Tag: fluorescence microscope

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Inhibition of SNARE-mediated membrane traffic impairs cell migration

Cell migration occurs as a highly-regulated cycle of cell polarization, membrane extension at the leading edge, adhesion, contraction of the cell body, and release from the extracellular matrix at the trailing edge. In this study, we investigated the involvement of SNARE-mediated membrane trafficking in cell migration. Using a dominant-negative form of the enzyme N-ethylmaleimide-sensitive factor as a general inhibitor of SNARE-mediated membrane traffic and tetanus toxin as a specific inhibitor of VAMP3/cellubrevin, we conducted transwell migration assays and determined that serum-induced migration of CHO-K1 cells is dependant upon SNARE function. Both VAMP3-mediated and VAMP3-independent traffic were involved in regulating this cell migration. Inhibition of SNARE-mediated membrane traffic led to a decrease in the protrusion of lamellipodia at the leading edge of migrating cells. Additionally, the reduction in cell migration resulting from the inhibition of SNARE function was accompanied by perturbation of a Rab11-containing alpha(5)beta(1) integrin compartment and a decrease in cell surface alpha(5)beta(1) without alteration to total cellular integrin levels. Together, these observations suggest that inhibition of SNARE-mediated traffic interferes with the intracellular distribution of integrins and with the membrane remodeling that contributes to lamellipodial extension during cell migration.

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http://www.sciencedirect.com/science/article/pii/S0014482704007244

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Canstatin, a Novel Matrix-derived Inhibitor of Angiogenesis and Tumor Growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the α2 chain of type IV collagen, was produced as a recombinant molecule inEscherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.

http://www.jbc.org/content/275/2/1209.long

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Direct Cell Adhesion to the Angiopoietins Mediated by Integrins

Genetic ablation of angiopoietin-1 (Ang-1) or of its cognate receptor, Tie2, disrupts angiogenesis in mouse embryos. The endothelial cells in growing blood vessels of Ang-1 knockout mice have a rounded appearance and are poorly associated with one another and their underlying basement membranes (Dumont, D. J., Gradwohl, G., Fong, G. H., Puri, M. C., Gertsenstein, M., Auerbach, A., and Breitman, M. L. (1994) Genes Dev. 8, 1897–1909; Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W., and Qin, Y. (1995) Nature 376, 70–74; Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T. N., and Yancopoulos, G. D. (1996) Cell 87, 1171–1180). It is therefore possible that Ang-1 regulates endothelial cell adhesion. In this study we asked whether Ang-1 might act as a direct substrate for cell adhesion. Human umbilical vein endothelial cells (HUVECs) plated for a brief period on different substrates were found to adhere and spread well on Ang-1. Similar results were seen on angiopoietin-2 (Ang-2)-coated surfaces, although cells did not spread well on Ang-2. Ang-1, but not Ang-2, supported HUVEC migration, and this was independent of growth factor activity. When the same experiments were done with fibroblasts that either lacked, or stably expressed, Tie2, results similar to those with HUVECs were seen, suggesting that adhesion to the angiopoietins was independent of Tie2 and not limited to endothelial cells. Interestingly, when integrin-blocking agents were included in these assays, adhesion to either angiopoietin was significantly reduced. Moreover, Chinese hamster ovary-B2 cells lacking the α5 integrin subunit did not adhere to Ang-1, but they did adhere to Ang-2. Stable expression of the human α5 integrin subunit in these cells rescued adhesion to Ang-1 and promoted an increase in adhesion to Ang-2. We also found that Ang-1 and Ang-2 bind rather selectively to vitronectin. These results suggest that, beyond their role in modulating Tie2 signaling, Ang-1 and Ang-2 can directly support cell adhesion mediated by integrins.

http://www.jbc.org/content/276/28/26516.long

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Lysophosphatidic acid induces cell migration through the selective activation of Akt1

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.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679274/