bandyopadhyay, Mausumi; Beeson, Craig

    2016-01-01

    One of the affected tissues in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), a tissue that consists of terminally differentiated cells and that accumulates damage over time. In all tissues, mitochondria (mt), which play an essential role in both cell health (energy) and death (initiator of apoptosis), undergo an aging process through the accumulation of mtDNA damage, changes in mitochondrial dynamics, a reduction in biogenesis, and mitophagy, leading to an overall reduction in mitochondrial energy production and other non-energy-related functions. Here we have compared energy metabolism in primary human RPE cells isolated from aborted fetus or aged donor eyes and grown as stable monolayers. H2O2 treatment resulted in the generation of reactive oxygen species and superoxide, an effect that was significantly augmented by age. Mitochondrial metabolism, as analyzed by Seahorse respirometry, revealed reduced mitochondrial oxygen consumption (ATP production) at baseline and a complete loss of reserve capacity in aged cells. Likewise, glycolysis was blunted in aged cells. Taken together, these studies showed that RPE cells derived from aged donor eyes are more susceptible to oxidative stress, and exhibit a loss in mitochondrial respiratory reserve capacity and a reduction in glycolysis. These data suggest that while old cells may have sufficient energy at rest, they cannot mount a stress response requiring additional ATP and reducing agents. In summary, these data support the hypothesis that mitochondria or energy metabolism is a valid target for therapy in AMD. PMID:26427491

  3. Chemokine-Dependent pH Elevation at the Cell Front Sustains Polarity in Directionally Migrating Zebrafish Germ Cells.

    PubMed

    Tarbashevich, Katsiaryna; Reichman-Fried, Michal; Grimaldi, Cecilia; Raz, Erez

    2015-04-20

    Directional cell migration requires cell polarization with respect to the distribution of the guidance cue. Cell polarization often includes asymmetric distribution of response components as well as elements of the motility machinery. Importantly, the function and regulation of most of these molecules are known to be pH dependent. Intracellular pH gradients were shown to occur in certain cells migrating in vitro, but the functional relevance of such gradients for cell migration and for the response to directional cues, particularly in the intact organism, is currently unknown. In this study, we find that primordial germ cells migrating in the context of the developing embryo respond to the graded distribution of the chemokine Cxcl12 by establishing elevated intracellular pH at the cell front. We provide insight into the mechanisms by which a polar pH distribution contributes to efficient cell migration. Specifically, we show that Carbonic Anhydrase 15b, an enzyme controlling the pH in many cell types, including metastatic cancer cells, is expressed in migrating germ cells and is crucial for establishing and maintaining an asymmetric pH distribution within them. Reducing the level of the protein and thereby erasing the pH elevation at the cell front resulted in abnormal cell migration and impaired arrival at the target. The basis for the disrupted migration is found in the stringent requirement for pH conditions in the cell for regulating contractility, for the polarization of Rac1 activity, and hence for the formation of actin-rich structures at the leading edge of the migrating cells.

  4. Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

    PubMed

    Piater, Birgit; Doerner, Achim; Guenther, Ralf; Kolmar, Harald; Hock, Bjoern

    2015-01-01

    The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding. PMID:26658271

  5. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    NASA Astrophysics Data System (ADS)

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-02-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

  6. Microfluidics-based devices: New tools for studying cancer and cancer stem cell migration

    PubMed Central

    Huang, Yu; Agrawal, Basheal; Sun, Dandan; Kuo, John S.; Williams, Justin C.

    2011-01-01

    Cell movement is highly sensitive to stimuli from the extracellular matrix and media. Receptors on the plasma membrane in cells can activate signal transduction pathways that change the mechanical behavior of a cell by reorganizing motion-related organelles. Cancer cells change their migration mechanisms in response to different environments more robustly than noncancer cells. Therefore, therapeutic approaches to immobilize cancer cells via inhibition of the related signal transduction pathways rely on a better understanding of cell migration mechanisms. In recent years, engineers have been working with biologists to apply microfluidics technology to study cell migration. As opposed to conventional cultures on dishes, microfluidics deals with the manipulation of fluids that are geometrically constrained to a submillimeter scale. Such small scales offer a number of advantages including cost effectiveness, low consumption of reagents, high sensitivity, high spatiotemporal resolution, and laminar flow. Therefore, microfluidics has a potential as a new platform to study cell migration. In this review, we summarized recent progress on the application of microfluidics in cancer and other cell migration researches. These studies have enhanced our understanding of cell migration and cancer invasion as well as their responses to subtle variations in their microenvironment. We hope that this review will serve as an interdisciplinary guidance for both biologists and engineers as they further develop the microfluidic toolbox toward applications in cancer research. PMID:21522502

  7. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells.

    PubMed

    Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A; Costa, Max

    2016-02-15

    The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. PMID:26780400

  8. A Novel Role of Cab45-G in Mediating Cell Migration in Cancer Cells

    PubMed Central

    Luo, Judong; Li, Zengpeng; Zhu, Hong; Wang, Chenying; Zheng, Weibin; He, Yan; Song, Jianyuan; Wang, Wenjie; Zhou, Xifa; Lu, Xujing; Zhang, Shuyu; Chen, Jianming

    2016-01-01

    Ca2+-binding protein of 45 kDa (Cab45), a CREC family member, is reported to be associated with Ca2+-dependent secretory pathways and involved in multiple diseases including cancers. Cab45-G, a Cab45 isoform protein, plays an important role in protein sorting and secretion at Golgi complex. However, its role in cancer cell migration remains elusive. In this study, we demonstrate that Cab45-G exhibited an increased expression in cell lines with higher metastatic potential and promoted cell migration in multiple types of cancer cells. Overexpression of Cab45-G resulted in an altered expression of the molecular mediators of epithelial-mesenchymal transition (EMT), which is a critical step in the tumor metastasis. Quantitative real-time PCR showed that overexpression of Cab45-G increased the expression of matrix metalloproteinase-2 and -7 (MMP-2 and MMP-7). Conversely, knock-down of Cab45-G reduced the expression of the above MMPs. Moreover, forced expression of Cab45-G upregulated the level of phosphorylated ERK and modulated the secretion of extracellular proteins fibronectin and fibulin. Furthermore, in human cervical and esophageal cancer tissues, the expression of Cab45-G was found to be significantly correlated with that of MMP-2, further supporting the importance of Cab45-G on regulating cancer metastasis. Taken together, these results suggest that Cab45-G could regulate cancer cell migration through various molecular mechanisms, which may serve as a therapeutic target for the treatment of cancers. PMID:27194945

  9. A Novel Role of Cab45-G in Mediating Cell Migration in Cancer Cells.

    PubMed

    Luo, Judong; Li, Zengpeng; Zhu, Hong; Wang, Chenying; Zheng, Weibin; He, Yan; Song, Jianyuan; Wang, Wenjie; Zhou, Xifa; Lu, Xujing; Zhang, Shuyu; Chen, Jianming

    2016-01-01

    Ca(2+)-binding protein of 45 kDa (Cab45), a CREC family member, is reported to be associated with Ca(2+)-dependent secretory pathways and involved in multiple diseases including cancers. Cab45-G, a Cab45 isoform protein, plays an important role in protein sorting and secretion at Golgi complex. However, its role in cancer cell migration remains elusive. In this study, we demonstrate that Cab45-G exhibited an increased expression in cell lines with higher metastatic potential and promoted cell migration in multiple types of cancer cells. Overexpression of Cab45-G resulted in an altered expression of the molecular mediators of epithelial-mesenchymal transition (EMT), which is a critical step in the tumor metastasis. Quantitative real-time PCR showed that overexpression of Cab45-G increased the expression of matrix metalloproteinase-2 and -7 (MMP-2 and MMP-7). Conversely, knock-down of Cab45-G reduced the expression of the above MMPs. Moreover, forced expression of Cab45-G upregulated the level of phosphorylated ERK and modulated the secretion of extracellular proteins fibronectin and fibulin. Furthermore, in human cervical and esophageal cancer tissues, the expression of Cab45-G was found to be significantly correlated with that of MMP-2, further supporting the importance of Cab45-G on regulating cancer metastasis. Taken together, these results suggest that Cab45-G could regulate cancer cell migration through various molecular mechanisms, which may serve as a therapeutic target for the treatment of cancers. PMID:27194945

  10. Collective mesendoderm migration relies on an intrinsic directionality signal transmitted through cell contacts.

    PubMed

    Dumortier, Julien G; Martin, Sandro; Meyer, Dirk; Rosa, Frederic M; David, Nicolas B

    2012-10-16

    Collective cell migration is key to morphogenesis, wound healing, or cancer cell migration. However, its cellular bases are just starting to be unraveled. During vertebrate gastrulation, axial mesendoderm migrates in a group, the prechordal plate, from the embryonic organizer to the animal pole. How this collective migration is achieved remains unclear. Previous work has suggested that cells migrate as individuals, with collective movement resulting from the addition of similar individual cell behavior. Through extensive analyses of cell trajectories, morphologies, and polarization in zebrafish embryos, we reveal that all prechordal plate cells show the same behavior and rely on the same signaling pathway to migrate, as expected if they do so individually. However, by using cell transplants, we demonstrate that prechordal plate migration is a true collective process, as isolated cells do not migrate toward the animal pole. They are still polarized and motile but lose directionality. Directionality is restored upon contact with the endogenous prechordal plate. This contact dependent orientation relies on E-cadherin, Wnt-PCP signaling, and Rac1. Importantly, groups of cells also need contact with the endogenous plate to orient correctly, showing an instructive role of the plate in establishing directionality. Overall, our results lead to an original model of collective migration in which directional information is contained within the moving group rather than provided by extrinsic cues, and constantly maintained in cells by contacts with their neighbors. This self-organizing model could account for collective invasion of new territories, as observed in cancer strands, without requirement for any attractant in the colonized tissue.

  11. Time-lapse cinematography of the capillary tube cell migration inhibition test.

    PubMed

    Bray, M A

    1980-01-01

    The kinetics of human and guinea pig cell migration inhibition have been studied using time-lapse cinematography of cells migrating from capillary tubes. Guinea pig and human cells exhibit markedly different kinetics in the absence of inhibitors. Specific antigen causes a dose-related inhibition of migration for up to 60 h using guinea pig cells and a peak of inhibition after 18 h using the human leucocyte system. The timing of measurement of maximum activity more critical for the latter test. The kinetics of lymphokine generation have been examined and the migration inhibitory activity of the plant mitogen (PHA), a Kurloff cell product and a continuous cell line supernatant have been compared with the inhibitory profiles of lymphokine preparations and specific antigen. PMID:7350125

  12. Time-lapse cinematography of the capillary tube cell migration inhibition test.

    PubMed

    Bray, M A

    1980-01-01

    The kinetics of human and guinea pig cell migration inhibition have been studied using time-lapse cinematography of cells migrating from capillary tubes. Guinea pig and human cells exhibit markedly different kinetics in the absence of inhibitors. Specific antigen causes a dose-related inhibition of migration for up to 60 h using guinea pig cells and a peak of inhibition after 18 h using the human leucocyte system. The timing of measurement of maximum activity more critical for the latter test. The kinetics of lymphokine generation have been examined and the migration inhibitory activity of the plant mitogen (PHA), a Kurloff cell product and a continuous cell line supernatant have been compared with the inhibitory profiles of lymphokine preparations and specific antigen.

  13. Controlled skeletal progenitor cell migration on nanostructured porous silicon/silicon micropatterns

    NASA Astrophysics Data System (ADS)

    Torres-Costa, V.; Sánchez-Vaquero, V.; Muñoz-Noval, Á.; González-Méndez, L.; Punzón-Quijorna, E.; Gallach-Pérez, D.; Manso-Silván, M.; Martínez-Muñoz, G.; Climent-Font, A.; García-Ruiz, J. P.; Martín-Palma, R. J.

    2011-10-01

    In this work nanostructured porous silicon (nanoPS) was used for the fabrication of surface micropatterns aiming at controlling cell adhesion and migration. In particular, surface patterns of nanoPS and Si were engineered by high-energy ion-beam irradiation and subsequent anodization. It was found that human skeletal progenitor cells are sensitive to oneand two-dimensional patterns and that focal adhesion is inhibited on nanoPS areas. In spite of this anti-fouling characteristics, studies on patterns with reduced Si areas show that cells conform to nanoPS pathways favoring migration through cell protrusion, body translocation and tail retraction from two parallel Si traction rails. Moreover, migration can be blocked and cells tend to arrange when grid patterns with the appropriate dimensions are fabricated. The experimental results confirm that progenitor cells are able to exploit nanoPS anti-fouling designs by adapting to it for migration purposes.

  14. Migration of Founder Epithelial Cells Drives Proper Molar Tooth Positioning and Morphogenesis.

    PubMed

    Prochazka, Jan; Prochazkova, Michaela; Du, Wen; Spoutil, Frantisek; Tureckova, Jolana; Hoch, Renee; Shimogori, Tomomi; Sedlacek, Radislav; Rubenstein, John L; Wittmann, Torsten; Klein, Ophir D

    2015-12-21

    The proper positioning of organs during development is essential, yet little is known about the regulation of this process in mammals. Using murine tooth development as a model, we have found that cell migration plays a central role in positioning of the organ primordium. By combining lineage tracing, genetic cell ablation, and confocal live imaging, we identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible. These Fgf8-expressing progenitors furnish the epithelial cells required for tooth development, and the progenitor population migrates toward a Shh-expressing region in the mandible, where the tooth placode will initiate. Inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to a failure of tooth development. These results demonstrate the importance of intraepithelial cell migration in proper positioning of an initiating organ.

  15. Crawling and Gliding: A Computational Model for Shape-Driven Cell Migration

    PubMed Central

    Niculescu, Ioana; Textor, Johannes; de Boer, Rob J.

    2015-01-01

    Cell migration is a complex process involving many intracellular and extracellular factors, with different cell types adopting sometimes strikingly different morphologies. Modeling realistically behaving cells in tissues is computationally challenging because it implies dealing with multiple levels of complexity. We extend the Cellular Potts Model with an actin-inspired feedback mechanism that allows small stochastic cell rufflings to expand to cell protrusions. This simple phenomenological model produces realistically crawling and deforming amoeboid cells, and gliding half-moon shaped keratocyte-like cells. Both cell types can migrate randomly or follow directional cues. They can squeeze in between other cells in densely populated environments or migrate collectively. The model is computationally light, which allows the study of large, dense and heterogeneous tissues containing cells with realistic shapes and migratory properties. PMID:26488304

  16. Hedgehog Is a Positive Regulator of FGF Signalling during Embryonic Tracheal Cell Migration

    PubMed Central

    Butí, Elisenda; Mesquita, Duarte; Araújo, Sofia J.

    2014-01-01

    Cell migration is a widespread and complex process that is crucial for morphogenesis and for the underlying invasion and metastasis of human cancers. During migration, cells are steered toward target sites by guidance molecules that induce cell direction and movement through complex intracellular mechanisms. The spatio-temporal regulation of the expression of these guidance molecules is of extreme importance for both normal morphogenesis and human disease. One way to achieve this precise regulation is by combinatorial inputs of different transcription factors. Here we used Drosophila melanogaster mutants with migration defects in the ganglionic branches of the tracheal system to further clarify guidance regulation during cell migration. By studying the cellular consequences of overactivated Hh signalling, using ptc mutants, we found that Hh positively regulates Bnl/FGF levels during embryonic stages. Our results show that Hh modulates cell migration non-autonomously in the tissues surrounding the action of its activity. We further demonstrate that the Hh signalling pathway regulates bnl expression via Stripe (Sr), a zinc-finger transcription factor with homology to the Early Growth Response (EGR) family of vertebrate transcription factors. We propose that Hh modulates embryonic cell migration by participating in the spatio-temporal regulation of bnl expression in a permissive mode. By doing so, we provide a molecular link between the activation of Hh signalling and increased chemotactic responses during cell migration. PMID:24651658

  17. Cognate antigen directs CD8+ T cell migration to vascularized transplants.

    PubMed

    Walch, Jeffrey M; Zeng, Qiang; Li, Qi; Oberbarnscheidt, Martin H; Hoffman, Rosemary A; Williams, Amanda L; Rothstein, David M; Shlomchik, Warren D; Kim, Jiyun V; Camirand, Geoffrey; Lakkis, Fadi G

    2013-06-01

    The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

  18. Optical control demonstrates switch-like PIP3 dynamics underlying the initiation of immune cell migration.

    PubMed

    Karunarathne, W K Ajith; Giri, Lopamudra; Patel, Anilkumar K; Venkatesh, Kareenhalli V; Gautam, N

    2013-04-23

    There is a dearth of approaches to experimentally direct cell migration by continuously varying signal input to a single cell, evoking all possible migratory responses and quantitatively monitoring the cellular and molecular response dynamics. Here we used a visual blue opsin to recruit the endogenous G-protein network that mediates immune cell migration. Specific optical inputs to this optical trigger of signaling helped steer migration in all possible directions with precision. Spectrally selective imaging was used to monitor cell-wide phosphatidylinositol (3,4,5)-triphosphate (PIP3), cytoskeletal, and cellular dynamics. A switch-like PIP3 increase at the cell front and a decrease at the back were identified, underlying the decisive migratory response. Migration was initiated at the rapidly increasing switch stage of PIP3 dynamics. This result explains how a migratory cell filters background fluctuations in the intensity of an extracellular signal but responds by initiating directionally sensitive migration to a persistent signal gradient across the cell. A two-compartment computational model incorporating a localized activator that is antagonistic to a diffusible inhibitor was able to simulate the switch-like PIP3 response. It was also able simulate the slow dissipation of PIP3 on signal termination. The ability to independently apply similar signaling inputs to single cells detected two cell populations with distinct thresholds for migration initiation. Overall the optical approach here can be applied to understand G-protein-coupled receptor network control of other cell behaviors.

  19. The oncoprotein HBXIP promotes migration of breast cancer cells via GCN5-mediated microtubule acetylation.

    PubMed

    Li, Leilei; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong

    2015-03-13

    We have documented that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. A subset of acetylated microtubules that accumulates in the cell leading edge is necessary for cell polarization and directional migration. In this study, we explored the hypothesis that HBXIP contributes to migration of breast cancer cells by supporting microtubule acetylation in breast cancer cells. We found that HBXIP could induce acetylated microtubules accumulating into the leading protrusion in wound-induced directional migration in breast cancer cells by immunofluorescence staining analysis. Interestingly, HBXIP was able to increase the acetylation of α-tubulin in the cells by immunofluorescence staining and Western blot analysis. Furthermore, we observed that acetyltransferase GCN5 was involved in the event that HBXIP induced increase of acetylated microtubules and their expansion in protrusions in breast cancer cells by Western blot analysis and immunofluorescence staining. Moreover, GCN5 was required for the HBXIP-enhanced migration of breast cancer cells by wound healing assay. Thus, we conclude that HBXIP promotes the migration of breast cancer cells through modulating microtubule acetylation mediated by GCN5. Therapeutically, HBXIP may serve as a novel target in breast cancer.

  20. Neural cell adhesion molecule modulates mesenchymal stromal cell migration via activation of MAPK/ERK signaling.

    PubMed

    Shi, Yu; Xia, Yin-Yan; Wang, Lei; Liu, Rui; Khoo, King-Shung; Feng, Zhi-Wei

    2012-10-15

    Mesenchymal Stromal Cells (MSCs) represent promising tools for cellular therapy owing to their multipotentiality and ability to localize to injured, inflamed sites and tumor. Various approaches to manipulate expression of MSC surface markers, including adhesion molecules and chemokine receptors, have been explored to enhance homing of MSCs. Recently, Neural Cell Adhesion Molecule (NCAM) has been found to be expressed on MSCs yet its function remains largely elusive. Herein, we show that bone marrow-derived MSCs from NCAM deficient mice exhibit defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. We further explore the mechanism governing NCAM mediated migration of MSCs by showing the interplay between NCAM and Fibroblast Growth Factor Receptor (FGFR) induces activation of MAPK/ERK signaling, thereby the migration of MSCs. In addition, re-expression of NCAM180, but not NCAM140, could restore the defective MAPK/ERK signaling thereby the migration of NCAM deficient MSCs. Finally, we demonstrate that NCAM180 expression level could be manipulated by pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α treatment. Overall, our data reveal the vital function of NCAM in MSCs migration and differentiation thus raising the possibility of manipulating NCAM expression to enhance homing and therapeutic potential of MSCs in cellular therapy.

  1. Contact-mediated control of radial migration of corneal epithelial cells

    PubMed Central

    Walczysko, Petr; Rajnicek, Ann M.

    2016-01-01

    Purpose Patients with a heterozygous mutation in the gene encoding the transcription factor, PAX6, have a degenerative corneal opacity associated with failure of normal radial epithelial cell migration across the corneal surface and a reported wound healing defect. This study investigated the guidance mechanisms that drive the directed migration of corneal epithelial cells. Methods In vivo corneal epithelial wounding was performed in adult wild-type and Pax6+/− mice, and the healing migration rates were compared. To investigate the control of the cell migration direction, primary corneal epithelial cells from wild-type and Pax6+/− mice were plated on grooved quartz substrates, and alignment relative to the grooves was assayed. A reconstructed corneal culture system was developed in which dissociated wild-type and genetically mutant corneal epithelial cells could be cultured on a de-epithelialized corneal stroma or basement membrane and their migration assayed with time-lapse microscopy. Results The Pax6+/− cells efficiently re-epithelialized corneal wounds in vivo but had mild slowing of healing migration compared to the wild-type. Cells aligned parallel to quartz grooves in vitro, but the Pax6+/− cells were less robustly oriented than the wild-type. In the reconstructed corneal culture system, corneal epithelial cells continued to migrate radially, showing that the cells are guided by contact-mediated cues from the basement membrane. Recombining wild-type and Pax6 mutant corneal epithelial cells with wild-type and Pax6 mutant corneal stroma showed that normal Pax6 dosage was required autonomously in the epithelial cells for directed migration. Integrin-mediated attachment to the substrate, and intracellular PI3Kγ activity, were required for migration. Pharmacological inhibition of cAMP signaling randomized migration tracks in reconstructed corneas. Conclusions Striking patterns of centripetal migration of corneal epithelial cells observed in vivo are

  2. Colonizing while migrating: how do individual enteric neural crest cells behave?

    PubMed Central

    2014-01-01

    Background Directed cell migration is essential for normal development. In most of the migratory cell populations that have been analyzed in detail to date, all of the cells migrate as a collective from one location to another. However, there are also migratory cell populations that must populate the areas through which they migrate, and thus some cells get left behind while others advance. Very little is known about how individual cells behave to achieve concomitant directional migration and population of the migratory route. We examined the behavior of enteric neural crest-derived cells (ENCCs), which must both advance caudally to reach the anal end and populate each gut region. Results The behavior of individual ENCCs was examined using live imaging and mice in which ENCCs express a photoconvertible protein. We show that individual ENCCs exhibit very variable directionalities and speed; as the migratory wavefront of ENCCs advances caudally, each gut region is populated primarily by some ENCCs migrating non-directionally. After populating each region, ENCCs remain migratory for at least 24 hours. Endothelin receptor type B (EDNRB) signaling is known to be essential for the normal advance of the ENCC population. We now show that perturbation of EDNRB principally affects individual ENCC speed rather than directionality. The trajectories of solitary ENCCs, which occur transiently at the wavefront, were consistent with an unbiased random walk and so cell-cell contact is essential for directional migration. ENCCs migrate in close association with neurites. We showed that although ENCCs often use neurites as substrates, ENCCs lead the way, neurites are not required for chain formation and neurite growth is more directional than the migration of ENCCs as a whole. Conclusions Each gut region is initially populated by sub-populations of ENCCs migrating non-directionally, rather than stopping. This might provide a mechanism for ensuring a uniform density of ENCCs along the

  3. Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo

    PubMed Central

    Melchionda, Manuela; Pittman, Jon K.

    2016-01-01

    Increasing evidence implicates Ca2+ in the control of cell migration. However, the underlying mechanisms are incompletely understood. Acidic Ca2+ stores are fast emerging as signaling centers. But how Ca2+ is taken up by these organelles in metazoans and the physiological relevance for migration is unclear. Here, we identify a vertebrate Ca2+/H+ exchanger (CAX) as part of a widespread family of homologues in animals. CAX is expressed in neural crest cells and required for their migration in vivo. It localizes to acidic organelles, tempers evoked Ca2+ signals, and regulates cell-matrix adhesion during migration. Our data provide new molecular insight into how Ca2+ is handled by acidic organelles and link this to migration, thereby underscoring the role of noncanonical Ca2+ stores in the control of Ca2+-dependent function. PMID:27002171

  4. Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo.

    PubMed

    Melchionda, Manuela; Pittman, Jon K; Mayor, Roberto; Patel, Sandip

    2016-03-28

    Increasing evidence implicates Ca(2+) in the control of cell migration. However, the underlying mechanisms are incompletely understood. Acidic Ca(2+) stores are fast emerging as signaling centers. But how Ca(2+) is taken up by these organelles in metazoans and the physiological relevance for migration is unclear. Here, we identify a vertebrate Ca(2+)/H(+)exchanger (CAX) as part of a widespread family of homologues in animals. CAX is expressed in neural crest cells and required for their migration in vivo. It localizes to acidic organelles, tempers evoked Ca(2+) signals, and regulates cell-matrix adhesion during migration. Our data provide new molecular insight into how Ca(2+) is handled by acidic organelles and link this to migration, thereby underscoring the role of noncanonical Ca(2+) stores in the control of Ca(2+)-dependent function. PMID:27002171

  5. APC binds intermediate filaments and is required for their reorganization during cell migration.

    PubMed

    Sakamoto, Yasuhisa; Boëda, Batiste; Etienne-Manneville, Sandrine

    2013-02-01

    Intermediate filaments (IFs) are components of the cytoskeleton involved in most cellular functions, including cell migration. Primary astrocytes mainly express glial fibrillary acidic protein, vimentin, and nestin, which are essential for migration. In a wound-induced migration assay, IFs reorganized to form a polarized network that was coextensive with microtubules in cell protrusions. We found that the tumor suppressor adenomatous polyposis coli (APC) was required for microtubule interaction with IFs and for microtubule-dependent rearrangements of IFs during astrocyte migration. We also show that loss or truncation of APC correlated with the disorganization of the IF network in glioma and carcinoma cells. In migrating astrocytes, vimentin-associated APC colocalized with microtubules. APC directly bound polymerized vimentin via its armadillo repeats. This binding domain promoted vimentin polymerization in vitro and contributed to the elongation of IFs along microtubules. These results point to APC as a crucial regulator of IF organization and confirm its fundamental role in the coordinated regulation of cytoskeletons.

  6. Collective migration models: Dynamic monitoring of leader cells in migratory/invasive disease processes

    NASA Astrophysics Data System (ADS)

    Dean, Zachary Steven

    Leader cells are a fundamental biological process that have only been investigated since the early 2000s. These cells have often been observed emerging at the edge of an artificial wound in 2D epithelial cell collective invasion, created with either a mechanical scrape from a pipette tip or from the removal of a plastic, physical blocker. During migration, the moving cells maintain cell-cell contacts, an important quality of collective migration; the leader cells originate from either the first or the second row, they increase in size compared to other cells, and they establish ruffled lamellipodia. Recent studies in 3D have also shown that cells emerging from an invading collective group that also exhibit leader-like properties. Exactly how leader cells influence and interact with follower cells as well as other cells types during collective migration, however, is another matter, and is a subject of intense investigation between many different labs and researchers. The majority of leader cell research to date has involved epithelial cells, but as collective migration is implicated in many different pathogenic diseases, such as cancer and wound healing, a better understanding of leader cells in many cell types and environments will allow significant improvement to therapies and treatments for a wide variety of disease processes. In fact, more recent studies on collective migration and invasion have broadened the field to include other cell types, including mesenchymal cancer cells and fibroblasts. However, the proper technology for picking out dynamic, single cells within a moving and changing cell population over time has severely limited previous investigation into leader cell formation and influence over other cells. In line with these previous studies, we not only bring new technology capable of dynamically monitoring leader cell formation, but we propose that leader cell behavior is more than just an epithelial process, and that it is a critical physiological

  7. Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

    SciTech Connect

    Li, Jie; Yang, Xi-fei; Ren, Xiao-hu; Meng, Xiao-jing; Huang, Hai-yan; Zhao, Qiong-hui; Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li; Liu, Jian-jun; Zou, Fei

    2014-10-10

    Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.

  8. MNT inhibits the migration of human hepatocellular carcinoma SMMC7721 cells

    SciTech Connect

    Wu, Jian; Zhou, Qi; Wang, Yafeng; Zhou, Xiangbing; Li, Jiaping

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer MNT is a member of the Myc/Max/Mad network that plays a role in cell proliferation. Black-Right-Pointing-Pointer Our study further emphasized the role of MNT in migration inhibition of SMMC7721 cells. Black-Right-Pointing-Pointer MNT might be a promising target for HCC chemotherapy. -- Abstract: Max binding protein (MNT) is a member of the Myc/Max/Mad network that plays a role in cell proliferation, differentiation and apoptosis. We previously observed that MNT was differentially expressed in hepatocellular carcinoma (HCC) and interacted with Nck1 by 2-DE. Nck family adaptor proteins function to couple tyrosine phosphorylation signals, regulate actin cytoskeletal reorganization and lead to cell motility. In order to investigate the regulatory role of MNT in HCC migration, we used transient transfection with a MNT expressing vector to overexpress MNT protein in SMMC7721 cells, and MNT siRNA to knockdown MNT expression. Rho Family Small GTPase activation assay, Western blots and transwell assay were used to determine the migration potential of cells. We found that knockdown of MNT expression might promote SMMC7721 cell migration, while the overexpressed MNT could significantly inhibit cell migration. It further emphasized the role of MNT in inhibition of cell migration that might be a promising target for HCC chemotherapy.

  9. Slits Affect the Timely Migration of Neural Crest Cells via Robo Receptor

    PubMed Central

    Giovannone, Dion; Reyes, Michelle; Reyes, Rachel; Correa, Lisa; Martinez, Darwin; Ra, Hannah; Gomez, Gustavo; Kaiser, Josh; Ma, Le; Stein, Mary-Pat; de Bellard, Maria Elena

    2013-01-01

    SUMMARY Background Neural crest cells emerge by delamination from the dorsal neural tube and give rise to various components of the peripheral nervous system in vertebrate embryos. These cells change from non-motile into highly motile cells migrating to distant areas before further differentiation. Mechanisms controlling delamination and subsequent migration of neural crest cells are not fully understood. Slit2, a chemorepellant for axonal guidance that repels and stimulates motility of trunk neural crest cells away from the gut has recently been suggested to be a tumor suppressor molecule. The goal of this study was to further investigate the role of Slit2 in trunk neural crest cell migration by constitutive expression in neural crest cells. Results We found that Slit gain-of-function significantly impaired neural crest cell migration while Slit loss-of-function favored migration. In addition, we observed that the distribution of key cytoskeletal markers was disrupted in both gain and loss of function instances. Conclusions These findings suggest that Slit molecules might be involved in the processes that allow neural crest cells to begin migration and transitioning to a mesenchymal type. PMID:22689303

  10. A Mathematical Model of Collective Cell Migration in a Three-Dimensional, Heterogeneous Environment

    PubMed Central

    Stonko, David P.; Manning, Lathiena; Starz-Gaiano, Michelle; Peercy, Bradford E.

    2015-01-01

    Cell migration is essential in animal development, homeostasis, and disease progression, but many questions remain unanswered about how this process is controlled. While many kinds of individual cell movements have been characterized, less effort has been directed towards understanding how clusters of cells migrate collectively through heterogeneous, cellular environments. To explore this, we have focused on the migration of the border cells during Drosophila egg development. In this case, a cluster of different cell types coalesce and traverse as a group between large cells, called nurse cells, in the center of the egg chamber. We have developed a new model for this collective cell migration based on the forces of adhesion, repulsion, migration and stochastic fluctuation to generate the movement of discrete cells. We implement the model using Identical Math Cells, or IMCs. IMCs can each represent one biological cell of the system, or can be aggregated using increased adhesion forces to model the dynamics of larger biological cells. The domain of interest is filled with IMCs, each assigned specific biophysical properties to mimic a diversity of cell types. Using this system, we have successfully simulated the migration of the border cell cluster through an environment filled with larger cells, which represent nurse cells. Interestingly, our simulations suggest that the forces utilized in this model are sufficient to produce behaviors of the cluster that are observed in vivo, such as rotation. Our framework was developed to capture a heterogeneous cell population, and our implementation strategy allows for diverse, but precise, initial position specification over a three- dimensional domain. Therefore, we believe that this model will be useful for not only examining aspects of Drosophila oogenesis, but also for modeling other two or three-dimensional systems that have multiple cell types and where investigating the forces between cells is of interest. PMID:25875645

  11. Role of Nonmuscle Myosin II in Migration of Wharton's Jelly-Derived Mesenchymal Stem Cells

    PubMed Central

    Arora, Sneha; Saha, Shekhar; Roy, Saheli; Das, Madhurima; Jana, Siddhartha S.

    2015-01-01

    It is the promise of regeneration and therapeutic applications that has sparked an interest in mesenchymal stem cells (MSCs). Following infusion, MSCs migrate to sites of injury or inflammation by virtue of their homing property. To exert optimal clinical benefits, systemically delivered MSCs need to migrate efficiently and in adequate numbers to pathological areas in vivo. However, underlying molecular mechanisms responsible for MSC migration are still not well understood. The Wharton's jelly (WJ) of the umbilical cord is an attractive source of MSCs for stem cell therapy because of its abundant availability and painless collection. In this study, we attempted to identify the role of nonmuscle myosin II (NMII), if any, in the migration of WJ-derived MSCs (WJ-MSCs). Expression of NMII isoforms, NMIIA, and NMIIB was observed both at RNA and protein levels in WJ-MSCs. Inhibition of NMII or its regulator ROCK, by pharmacological inhibitors, resulted in significant reduction in the migration of WJ-MSCs as confirmed by the scratch migration assay and time-lapse microscopy. Next, trying to dissect the role of each NMII isoform in migration of WJ-MSCs, we found that siRNA-mediated downregulation of NMIIA, but not NMIIB expression, led to cells failing to retract their trailing edge and losing cell–cell cohesiveness, while exhibiting a nondirectional migratory pathway. Migration, moreover, is also dependent on optimal affinity adhesion, which would allow rapid attachment and release of cells and, hence, can be influenced by extracellular matrix (ECM) and adhesion molecules. We demonstrated that inhibition of NMII and more specifically NMIIA resulted in increased gene expression of ECM and adhesion molecules, which possibly led to stronger adhesions and, hence, decreased migration. Therefore, these data suggest that NMII acts as a regulator of cell migration and adhesion in WJ-MSCs. PMID:25923805

  12. Interplanetary Migration of Eucaryotic Cell, Spore of Schizosaccharomyces Pombe

    NASA Astrophysics Data System (ADS)

    Hayashi, N.; Nosaka, J.; Ando, R.; Hashimoto, H.; Yokobori, S.; Narumi, I.; Nakagawa, K.; Yamagishi, A.; Tohda, H.

    2013-11-01

    The Tanpopo mission to examine possible interplanetary migration of microbes is progressing. Spore of Schizosaccharomyces pombe are considered as the exposed samples. In this paper, results of preliminary experiments for the exposure are shown.

  13. Elucidating the Role of Matrix Stiffness in 3D Cell Migration and Remodeling

    PubMed Central

    Ehrbar, M.; Sala, A.; Lienemann, P.; Ranga, A.; Mosiewicz, K.; Bittermann, A.; Rizzi, S.C.; Weber, F.E.; Lutolf, M.P.

    2011-01-01

    Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena. PMID:21244824

  14. Cross-talk between calcium and protein kinase A in the regulation of cell migration.

    PubMed

    Howe, Alan K

    2011-10-01

    Calcium (Ca(2+)) and the cAMP-dependent protein kinase (PKA) are pleiotropic cellular regulators and both exert powerful, diverse effects on cytoskeletal dynamics, cell adhesion, and cell migration. Localization, by A-kinase-anchoring proteins (AKAPs), of PKA activity to the protrusive leading edge, integrins, and other regulators of cytoskeletal dynamics has emerged as an important facet of its role in cell migration. Additional recent work has firmly established the importance of Ca(2+) influx through mechanosensitive transient receptor potential (TRP) channels and through store-operated Ca(2+) entry (SOCE) in cell migration. Finally, there is considerable evidence showing that these mechanisms of Ca(2+) influx and PKA regulation intersect--and often interact--and thus may work in concert to translate complex extracellular cues into the intracellular biochemical anisotropy required for directional cell migration.

  15. Neurotrophins regulate Schwann cell migration by activating divergent signaling pathways dependent on Rho GTPases

    PubMed Central

    Yamauchi, Junji; Chan, Jonah R.; Shooter, Eric M.

    2004-01-01

    Neurotrophins are recognized widely as essential factors in the developing nervous system. Previously, we demonstrated that neurotrophin 3 activation of TrkC inhibits Schwann cell myelination and enhances the migration of primary Schwann cells through the signaling pathway regulated by the Rho GTPases Rac1 and Cdc42. Here, we show that neurotrophins activate divergent signaling pathways to promote or inhibit Schwann cell migration. Endogenous brain-derived neurotrophic factor acting through p75NTR inhibits Schwann cell migration dramatically by Src kinase-dependent activation of the guanine-nucleotide exchange factor Vav2 and RhoA. Together, the