Journal of Cell Science 108, 3473-3483 (1995) 3473 Printed in Great Britain © The Company of Biologists Limited 1995 JCS6965 Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: implications for the role of dynamic microtubules in cell locomotion Guojuan Liao1, Takayuki Nagasaki1 and Gregg G. Gundersen1,2,* 1Departments of Anatomy & Cell Biology and 2Pathology, Columbia University, New York, NY 10032, USA *Author for correspondence SUMMARY The role of microtubules (MTs) in cell locomotion is polymer levels by cell extraction and western blotting uncertain: while MTs are not essential for motility of certain yielded results similar to those obtained by quantification of cells, MTs are necessary for the directed translocation of MT fluorescence. A comparison of the locomotion rate mea- large cells such as fibroblasts, endothelial cells and neuronal surements with the MT level measurements indicated that growth cones. Based on previous studies, we hypothesize that over half of the cell locomotion rate could be blocked by cell locomotion may involve MTs in two possible ways: (1) nocodazole without significantly affecting MT levels in the the rate of cell locomotion is proportional to MT level; or (2) cell; the remaining locomotion rate was reduced propor- cell locomotion is not proportional to MT level but requires tionally to MT levels. These results do not support the notion a critical level of MTs to proceed. To test these hypotheses, that a critical level of MTs is required for cell locomotion we measured the rate of locomotion of NRK fibroblasts and suggest that only a portion (<50%) of the speed of the migrating into an in vitro wound, before and after treatment cells is proportional to MT levels. Rather, by analogy with with different concentrations of nocodazole to generate cells studies of MT antagonists on the mitotic spindle, they with different levels of MTs. Locomotion of cells was suggest a third possibility: that low concentrations of noco- monitored directly using timelapse recording and analyzed dazole interfere with MT dynamics and thus, MT dynamics with an Image-1 image analysis program. Addition of noco- are critical for the maximal speed of cell locomotion. This dazole (>50 nM) resulted in a rapid reduction in locomotion notion was further supported by analogous effects of taxol to a new rate that was maintained for >2 hours. We found and vinblastine on cell locomotion: at concentrations that that addition of as little as 100 nM nocodazole decreased the reportedly cause little change in the level of MTs, taxol and rate of locomotion by more than 60%; and that 300 nM vinblastine also dramatically decreased the rate of locomo- nocodazole completely stopped cell locomotion. Although tion of NRK cells. In summary, our results establish the rela- 100 nM nocodazole decreased locomotion over 60%, we tionship between microtubule levels and locomotion rate and detected no qualitative change in MT distribution by suggest that dynamic MTs are rate-limiting for fibroblast immunofluorescence. Quantitative analysis of MT fluores- locomotion. cence in immunofluorescently stained preparations showed that 100 nM nocodazole had no detectable effect on MT levels and that 300 nM nocodazole only decreased MT levels Key words: cell motility, wound healing, cytoskeleton, video to ~40% of controls. Quantitative analysis of tubulin microscopy, timelapse recording, taxol, vinblastine INTRODUCTION trophils (Zigmond et al., 1981), MTs do not seem to be imme- diately necessary for locomotion, since complete breakdown Microtubules (MTs) are major components of the cytoskeleton of MTs does not affect the short term motility of the cells. From that are present in nearly every eukaryotic cell. Previous these studies, it seems unlikely that intact MTs are necessary studies have found that MTs play a role in the directed cell for producing the driving force for motility. Nonetheless, the locomotion of some, but not all cell types. Intact arrays of MTs essential requirement for intact MTs in locomotion of large are clearly necessary for the directed migration of larger cells cells suggests that MTs do play an important role. The corre- such as fibroblasts (Vasiliev et al., 1970; Goldman, 1971; Gail lation between cell size and MT dependence of cell locomo- and Boone, 1971), endothelial cells (Gotlieb et al., 1983), tion (Schliwa and Honer, 1993) suggests that MTs may monocytes (Zakhireh and Malech, 1980), and nerve growth become essential in organizing or contributing to the motile cones (Bamburg et al., 1986). However, in some smaller cells apparatus of the cell as it becomes larger. such as fish keratocytes (Euteneuer and Schliwa, 1986) or neu- Previous studies on how MTs contribute to cell locomotion 3474 G. Liao, T. Nagasaki and G. G. Gundersen were done almost exclusively by MT ‘knock out’ experiments The hypothesis that MTs may be involved in delivering in which complete MT breakdown was induced by high con- membrane components to the leading edge is supported by sub- centrations of MT antagonists. Breakdown of the bulk of the stantial structural data. Yet, despite some effort, there are a MTs results in the loss of polarization of the cell as assessed number of features of this model that have not been demon- by the presence of ruffling around the entire circumference of strated. One concern is that it is not particularly clear that the cell rather than in a localized area (Vasiliev et al., 1970; newly synthesized membrane is necessary for cell locomotion; Goldman, 1971). These results are consistent with the idea that it is possible that membraneous precursors may come from without MTs, the cell is incapable of polarizing its actin recycled or stored sources (Erickson and Trinkaus, 1976). activity and consequently attempts to go in every direction at Also, studies have failed to detect a population of vesicles near once (Vasiliev, 1991). Similar conclusions were drawn from the leading edge that might be candidates for the Golgi-derived experiments using epithelial cells in which the effect of MT insertion vesicles. Other studies designed to measure a antagonists was to cause the misexpression of actin activity (in polarized ‘flow’ of membrane material rearward from the this case microvilli rather than ruffles) on basolateral surfaces leading edge, as would be predicted if components were being of the cell where they are not normally assembled (Achler et inserted selectively at the leading edge, have generally failed al., 1989). At this level, one function of MTs in directed cell to produce evidence that such a flow exists (Holifield et al., locomotion seems to be the establishment and maintenance of 1990; Sheetz et al., 1989; Lee et al., 1990). Finally, additional cell polarity, so that the cell can utilize its actin-based driving ideas need to be added to the polarized membrane insertion force to locomote itself in a unidirectional manner. model to account for the clear effect that MT depolymeriza- The studies described above have explained the role of MTs tion has on polarized actin activity, an activity which is clearly in cell locomotion by emphasizing the depolarizing effect that important for locomotion. depletion of MTs has on actin-based activity. This has been a Is the role of MTs in cell locomotion explained by the idea useful idea for considering the role of MTs in cell locomotion, that MTs serve as tracks for vesicle delivery? Or is this role however, it does not suggest a molecular mechanism by which instead explained by the ability of MTs to establish intracel- MTs contribute to cell motility. It also cannot explain the lular polarity, a function that may require a critical level of ability of MT antagonists to completely block locomotion of MTs? Such questions could be addressed by determining the cells at the edge of a wound (see Gotlieb et al., 1983; also this relationship between MT levels and the rate of cell locomo- study). In such cells, actin activity is naturally constrained to tion. If there is a positive correlation between the MT level the portion of the cell not in contact with surrounding cells. If and the rate of cell locomotion, it would suggest that MTs play MT antagonists redistribute the actin activity that is polarized a direct role in determining the migration rate. Such a role at the front of such cells, it should lead to a reduction in the would be consistent with the notion that MTs serve in the speed of cell locomotion but not a complete inhibition, since transport of rate-limiting materials to the leading edge. An some actin activity would still be found at the front of the cells. alternative hypothesis is that there is a particular level of MTs Indeed, measurements of the effect of MT antagonists on the above which, but not below which, cells can migrate. This membrane ruffling of wound-edge fibroblasts have shown that ‘critical level’ of MTs may be important for the gross organ- ruffling is decreased 4-6 fold over that in controls, but it is not ization and/or maintenance of cellular elements or organelles reduced to zero (Bershadsky et al., 1991). necessary for the locomotion of a cell. For example, this might Other explanations for MT function in cell locomotion include polarizing actin activity (Vasiliev, 1991) or clustering suggest that MTs may polarize the delivery of membrane of the Golgi apparatus so that a functional polarity is main- proteins to the leading edge. This hypothesis is supported by tained (Kreis, 1990; Singer and Kupfer, 1986). numerous studies which have shown that locomoting cells In this study, we have determined the relationship between have a propensity for reorienting their microtubule organizing cellular MT levels and the rate of fibroblast locomotion. To center (MTOC) to a position between the nucleus and the generate cells with varying levels of MTs, we treated wounded leading edge of the cell (Gotlieb et al., 1981, 1983; Kupfer et monolayers of NRK fibroblasts with nocodazole, a MT depoly- al., 1982; Albrecht-Buehler and Bushnell, 1979; Gundersen merizing agent, over a range of concentrations.