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The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of alpha(v)beta(5) to focal contact sites, but not of alpha(v)beta(3) or alpha(5)beta(1), was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with alpha(v)beta(5) and paxillin but not with alpha(v)beta(3) that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the alpha(v)beta(5) integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-alpha(v)beta(5) antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the alpha(v)beta(5) integrin that supports tumor cell invasion.

Human embryonic stem cells (hESCs) can proliferate extensively in culture and give rise to progeny of the three germ layers. Several reports suggested that mouse and hESCs may attenuate immune responses. In this study, we focused on the mechanism by which hESCs inhibit T cell responses. Using coculture experiments, we demonstrate that hESCs inhibit cytokine secretion and T cell proliferation in response to potent T cell activators. Furthermore, we show that hESCs downmodulate the TCR-associated CD3-ζ chain. These effects are maintained when hESCs are replaced by their conditioned media and can be restored by the addition of l-arginine to hESC-conditioned media or by treatment of hESCs with a specific arginase inhibitor. Moreover, we show arginase-I expression and activity in hESCs. We further demonstrate that mouse ESCs (mESCs) similarly inhibit T cell activation via arginase I, suggesting an evolutionary conserved mechanism of T cell suppression by ESCs. In addition, we demonstrate that arginase I expression is not limited to ESCs in culture, but can also be detected in the inner cell mass and the trophectoderm of preimplantation mouse embryos and hESC-derived trophectoderm cells. Finally, T cells infiltrating ESC-derived teratomas have significantly lower levels of CD3-ζ chain. Collectively, the data indicate a role for ESC-arginase I activity in the attenuation of T cell activation.

Non-muscle myosin II has diverse functions in cell contractility, morphology, cytokinesis and migration. Mammalian cells have three isoforms of non-muscle myosin II, termed IIA, IIB and IIC, encoded by three different genes. These isoforms share considerable homology and some overlapping functions, yet they exhibit differences in enzymatic properties, subcellular localization, molecular interaction and tissue distribution.(1-6) Our studies have focused on the IIA isoform, and they reveal unique regulatory roles in cell-cell adhesion and cell migration that are associated with cross-talk of the actomyosin system with microtubules. In humans, various mutations in the MYH9 gene that encodes the myosin IIA heavy chain cause autosomal dominant disease, whereas in mice, the complete deficiency is embryonic lethal but heterozygous mice are nearly normal. We discuss here the differences between mouse and human phenotypes and how the wealth of mechanistic knowledge about myosin II based on in vitro studies and mouse models can help us understand the molecular and cellular pathophysiology of myosin IIA deficiency in humans.

Preface Contributors I. Molecular Biology Retroviral delivery of ECM genes Vitali Alexeev and Olga Igoucheva Tissue-Specific KO of ECM Proteins Mara Brancaccio, Emila Turco and Emilio Hirsch Recombinant Collagen Trimers from Insect Cells and Yeast- Johanna Myllyharju Eukaryotic Expression and Purification of Recombinant Extracellular Matrix Proteins Carrying the Strep II Tag Neil Smyth, Uwe Odenthal, Barbara Merkl, and Mats Paulsson Tissue Recombinants to Study Extracellular Matrix Targeting to Basement Membranes Patricia Simon-Assmann, Anne-Laure Bolcato-Bellemin, Annick Klein and Michele Kedinger Preparation of recombinant fibronectin fragments for functional and structural studies David Staunton, Christopher J Millard, Radu Aricescu and Iain D Campbell II. Biochemical and biophysical analysis Quantitative Determination of Collagen Cross-links Nicholas C Avery, Trevor J Sims and Allen J Bailey ECM Macromolecules: Height-mapping and Nano-mechanics Using Atomic Force Microscopy Nigel W. Hodson, Cay M. Kielty and Michael J. Sherratt Atomic Force Microscopy Measurements of Intermolecular Binding Forces- Gradimir N. Misevic, Yannis Karamanos and Nikola J. Misevic Mass-Mapping of ECM Macromolecules by Scanning Transmission Electron Microscopy Michael J. Sherratt, Helen K. Graham, Cay M. Kielty and David F. Holmes Chemical Microscopy of Biological Samples by Dynamic Mode Secondary Ion Mass Spectrometry (SIMS) Gradimir N. Misevic, Bernard Rasser, Vic Norris, Cedric Derue, David Gibouin, Fabrice Lefebvre, Marie-Claire Verdus, Anthony Delaune, Guillaume Legent and CamilleRipoll ECM Macromolecules: Rotary Shadowing and Transmission Electron Microscopy Michael J. Sherratt, Roger S. Meadows, Helen K. Graham, Cay M. Kielty and David F. Holmes Using Self-Assembled Monolayers to Pattern ECM Proteins and Cells on Substrates Emanuele Ostuni, George M. Whitesides, Donald E. Ingber and Christopher S. Chen Solid Phase Assays for Studying ECM Protein-Protein Interactions Paul A Mould III. Cell biology assays Cell Adhesion Assays Martin J. Humphries ECM Degradation Assays for Analyzing Local Cell Invasion Vira V. Artym, Kenneth M. Yamada, Susette C. Mueller Fluorescence-Based Assays for In Vitro Analysis of Cell Adhesion and Migration Paola Spessotto, Katia Lacrima, Pier Andrea Nicolosi, Eliana Pivetta, Martina Scapolan and Roberto Perris Fibrin Gel Model for Assessment of Cellular Contractility Sharona Even-Ram Fluorescent labeling techniques for investigation of fibronectin fibrillogenesis ( Labeling fibronectin fibrillogenesis) Roumen Pankov and Albena Momchilova Stromagenesis During Tumorigenesis: Characterization of Tumor-associated Fibroblasts and Stroma-derived 3D Matrices Remedios Castello-Cros and Edna Cukierman IV. Organ models ECM and FGF-dependent assay of embryonic SMG epithelial morphogenesis: investigating growth factor /matrix regulation of gene expression during submandibular gland development Ivan T. Rebustini and Matthew P. Hoffman Analyzing Cell-ECM Interactions in Adult Mammary Gland by Transplantation of Embryonic Mammary Tissue from Knockout Mice Teresa C. M. Klinowska and Charles H. Streuli V. Tissue

Previous work has shown that ablation or mutation of nonmuscle myosin heavy chain II-B (NMHC II-B) in mice results in defects in the heart and brain with death occurring between embryonic day 14.5 (E14.5) and birth (Tullio, A. N., Accili, D., Ferrans, V. J., Yu, Z. X., Takeda, K., Grinberg, A., Westphal, H., Preston, Y. A., and Adelstein, R. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12407-12412). Here we show that mice ablated for NMHC II-A fail to develop a normal patterned embryo with a polarized visceral endoderm by E6.5 and die by E7.5. Moreover, A(-)/A(-) embryoid bodies grown in suspension culture constantly shed cells. These defects in cell adhesion and tissue organization are explained by loss of E-cadherin and beta-catenin localization to cell adhesion sites in both cell culture and in the intact embryos. The defects can be reproduced by introducing siRNA directed against NMHC II-A into wild-type embryonic stem cells. Our results suggest an essential role for a single, specific nonmuscle myosin isoform in maintaining cell-cell adhesions in the early mammalian embryo.

Thrombin receptor (ThR) plays a significant role in myocyte contractility and hypertrophy. Heart myocyte ischemic damage, caused by insufficient blood supply, is the leading cause of heart infarction. Here we demonstrate that when primary myocyte cultures are subjected to hypoxic stress, ThR mRNA levels are reduced markedly. This takes place also in vivo in a model of ischemic pig heart, exhibiting reduced levels of ThR compared with normal heart sections. Prior activation of ThR however, by either thrombin receptor-activating peptide (TRAP) or by alpha-thrombin resulted in full protection of ThR mRNA levels under hypoxia. The effect appeared specific to ThR because the addition of TRAP did not affect the hypoxic damage as shown by the levels of lactic dehydrogenase release and up-regulated GLUT-1, a glucose transporter gene. This protection effect took place not only in primary myocytes but also in NIH3T3 fibroblasts. ThR protection occurs via specific cell signaling events because activation of the receptor by TRAP, following interruption of the signaling cascade by calphostin C, a protein kinase C inhibitor, resulted in loss of ThR mRNA protection. Because Ras and Src are part of the ThR signaling cascade, the introduction of either dominant ras or src oncogenes to NIH3T3 murine fibroblasts gave rise to similar protection of ThR mRNA levels under hypoxic conditions without the exogenous addition of TRAP. Likewise, ThR mRNA protection was obtained after transfection with proto-oncogene vav. The 95-kDa protein Vav undergoes tyrosine phosphorylation after ThR activation, serving thus as part of the receptor machinery cascade. We therefore conclude that the initiation of the signaling cascades either exogenously by TRAP or within the cell via src or ras, as well as via vav oncogene interconnecting G-binding protein to the tyrosine kinase pathway, ultimately results in ThR protection under hypoxia. We present hereby, a novel concept of activated receptors, which under minimal oxygen tension protect their otherwise decaying mRNA. Maintaining the level of ThR that plays an active role in normal myocyte function may provide a significant repair mechanism in ischemic tissue, assisting in the regaining of normal myocyte functions.

Although the involvement of soluble and matrix-immobilized proteases in tumor cell invasion and metastasis is well recognized, the role of proteolytically activated cell surface receptors has not been elucidated. We report here that thrombin receptor, a member of the protease-activated receptor family, is preferentially expressed in highly metastatic human breast carcinoma cell lines and breast carcinoma biopsy specimens. Introduction of thrombin receptor antisense cDNA considerably inhibited the invasion of metastatic breast carcinoma cells in culture through a reconstituted basement membrane. During placental implantation of the human embryo, thrombin receptor is transiently expressed in the invading cytotrophoblasts. These results emphasize the involvement of thrombin receptor in cell invasion associated with tumor progression and normal embryonic development.

The yeast mitochondrial and cytosolic isoenzymes of fumarase, which are encoded by a single nuclear gene (FUM1), follow a unique mechanism of protein subcellular localization and distribution. Translation of all FUM1 messages initiates only from the 5'-proximal AUG codon and results in a single translation product that contains the targeting sequence located within the first 32 amino acids of the precursor. All fumarase molecules synthesized in the cell are processed by the mitochondrial matrix signal peptidase; nevertheless, most of the enzyme (80 to 90%) ends up in the cytosol. The translocation and processing of fumarase are cotranslational. We suggest that in Saccharomyces cerevisiae, the single type of initial translation product of the FUM1 gene is first partially translocated, and then a subset of these molecules continues to be fully translocated into the organelle, whereas the rest are folded into an import-incompetent state and are released by the retrograde movement of fumarase into the cytosol.