We have found that motility of human glioblastoma cells proceeds in the absence of intact actin polymers. Suppression of actin assembly with DCB or LatA at concentrations sufficient for complete actin disassembly and more than sufficient to block cell cleavage is permissive for extension of cell protrusions and for sustained motility. In contrast, motility ceases when cells are exposed to the microtubule inhibitors NOC and VBL at minimal concentrations required for mitotic arrest.
To our knowledge, the present work is the first demonstration that interphase cell migration can function independent of actin polymers. These results are clearly divergent from the standard model of cell motility, in which actin-dependent extensions of lamellipodia are the principal means of cell motility, whereas microtubules orient the cell toward a chemotactic gradient (Pollard and Borisy, 2003
Although microtubules have not been demonstrated previously to move cells independent of actin, previous work has shown that microtubules are important to cell shape changes and to various aspects of cell motility. For example, microtubules generate protrusions in astrocytes (Etienne-Manneville and Hall, 2001
) and move the nuclei of neurons following actin-dependent leading-edge migration (Lambert de Rouvroit and Goffinet, 2001
Gerashchenko et al., 2009
Moore et al., 2009
). Microtubules are also involved in movement of the cell body toward the lamellipodial leading edge in melanoma cells (Ballestrem et al., 2000
), and they have been demonstrated to create cell surface protrusions in MAP2-transfected hepatoma cells following actin disassembly (Edson et al., 1993
). Microtubules also are involved in creating cell surface deformations in neural cells and thus are implicated in creating neural extensions (Kaech et al., 1996
Witte et al., 2008
). Furthermore, preexisting neurite extensions retract in the presence of microtubule inhibitors (Bray et al., 1978
) and have been reported to form in the presence of cytochalasin (Bradke and Dotti, 1999
). Thus, microtubules are clearly involved in the motility of intracellular elements in disparate systems. However, the apparent role for microtubules in all aspects of cell motility in glioblastoma in the absence of actin polymer as suggested by our results, is unprecedented. It will be of great interest to determine its mechanism. If microtubules function in this manner, they may do so by interacting with a unique set of proteins. One candidate is the gap junction protein connexin43, a microtubule-anchoring protein (Giepmans et al., 2001
) implicated in control of astrocyte motility (Olk et al., 2010
). Although it is clear that cells move in an actin-independent manner when exposed to actin assembly inhibitors, it remains to be unequivocally established that glioblastoma cells move in an actin-independent manner when actin polymer is present. Given that Rho GTPase requirements are unusual, we anticipate this may be the case.
The response of U87MG cells to Rho family GTPase dominant mutants also appears to vary substantially from the response of other cell types. In primary fibroblasts dominant negative Rac and Cdc42 suppress motility (Nobes and Hall, 1999
). In accord with the unique mode of motility in glioblastoma cells, we found, strikingly, that dominant negative Rac does not affect motility and dominant negative Cdc42 has only a modest effect ().
Microtubule disassembly by NOC has been shown to activate RhoA, increasing myosin contractility (Chang et al., 2008
) and inducing surface blebbing instead of lamellipodia (Takesono et al., 2010
), in part through release of GEF-H1 (Chang et al., 2008
). We have observed that NOC induces blebbing, which is reversed by blebbistatin (Supplemental Video 8). However, failure of dominant negative GEF-H1 Y393A to restore motility in NOC-treated cells () suggests that RhoA hyperactivation through release of GEF-H1 from disassembled microtubules (Krendel et al., 2002
) is not a likely explanation of the failure of U87MG cells to move in NOC. Furthermore, we found that EGFP-dynamitin, which effectively suppresses dynein motor function (Etienne-Manneville and Hall, 2001
), also significantly decreased U87MG motility. These results are consistent with an interpretation that motility is directly dependent on dynamic microtubules even when actin polymer is present. However, other interpretations are possible, and further work is required to resolve this issue.
We found that the constitutively active Rac1 Q61L mutant completely suppresses U87MG motility with or without suppression of actin assembly. However, locking Rac1 in a GTP-bound state can have effects at multiple levels in cells and could thus suppress motility through other mechanisms. We therefore tested the more physiological Rac1 mutant F28L, which is spontaneously activated and rapidly cycling and is capable of transforming cells (Lin et al., 1999
). We found the F28L mutant also suppressed motility in U87MG cells. As we have also found a lack of effect of dominant negative Rac1 on U87MG motility, taken together, our results demonstrate that U87MG motility requires little or no Rac1 activity and is actually suppressed by elevated Rac1 activity. Overall, our data are consistent with the lack of a major role for Rho GTPases in glioblastoma motility, with the possible exception of a small but statistically significant effect of Cdc42.
In summary, we have established that complex subcellular systems have the capacity to integrate to enable cell motility in a manner that is independent of assembled actin. Our results further suggest that this motility depends on microtubules. In cells where leading-edge protrusion depends on actin assembly, actin polymerization has been regarded as the motor that converts chemical energy to mechanical energy to drive the cell forward (Theriot, 2000
). In the case that microtubules may drive glioblastoma cells forward, one might consider that microtubule dynamics possesses the same capacity for force generation as actin to create motility (Margolis and Wilson, 1981
Inoue and Salmon, 1995
It is unlikely that such complex actin-independent behavior would have arisen solely in one particular cell type. This opens the question of whether other cells may use an actin-independent motility mechanism similar to that uncovered here. It is further important to note that the actin-independent motility of glioblastoma cells reported here could have implications for tumor therapy, in that it may offer unique targets to suppress aggressive glioblastoma infiltration within the brain parenchyma.