News and Publications

Dynamic Cytoskeletal Interactions in Cell Motility and Cell-Cell Adhesion

C.M. Waterman-Storer, T. Wittmann, N. Prigozhina, W.C. Salmon, J. Lam

Cell motility is crucial to development, wound healing, and tissue repair. The loss of regulation of cell-cell adhesion and motility, or metastasis, in cancer cells is a major cause of death due to cancer. The locomotion of vertebrate tissue cells requires complex and dynamic interactions between the microtubule and actin cytoskeletal polymers. We hypothesize that these interactions are both structural and regulatory: microtubules and F-actin may physically interact to promote cell shape change, and microtubules may localize signaling molecules to spatially regulate actin. We use microscopy of living cells and in vitro biochemistry to test this hypothesis.

To aid our studies of cytoskeletal dynamics, we pioneered a powerful method called fluorescent speckle microscopy (FSM), which allows visualization of the dynamics of macromolecular assemblies in vivo. When a few fluorescently labeled proteins are coassembled with many unlabeled proteins into a macromolecular structure such as the cytoskeleton, a pattern of "speckled" fluorescence results that can be viewed with a high-resolution microscope and recorded with a digital camera. Changes in the speckle pattern over time indicate the assembly or disassembly, movement, and turnover of the structure.

In the past year, we improved FSM by adding multiwavelength capability to follow multiple proteins and by combining FSM with real-time spinning disc laser confocal microscopy to improve clarity and spatial resolution. We are collaborating to develop image analysis software to easily extract quantitative descriptions of protein behavior from FSM images.

Evidence for structural interactions between microtubules and actin comes from our recent studies of the dynamic interactions between actin and microtubules both in vitro and in vivo. We used time-lapse dual-wavelength FSM of fluorescently labeled actin and microtubules in cytoplasmic extracts to show that microtubules and actin bind to each another and generate force against each other to affect each other's dynamic organization. We are using this approach with living, moving cells and will also dissect the molecular mechanism of microtubule-actin interaction by using a biochemical approach.

We are also characterizing the differences in microtubule and F-actin dynamics between motile cells and immotile cells within tissues. In motile cells, microtubules constantly remodel, whereas in immotile cells, these dynamics are inhibited. Similarly, actin in motile cells is continuously polymerized along the cell edge facing the direction of migration, whereas in nonmotile cells, this polarized actin assembly is shut down. We will expand these studies to observe microtubule and actin and their regulators as motile cells collide during wound healing.

PUBLICATIONS

Bulinski, J.C., Odde, D.J., Howell, B., Salmon, E.D., Waterman-Storer, C.M. Rapid dynamics of ensconsin (E-MAP-115)-microtubule binding in vivo. J. Cell Sci., in press.

Nachury, M.V., Maresca, T.J., Salmon, W.C., Waterman-Storer, C.M., Heald, R., Weis, K. Importin ß is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104:95, 2001.

Waterman-Storer, C., Duey, D.Y., Weber, K.L., Keech, J., Cheney, R.E., Salmon, E.D., Bement, W.M. Microtubules remodel actomyosin networks in Xenopus egg extracts via two mechanisms of F-actin transport. J. Cell Biol. 150:361, 2000.

Waterman-Storer, C.M. Fluorescent speckle microscopy (FSM) of microtubules and actin in living cells. In: Current Protocols in Cell Biology. Bonifacino, J.S., et al. (Eds.). Wiley & Sons, New York, in press.

Waterman-Storer, C.M., Salmon, W.C., Salmon, E.D. Feedback interactions between cell-cell adherens junctions and cytoskeletal dynamics in newt lung epithelial cells. Mol. Biol. Cell 11:2471, 2000.

Wittman, T., Waterman-Storer, C.M. Interactions of microtubules, actin, and signal transduction in cell motility. J. Cell Sci., in press.

Waterman-Storer Website