Sharona Even-Ram

A Rac switch regulates random versus directionally persistent cell migration

Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3′-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

Myosin IIA regulates cell motility and actomyosin- microtubule crosstalk

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.

Integrin regulation of growth factor receptors

Integrins are receptors for extracellular matrix proteins that engage in reciprocal crosstalk with growth factor receptors. Recent work identifies a unique mechanism for the regulation of growth factor receptor phosphorylation by integrins, indicating multiple ways of achieving cooperation between these major signalling systems.

Human Protease-Activated Receptor 1 Expression in Malignant Epithelia: A Role in Invasiveness

While protease-activated receptors (PARs) play a traditional role in vascular biology, they emerge with surprisingly new assignments in tumor biology. PAR1 expression correlates with the invasion properties of breast carcinoma, whereas human PAR1 antisense reduces their ability to migrate through Matrigel. Part of the molecular mechanism of PAR1 invasion involves the formation of focal contact complexes on PAR1 activation. PAR1 induces angiogenesis in animal models in vivo and exhibits an oncogenic phenotype of enhanced ductal complexity when overexpressed in mouse mammary glands.

The pattern of expression of protease-activated receptors (PARs) during early trophoblast development

Human fetal development depends on the ability of the embryo to gain access to the maternal circulation. Thus, specialized stem cells of the newly formed placenta, trophoblast, invade the uterus and its arterial network to establish an efficient feto‐maternal molecular exchange. To accomplish this task, trophoblast differentiation during the first trimester of pregnancy involves cell proliferation, invasion, and extracellular matrix (ECM) remodelling. Trophoblast invasion shares many features with tumour cell invasion, with the distinction that it is strictly spatially and temporally controlled. We have previously demonstrated that PAR1, the first member of the protease‐activated receptor (PAR) family, plays a central role in tumour cell invasion. In the present study we have examined the pattern of expression of PAR1 and other PAR family candidates during early human placental development. We show that PAR1 and PAR3 are highly and spatially expressed between the 7th and 10th weeks of gestation but not at the 12th week and thereafter. Likewise, high expression levels of PAR1 and PAR3 were observed in the cytotrophoblast cells of complete hydatidiform mole as compared to minimal levels in normal age‐matched placenta. Together, our data suggest the involvement of PAR1 and PAR3 in restricted and unrestricted pathological trophoblast invasion. Copyright

Of Mice and Men: Relevance of Cellular and Molecular Characterizations of Myosin IIA to MYH9-Related Human Disease

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.

Extracellular matrix protocols

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

Protection of Thrombin Receptor Expression under Hypoxia

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 α-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 viavav 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.

Inhibition of rho GTPases by RNA interference.

Selective down-modulation or silencing of individual members of the Rho-GTPase family is now practical using RNA interference. Transfection of mammalian cells with an individual siRNA duplex or siRNA pools can suppress expression of a specific isoform to understand its function. By adjusting the dose of siRNA, intermediate levels of suppression can be attained to test the biological role of different levels of a GTPase such as Rac. Nevertheless, there are significant potential pitfalls, including off-target effects of the siRNA on other genes. Besides demonstrating successful, noncytotoxic suppression of protein and activity levels of a specific GTPase, controls are essential to establish specificity. In this chapter, we provide methods for selective knockdown of expression by siRNA and confirmation of the effectiveness of Rho GTPase silencing, as well as descriptions and some examples of controls for specificity that include evaluations of dose-response, negative and positive controls, GTPase specificity, confirmation by using more than one siRNA for the same gene, rescue by a mutated siRNA-resistant cDNA encoding the target gene, and complementary supporting evidence. Selective silencing of specific Rho family GTPases should provide increasing insight into the regulatory and functional roles of each isoform in a wide variety of biological processes.