to integrate comes from in vitro studies, mainly concerning movement across two-dimensional substrata. We *Center for Biomedical Engineering and Department of Chemical Engineering nonetheless believe that much of the mechanistic understanding is relevant and useful for in vivo situations even Massachusetts Institute of Technology Cambridge, Massachusetts 02139 in three dimensions. It is likely that cells interact with their surroundings by means of the same types of recep- † Department of Cell and Structural Biology University of Illinois at Urbana-Champaign tors in vivo as in vitro, and that physical interactions of cells with their environment play important roles in Urbana, Illinois 61801 regulating function in both cases. We further attempt to suggest generalizations across Perspective a wide spectrum of migratory cell types, including amoe-Cell migration plays a central role in a wide variety of bae, leukocytes, fibroblasts, and neurons, looking for biological phenomena. In embryogenesis, cellular mibroad similarities among physical mechanisms. Obsergrations are a recurring theme in important morphogenic vations of various cells demonstrating rapid, slow, or processes ranging from gastrulation to development of negligible locomotion on particular substrata may be

Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesions (adhesion turnover). Adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. Despite the importance of these processes in migration, the mechanisms that regulate adhesion formation and disassembly remain largely unknown. Here we develop quantitative assays to measure the rate of incorporation of molecules into adhesions and the departure of these proteins from adhesions. Using these assays, we show that kinases and adaptor molecules, including focal adhesion kinase (FAK), Src, p130CAS, paxillin, extracellular signal-regulated kinase (ERK) and myosin light-chain kinase (MLCK) are critical for adhesion turnover at the cell front, a process central to migration.

Migration of cells in higher organisms is mediated by adhesion receptors, such as integrins, that link the cell to extracellular-matrix ligands, transmitting forces and signals necessary for locomotion. Whether cells will migrate or not on a given substratum, and also their speed, depends on several variables related to integrin-ligand interactions, including ligand levels, integrin levels, and integrin-ligand binding affinities. These and other factors affect the way molecular systems integrate to effect and regulate cell migration. Here we show that changes in cell migration speed resulting from three separate variables-substratum ligand level, cell integrin expression level, and integrin-ligand binding affinity-are all quantitatively predictable through the changes they cause in a single unifying parameter: short-term cell-substratum adhesion strength. This finding is consistent with predictions of a mathematical model for cell migration. The ligand concentration promoting maximum migration speed decreases reciprocally as integrin expression increases. Increases in integrin-ligand affinity similarly result in maximal migration at reciprocally lower ligand concentrations. The maximum speed attainable, however, remains unchanged as ligand concentration, integrin expression, or integrin-ligand affinity vary, suggesting that integrin coupling with intracellular motors remains unaltered.

Cell migration is an integrated process that requires the continuous, coordinated formation and disassembly of adhesions. These processes are complex and require a regulated interaction of numerous molecules, and the activation of specific signalling pathways. Even though understanding these processes is challenging, important insights are beginning to emerge, and the technology to facilitate significant advances in this area is now in place.

We describe a technique based on moment-analysis for the measurement of the average number of molecules and brightness in each pixel in fluorescence microscopy images. The average brightness of the particle is obtained from the ratio of the variance to the average intensity at each pixel. To obtain the average number of fluctuating particles, we divide the average intensity at one pixel by the brightness. This analysis can be used in a wide range of concentrations. In cells, the intensity at any given pixel may be due to bright immobile structures, dim fast diffusing particles, and to autofluorescence or scattering. The total variance is given by the variance of each of the above components in addition to the variance due to detector noise. Assuming that all sources of variance are independent, the total variance is the sum of the variances of the individual components. The variance due to the particles fluctuating in the observation volume is proportional to the square of the particle brightness while the variance of the immobile fraction, the autofluorescence, scattering, and that of the detector is proportional to the intensity of these components. Only the fluctuations that depend on the square of the brightness (the mobile particles) will have a ratio of the variance to the intensity >1. Furthermore, changing the fluorescence intensity by increasing the illumination power, distinguishes between these possible contributions. We show maps of molecular brightness and number of cell migration proteins obtained using a two-photon scanning microscope operating with a photon-counting detector. These brightness maps reveal binding dynamics at the focal adhesions with pixel resolution and provide a picture of the binding and unbinding process in which dim molecules attach to the adhesions or large molecular aggregates dissociate from adhesion.