Biomedical subjects
Publications and source records attributed to Sharona Even-Ram.
A key challenge in stem cell research is to learn how to direct the differentiation of stem cells toward specific fates. In this issue of Cell, Engler et al. (2006) identify a new factor regulating stem cell fate: the elasticity of the matrix microenvironment. By changing the stiffness of the substrate, human mesenchymal stem cells could be directed along neuronal, muscle, or bone lineages.
Thymosin beta4 is a 43-amino acid actin-binding protein that promotes cell migration and is important in angiogenesis, wound healing, and tumor metastasis. We searched for genes upregulated by thymosin beta4 and identified zyxin as increased in SiHa cells in the presence of exogenously added thymosin beta4 and when thymosin beta4 is overexpressed using adenoviral vectors. Both zyxin and thymosin beta4 show increased localization in the nucleus. We conclude that thymosin beta4 may exert some of its migration promoting activity via increased zyxin expression.
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.
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.
The ability of cells to migrate within the extracellular matrix and to remodel it depends as much on the physical and biochemical characteristics of a particular matrix as on cellular properties. Analyzing the different modes of migration of cells in matrices, and how cells switch between these modes, is vital for understanding a variety of physiological and pathological processes. Recent work provides new insights, but also raises some debates about the mechanisms and regulation of cell migration in three-dimensional matrices.
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.
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