3385_Ch15 - Chapter 15 Tracking IntegrinMediated Adhesion...

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15 © 1999 by CRC Press LLC Chapter Tracking Integrin- Mediated Adhesion Using Green Fluorescent Protein and Flow Cytometry Wendy J. Kivens and Yoji Shimizu Contents I. Overview II. Protocols III. Materials IV. Buffers V. Summary and Future Directions Acknowledgments References I. Overview It is well established that the functional activity of integrin receptors can be regulated by activation of intracellular signaling pathways. This is particularly evident in hematopoietic cells, which undergo cycles of adhesive and non-adhesive states that are dependent on the activation state of the cells. For example, T lymphocytes express β 1 and β 2 integrins on the cell surface, but adhere poorly to relevant cell surface counter-receptors and extracellular matrix ligands. However, T cells can exhibit increases in integrin-mediated adhesion within seconds to minutes following activa-
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© 1999 by CRC Press LLC tion. 1-4 This activation-dependent regulation of integrin-mediated adhesion is critical to both T cell migration and T cell recognition of foreign antigen. 5 A number of activation stimuli have been identified that can regulate integrin adhesiveness on T cells, including: (1) pharmacological agents, such as the phorbol ester PMA and Ca 2+ ionophore; 1,3,6 (2) ligand engagement of the CD3/T cell receptor and other immunoglobulin superfamily member co-receptors; 1,3,6 (3) ligand engagement of G protein-coupled chemokine receptors; 4 (4) activation by integrin-specific antibod- ies; 7 and (5) certain divalent cations, particularly Mn 2+ . 8 A number of technical approaches can be used to analyze the intracellular signaling pathways that regulate integrin activity. One approach is the use of inhib- itors that block the activity of specific signaling pathways. Inhibitors have been used to implicate a number of signaling molecules in activation of integrin function, notably protein kinase C (PKC) and phosphoinositide 3-OH kinase (PI 3-K). 1,9,10 The advantage of inhibitors is their ease of use and applicability to the analysis of integrin activation in many different cell types. However, the investigator assumes a high degree of specificity in the molecular target of the inhibitor, an assumption that is often not warranted as the inhibitory mechanisms are investigated in more detail. One example of this inverse relationship between specificity and degree of use in the field is wortmannin, a fungal metabolite that blocks PI 3-K activity, but has recently been shown to block the activity of other enzymes, particularly when used at concentrations well above the half-maximal inhibitory concentration of wortmannin for PI 3-K. 11,12 Another approach for disrupting signaling pathways is to overexpress wild-type or mutant forms of kinases and adapter proteins in cells, and determine the effects of the expression of these proteins on the functional response of interest. This approach has been particularly illuminating in many signaling studies, due to the identification of mutations that either block the function of an endogenous molecule
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This note was uploaded on 05/06/2010 for the course MECH. 28197 taught by Professor Dr.shafii during the Spring '10 term at Sharif University of Technology.

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3385_Ch15 - Chapter 15 Tracking IntegrinMediated Adhesion...

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