Our laboratory is interested in two basic problems in cell biology. The first concerns the fundamental microstructure of membranes, what factors determine the lateral mobility of membrane proteins and lipids, and how such mobility is related to the functions that membranes carry out. To investigate this problem we use a combination of video microscopic and molecular biology techniques. By tracking the individual movements of single lipids tagged with 30 nm gold particles, we have found that the two-dimensional Brownian motion of lipids in the plane of the membrane can be directly observed by video microscopy. This technology, as applied to membrane proteins, reveals subtle features of the lateral organization and dynamics of individual membrane proteins. For example, the current idea that microdomains, termed lipid rafts, exist in the plane of the membrane is both attractive and controversial. Attractive because such domains could have important functional significance such as being hot spots for signal transduction. Controversial because the concept is derived from biochemical extraction data and the in vivo correlate of these procedures is not known. Our current interest is to use microscopy techniques, including the laser tweezers, to determine whether rafts exist in the membranes of living cells and to determine their size and dynamics. We have found that slightly cross-linked raft resident proteins can be found in 'transient confinement zones' about 100 nm in dimension that fit the operational definition of rafts. More robust cross-linking connects the cluster to the cytoskeleton underlying the membrane at sites from which signal transduction may occur. We have also found that raft-like domains can be reconstituted in various lipid bilayer model membranes that remarkably recapitulate the properties hypothesized for lipid rafts. Papers describing or reviewing this new technology and research have been published in Science, Biochemistry, Biophys. J., Current Opinion in Cell Biology, Trends in Cell Biol. and Proc. Natl. Acad. Sci. On going current research utilizes quantum dots, fluorescence correlation microscopy and pattern recognition to study domain organization in the living cell membraneWe are now also augmenting the laser tweezers with a technique we term lateral magnetophoresis. This technique will enable differences in lateral mobility to be discerned in different regions of the surface of a single cell.

The second area of research is the problem of how cells move. This research is relevant, for example, to the aberrant cell motility exhibited in metastasis and to transendothelial cell migration involved in aspects of the inflammatory response. One major intellectual challenge is to relate global descriptions of cell movement and force production to molecular mechanisms. We have completed a kinematic description accounting for how locomoting fish scale keratocytes maintain constant shape and speed. This model accounts for not only dynamic morphological behavior but also the behavior of the cytoskeletal meshwork and cell surface receptors. And we have developed the first quantitative assay for the strength and pattern of the traction forces exerted by moving cells. Our current work involves locally perturbing cell locomotion using single cell photomanipulative techniques including chromophore assisted laser inactivation [CALI] to knock out cell adhesion molecules and laser-mediated photoactivation to quickly increase the concentration of proteins that regulate the cytoskeleton. Work has recently been published demonstrating that local photorelease of caged-thymosin beta 4, a G-actin sequestering protein, near one margin of the cell causes specific turning of that cell. Theoretical models of cell migration are now being constructed by Alex Mogilner and their validity will gauged on how well they can describe the results of these local perturbations. In the course of these investigations, we have studied the role of calcium in regulating cell contractility, adhesion and traction force development and the role of paxillin phosphorylation in the adhesion turnover that must accompany rapidly moving cells. Papers describing these studies have been published in Nature, J. Cell Biol., Cell Motility and the Cytoskeleton and Biophys. J. On going work is aimed at using fluorescent biosensors, in collaboration with Klaus Hahn, to understand the pathways by which mechanochemical signal transduction occurs to allow the cell to sense and respond to forces as well as produce them.

Huang, C., Jacobson, K., and Schaller, M. MAP kinases and cell migration. J. Cell Science. 117:4619-4628 (2004).

Huang, C., Borchers, C.H., Schaller, M. and Jacobson, K. “Phosphorylation of paxillin by p38MARK is involved in the neurite extention of PC-12 cells”, J. Cell Biol., 164:593-602, (2004).

P. Roy, K. Jacobson. “Overexpression of profilin reduces the migration of invasive breast cancer cells”. Cell Motility and the Cytoskeleton, 57:84-95 (2004).

Pomorski, P., Watson, J.M., Haskill, S. and Jacobson, K. “How Adhesion, Migration, and Cytoplasmic Calcium Transients In.uence Interleukin-1_mRNA Stabilization in Human Monocytes”, Cell Motility and the Cytoskeleton 57:143–157 (2004).

Huang, C., Rajfur, Z., Borchers, D., Schaller, M. and Jacobson, K. “JNK phosphorylates paxillin and regulates cell migration". Nature, 424:219 - 223 (2003).

Khan, T.K., Yang, B., Thompson, N.L., Maekawa, S., Epand, R.M., and Jacobson, K. “Binding of NAP-22, a Calmodulin-Binding Neuronal Protein, to Raft-like Domains in Model Membranes”. Biochemistry. 42:4780-4786 (2003).

Anderson, R. and Jacobson, K. “ A Role for Lipid Shells in Targeting Proteins to Caveolae, Rafts and Other Lipid Domains”, Science, 296: 1821-1825 (2002).

Dietrich, C., Yang, B., Fujiwara, T., Kusumi, A. and Jacobson, K. "The relationship of lipid rafts to transient confinement zones detected by single particle tracking." Biophys. J, 82: 274-284 (2002).

Rajfur, Z., Roy, P., Romer, L., Otey, C. and Jacobson, K. "Dissecting the link between stress fibers and focal adhesions by CALI with EGFP fusion proteins", Nature Cell Biol., 4: 286-293 (2002).

Dietrich, C., Bagatolli, L., Volovyk, Z., Gratton, E., Thompson,N., Levi, M., Jacobson, K., and Gratton, E. 'Lipid Rafts' Reconstituted in Model Membranes." Biophys. J., 80: 1417-1428 (2001).

Roy, P., Rajfur, Z., Jones, D., Marriott, G., Loew, L. and Jacobson, K. "Local photorelease of caged thyomsin beta 4 in locomoting keratocytes causes cell turning" J. Cell Biol., 153: 1035-1047 (2001).

Jacobson, K and Dietrich, C. "Looking at lipid rafts" Trends in Cell Biol. , 9:87-91 (1999).

Oliver, T., Dembo, M. and Jacobson, K. "Separation of Propulsive and Adhesive Traction Stresses in Locomoting Keratocytes", J. Cell Biol.,145: 589-604 (1999).

Lee, J., Ishihara, A., Oxford, G. Johnson, B. and Jacobson, K "The regulation of cell movement is mediated by stretch-activated calcium channels", Nature, 400: 382-386 (1999).