News and Publications

Microscopes and Motility: Dynamic Cytoskeletal Interactions in Cell Migration

* State University of New York, Buffalo, New York

Cell motility is crucial to development, wound healing, and the immune response. In cancer cells, loss of regulation of cell-cell adhesion and cell motility results in deadly metastasis. The locomotion of vertebrate tissue cells is thought to require 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 motility, and microtubules may localize signaling molecules to spatially regulate actin dynamics during locomotion. We use quantitative microscopy of the cytoskeletal systems in living cells and in vitro biochemistry to test these hypotheses.

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 neurons. We used time-lapse multispectral fluorescent speckle microscopy (FSM) of fluorescent actin and microtubules in living, moving cells to show that microtubules and actin indeed bind to each other to promote a mutually dependent dynamic organization of the two systems. In tissue cells, we found that spatially modulated contraction of the cortical actin meshwork promotes the turnover of microtubule polymers in specific cellular regions, whereas in neurons, microtubule interactions with filopodial actin bundles direct growth cones toward chemoattractants. We are using a biochemical approach to dissect the molecular mechanism of these structural microtubule-actin interactions.

In support of the hypothesis that microtubules affect signaling cascades that direct actin dynamics and organization, we previously showed that growth of microtubules activated 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 (Fig. 1). We showed that 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 in the leading edge of migrating cells and that may be required for cell motility. We are exploring the molecular mechanism by which microtubule growth activates Rac1.

To aid our studies of cytoskeletal dynamics, we pioneered FSM, a powerful method that allows quantitative analysis of the dynamics of macromolecular assemblies in living cells. In the past year, we enhanced this technique by adding multispectral capabilities, allowing the first-ever simultaneous kinetic analysis of microtubules and actin in living, migrating cells. Via our collaboration with G. Danuser, Department of Cell Biology, we made huge strides in our ability to extract molecular kinetic measurements from in vivo FSM images by using computer vision image analysis algorithms.

Using our image analysis software, we can obtain maps of cytoskeleton transport and rates of assembly and disassembly at high spatial and temporal resolutions. We verified the sensitivity of the software to artifacts of FSM imaging such as focus drifts and fluorescence photobleaching.

Our analysis software will allow FSM to become the tool of choice for deciphering the link between cytoskeletal dynamics and cell behavior and will serve as a quantitative readout in molecular, chemical, or genetic interventions that affect dynamic, morphogenic cell processes. We are expanding FSM technology for application to other macromolecular structures besides the cytoskeleton and are combining FSM technology with total internal reflection fluorescence microscopy for quantitative analysis of molecular kinetics within structures such as focal adhesions during 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-disk confocal microscope system for fluorescent speckle microscopy of living cells. Methods 29:29, 2003.

Danuser, G., Waterman-Storer, C.M. Fluorescent speckle microscopy: where it came from and where it is going. J. Microsc., in press.

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. 84:3336, 2003.

Rodriguez, O., Schaefer, A., Mandato, C.M., Forscher, P.M., Bement, W.M., Waterman-Storer, C.M. Dynamic microtubule-actin interactions: conserved mechanisms underlying directed cell movement and polarized morphogenesis. Nat. Cell Biol., in press.

Vallotton, P., Salmon, E.D., Waterman-Storer, C.M., Danuser G. Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study. Biophys. J., in press.

Wittmann, T., Bokoch, G.M., Waterman-Storer, C.M. Regulation of leading edge microtubule and actin dynamics downstream of Rac1. J. Cell Biol. 161:845, 2003.

Wittmann, T., Littlefield, R., Waterman-Storer, C.M. Fluorescent speckle microscopy of cytoskeletal dynamics in living cells. In: Live Cell Imaging: A Laboratory Manual. Spector, D.L., Goldman, R.D. (Eds.). Cold Spring Harbor Press, Cold Spring Harbor, NY, in press.

Wittmann, T., Waterman-Storer, C.M. Regulation of microtubule dynamics in migrating cells: a new role for Rho GTPases. In: Cell Motility. Ridley, A., Peckham, M., Clark, P. (Eds.). Wiley & Sons, New York, in press.