News and Publications

Dynamic Cytoskeletal Interactions in Cell Migration

C.M. Waterman-Storer, T. Wittmann, N. Prigozhina, O. Rodriguez, M. Adams, S. Gupton, R. Littlefield, J. DeRooij, K. Kita

Cell motility is crucial to development, wound healing, and tissue repair. 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 changes in cell shape, 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. In the past year, we improved FSM by increasing the speed and power of our spinning-disc laser confocal microscope system. We used FSM for the first time to study the binding kinetics of microtubule-associated proteins interacting with microtubules in living cells and in microtubules in spindle assembly during regulation by the Ran GTPase. We are collaborating to develop image analysis software to automatically extract quantitative descriptions of protein behavior from FSM images.

Evidence for structural interactions between microtubules and actin comes from our studies of the dynamic interactions between actin and microtubules in vivo during migration of tissue cells and neuronal growth cone guidance. We used time-lapse dual-wavelength FSM of fluorescent actin and microtubules in living, moving cells to show that microtubules and actin indeed bind to each other to affect each other's dynamic organization. We are using a biochemical approach to dissect the molecular mechanism of interactions between microtubules and actin.

In support of the hypothesis that microtubules affect signaling cascades that direct actin dynamics and organization, we previously showed that growth of microtubules regulated the GTPase signaling protein Rac1 to promote actin polymerization, which drives forward cell movement. We recently found that this same Rac1 signaling cascade also promotes growth of microtubules via a feedback mechanism. We showed that this growth promotion occurs via a pathway in which Rac1 interacts directly with and activates the serine/threonine kinase Pak1, which directly phosphorylates and inactivates the microtubule-destabilizing protein Op18/stathmin. The inactivation of Op18/stathmin promotes microtubule growth, which in turn, activates Rac1 and perpetuates a positive feedback loop that regulates microtubules and actin and that is required for cell motility.

PUBLICATIONS

Adams, M.C., Salmon, W.C., Gupton, S.L., Cohan, C.S., Wittmann, T., Prigozhina, N., Waterman-Storer, C.M. A high-speed multispectral spinning disc confocal microscope system for fluorescent speckle microscopy of living cells. Methods, in press.

Bulinski, J.C., Odde, D.J., Howell, B.J., Salmon, E.D., Waterman-Storer, C.M. Rapid dynamics of the microtubule binding of ensconsin in vivo. J. Cell Sci. 114:3885, 2001.

Gupton, S.L., Salmon, W.C., Waterman-Storer, C.M. Converging populations of F-actin promote breakage of associated microtubules to spatially regulate microtubule turnover in migrating cells. Curr Biol. 12:1891, 2002.

Ponti, A., Vallotton, P., Salmon, W.C., Waterman-Storer, C.M., Danuser, G. Computational analysis of F-actin turnover in cortical actin meshworks using fluorescent speckle microscopy. Biophys. J., in press.

Salmon, W.C., Adams, M.C., Waterman-Storer, C.M. Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells. J. Cell Biol. 158:31, 2002.

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. 2002, Unit 4.10.

Waterman-Storer, C.M., Danuser, G. New directions for fluorescent speckle microscopy. Curr. Biol. 12:R633, 2002.

Wittman, T., Waterman-Storer, C.M. Cell motility: can Rho GTPases and microtubules point the way? J. Cell Sci. 114:3795, 2001.

Zhou, F.Q., Waterman-Storer, C.M., Cohan, C.S. Focal loss of actin bundles causes microtubule redistribution and growth cone turning. J. Cell Biol. 157:839, 2002.