Integrin ␣ 4 ␤ 1 is a receptor for vascular cell adhesion molecule-1 and fibronectin. It is important in lymphopoiesis, inflammatory recruitment of leukocytes, and other situations that require cell adhesion to the vascular endothelium. The avidity of the cells expressing ␣ 4 ␤ 1 integrin can be rapidly changed by chemokines and chemoattractants. Different mechanisms, including changes in the number of interacting molecules due to the alteration of the receptor topology or changes in the affinity of the individual bonds, have been proposed to explain the nature of these fast changes in avidity. Recently, we described a fluorescent LDV-containing small molecule, which we used to monitor the affinity changes on live cells in real time (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S. et al. (2001) J. Biol. Chem. 276, 48670 -48678). Here we show that the affinity of the small molecule probe as well as the native ligand vascular cell adhesion molecule-1 varies in parallel when the integrin is modulated with divalent cations and that the affinity modulation leads to the changes in cell avidity. Using formyl peptide receptor-transfected U937 cells, we further show that the time course of avidity changes in response to the receptor activation coincides with the time course of the affinity changes. Taken together, these data are consistent with the idea that affinity regulation is a major factor that governs the avidity of cell adhesion mediated by the ␣ 4 integrin.Cell adhesion to the vascular endothelium is important for inflammation, hemostasis, cancer cell metastasis, and hostparasite interaction. Integrins and their counterstructures, the cell adhesion molecules (VCAM-1 and ICAMs), 1 together with selectins and their counterstructures (cellular mucins) determine the cell adhesive properties in these processes (1). The adhesion between endothelial cells and leukocytes is regulated by changes in the number of interacting adhesion molecules due to the difference in the expression level, molecular trafficking, and/or internalization; changes in topographical distribution due to clustering, dimerization, and other forms of molecular assembly; and changes in affinity of the individual molecules to their counterstructures (2-7). ␣ 4 ␤ 1 integrin (very late antigen-4 (VLA-4), CD49d/CD29) is one of the integrins that can mediate initial capture, rolling, and firm cell attachment to the endothelial cells (8, 9). VLA-4 is expressed on several classes of blood cells. It mediates binding to the CS-1 domain of fibronectin and to the vascular cell adhesion molecule 1 (VCAM-1), an immunoglobulin superfamily member induced by cytokines on endothelium (10, 11). ␣ 4 ␤ 1 -Integrin adhesive properties can be modulated by cytokines and chemokines, but the mechanism controlling the regulation of integrin avidity is poorly understood. In particular, it has been suggested that the changes in VLA-4-dependent adhesion are due to either th...

Rapid activation of integrins in response to chemokine-induced signaling serves as a basis for leukocyte arrest on inflamed endothelium. Current models of integrin activation include increased affinity for ligand, molecular extension, and others. In this study, using real-time fluorescence resonance energy transfer to assess α4β1 integrin conformational unbending and fluorescent ligand binding to assess affinity, we report at least four receptor states with independent regulation of affinity and unbending. Moreover, kinetic analysis of chemokine-induced integrin conformational unbending and ligand-binding affinity revealed conditions under which the affinity change was transient whereas the unbending was sustained. In a VLA-4/VCAM-1-specific myeloid cell adhesion model system, changes in the affinity of the VLA-4-binding pocket were reflected in rapid cell aggregation and disaggregation. However, the initial rate of cell aggregation increased 9-fold upon activation, of which only 2.5-fold was attributable to the increased affinity of the binding pocket. These data show that independent regulation of affinity and conformational unbending represents a novel and fundamental mechanism for regulation of integrin-dependent adhesion in which the increased affinity appears to account primarily for the increasing lifetime of the α4β1 integrin/VCAM-1 bond, whereas the unbending accounts for the increased capture efficiency.

The primary cilium is a ubiquitous organelle present on most mammalian cells. Malfunction of the organelle has been associated with various pathological disorders, many of which lead to cystic disorders in liver, pancreas, and kidney. Primary cilia have in kidney epithelial cells been observed to generate intracellular calcium in response to fluid flow, and disruption of proteins involved in this calcium signaling lead to autosomal dominant polycystic kidney disease, implying a direct connection between calcium signaling and cyst formation. It has also been shown that there is a significant lag between the onset of flow and initiation of the calcium signal. The present study focuses on the mechanics of cilium bending and the resulting calcium signal. Visualization of real-time cilium movements in response to different types of applied flow showed that the bending is fast compared with the initiation of calcium increase. Mathematical modeling of cilium and surrounding membrane was performed to deduce the relation between bending and membrane stress. The results showed a delay in stress buildup that was similar to the delay in calcium signal. Our results thus indicate that the delay in calcium response upon cilia bending is caused by mechanical properties of the cell membrane.