PROCEEDINGS |
The cytoskeleton is important for cell structure and cellular activities such as polarization, differentiation, locomotion and division. We studied the dynamics of cytoskeletal structures in motile fibroblasts by imaging cells expressing green fluorescent protein (GFP)-chimeras that label adhesion plaques (APs), and intermediate filaments (IFs). To study the assembly and turnover of Aps we prepared stable 3T3 cell lines expressing a GFP-beta1 integrin chimera in which GFP was fused to the transmembrane and cytoplasmic portions of beta1 integrin. The GFP-beta1 integrin labeled all the APs in stable cell lines and the GFP was exposed on the cell exterior. The cell lines had normal morphology, adhesion, locomotion and growth. Surprisingly, the APs in stationary cells were highly motile, moving linearly for many plaque lengths from the cell periphery toward the cell center. In contrast, almost all plaques in migrating cells were stationary and only moved when in the tail of the cell. The AP movement appeared to be driven by actin. In cells microinjected with rhodamine-actin, contracting actin filaments were associated with the motile APs, and the myosin inhibitor, BDM, inhibited AP movement. By interference reflection microscopy we also observed moving Aps in parental 3T3 cells. Thus the adherence of APs to the extracellular matrix or the tension exerted on APs by actin filaments may be regulated differently in stationary and migrating cells. To study the dynamics of IFs, we prepared stable 3T3 cell lines expressing a chimera of GFP fused to the N-terminus of vimentin. The GFP-vimentin labeled all of the endogenous IFs and revealed that the bulk of the IFs underwent wavy motions. In migrating cells, the ends of IFs extended out toward the cell periphery, apparently as intact filaments. Fragments of IFs were observed moving toward the cell periphery along linear courses. These IF fragments might be intermediates of IF turnover, since we observed the apparent joining of the fragments into a filamentous IF bundle and the formation of smaller fragments from a larger IF segment. Nocodazole rapidly inhibited the extension of the filaments and the movements of the fragments, indicating that these movements are dependent on microtubules. The IFs were observed to move toward the nucleus as intact filaments after microinjection of antibodies to Ifs. This shows that the redistribution does not involve the assembly-disassembly of IFs. These results are consistent with the idea that microtubules play a critical role in IF extension and distribution.