We hypothesized that a MAP interaction network may change state when cells alternate between interphase and mitosis, resulting in different MT dynamics. To test this hypothesis, we developed VIP and used it in Xenopus
egg extracts in which the cytoplasm is unperturbed [26
]. Such extracts also support the assembly of nuclei as well as spindles around added DNA in a cell cycle–dependent manner [27
Here, we have shown that VIP allows the visualization of interactions “in extract” that have been reported to occur by classical IPs. This, together with other controls involving FRET techniques, validates the VIP method. However, VIP has some limitations. The epitopes of some molecules may not be accessible to the antibodies used. It is therefore desirable to check interactions with several antibodies and to include positive controls in the assay whenever possible. VIP does not tell whether the interaction is direct or indirect, and it requires purified recombinant proteins and specific antibodies, as well as fluorescently labeled proteins. However, unlike FRET assays, VIP does not require that both interaction partners be recombinant, fluorescently labeled proteins. Furthermore, multiple interaction pairs can be visualized simultaneously in the same image. The use of VIP can easily be extended to the study of spatial regulation of interaction networks, since interaction signals are stored together with bead coordinates. Most importantly, measurements are made in situ, so that molecular interactions can be monitored in extracts shifting between cell-cycle states.
Using this technique, we have revealed a systematic change in the configuration of MAPs interactions between interphase and mitosis. Most strikingly, a major MT destabilizer, XKCM1, was found to interact with EB1, XMAP215, APC, and CLIP170 in interphase, interactions that are all lost or considerably reduced in the metaphase extract. This change in interactions may be caused by still-unknown phosphorylation events. By contrast, we found a robust interaction between XMAP215 and EB1 in metaphase that could not be detected in the interphase cytoplasm. This is probably also due to phosphorylation changes since this interaction cannot be revealed by classical IP (D), which suggests that the phosphorylation state of these molecules is dynamic and lost quickly when the extract is diluted. An interesting future challenge will be to determine what kind of phosphorylation events are required to induce the switch in the overall MAP interaction pattern.
Because VIP does not allow the determination of whether the interactions detected are direct, it is likely that the network of protein interactions involved in the regulation of MT dynamics is larger than the one we have revealed. In effect, the action of the C-EB1 on MT dynamics in metaphase (A) may be the result, not only of an inhibition of the XMAP215-EB1 interaction, (A, inset), but also of a more complex change in a series of interactions between partners of the network.
We have provided experimental results in support of the idea that there is a causal relationship between the two different states of this small network of interactions and the corresponding state of MT dynamics. In this context, it is interesting that XMAP215 interacts with tubulin both in interphase and mitosis. This suggests that in extracts, MT growth occurs by incorporation of tubulin-XMAP215 complexes. XMAP215 is supposed to stabilize tubulin oligomers and may lead to the incorporation of short protofilament segments into MTs, which would explain its effect on the MT growth rate [28
]. In connection with this and in relation to the regulation of MT dynamics between interphase and mitosis, it is interesting to note that in interphase, XMAP215 interacts with XKCM1, which is surrounded by EB1, CLIP170, and APC, whereas in mitosis, it does not interact with XKCM1, but rather with EB1. If we assume that XMAP215 is incorporated into MTs together with tubulin subunits, one could imagine that these other molecules come along as well. In interphase, XKCM1 catastrophe activity seems to be buffered out through its interactions with the other MAPs. It may be incorporated in the growing MT in an inactive form, whereas in metaphase it may be free to act directly at the tip of MTs or diffuse passively along the MT lattice toward the plus end to induce catastrophes [29
]. In mitosis, the tubulin-XMAP215-EB1 complex seems important to determine the exact MT dynamics of the mitotic state because removal of EB1 eliminates all MT growth. It is not surprising that an overexpression of XMAP215 overcomes EB1 depletion if we assume that the tubulin-XMAP215 complex is the unit that is incorporated into forming MTs. Indeed, by increasing the concentration of this complex, one increases the concentration of subunits available for MT assembly, thereby favoring it. Another interesting outcome of this study concerns the function of APC and EB1. It has been suggested on the basis of in vitro results that these two molecules stabilize MTs when they interact [6
]. Here we found that under physiological conditions, they do not interact—at least not in the cytoplasm. Moreover, EB1 is neither essential for MT growth in interphase nor in mitosis, in which it seems to have a regulatory role. APC is also not required for MT growth either in mitosis or interphase. However, double depletion of APC and EB1 does result in very short MTs in interphase (C and D). If one looks at the interactions between EB1, XKCM1, and APC in interphase (A), this is entirely consistent with the idea that the catastrophe-promoting activity of XKCM1 is inhibited by its interaction with XMAP215, APC, and EB1. Indeed, when both EB1 and APC are depleted, the steady-state concentration of free XKCM1 probably increases, resulting in MT destabilization. We actually demonstrated this by doing a triple depletion experiment of APC, EB1, and XKCM1 from interphase extracts that did not result in MT shortening. On the contrary, upon depletion of XKCM1 in the ΔAPC and ΔEB1 extract, MTs grew from rather short ones (~9 μm) to almost control levels (~18 μm), demonstrating that XKCM1 was responsible for MT destabilization in the ΔAPC and ΔEB1 extract. The observation that EB1 or APC single depletion does not significantly destabilize MTs in interphase does not conflict with our model. It rather indicates that there is an excess of “XKCM1 buffering capacity” present in interphase extracts constituted by a variety of MAPs whose individual functions are somewhat interchangeable as far as inactivation of XKCM1 is concerned. This may explain how the inhibition of XKCM1 activity in interphase can be maintained at a constant level even if one specific MAP is removed. Thus, the inhibitory effect of individual MAP depletion on XKCM1 activity only becomes obvious when the “total buffering capacity” is overloaded (e.g., by adding exogenous XKCM1).
It seems that we are getting closer to an understanding of how MT growth is regulated in interphase and mitosis: MT assembly in vivo probably does not only involve tubulin assembly into tubes. Rather, tube assembly may involve oligomeric complexes of tubulin and other MAPs. It is conceivable that XMAP215 has evolved as a major linker and essential protein between tubulin and these other MAPs. Regulation of MT assembly between interphase and metaphase then largely occurs through a regulation of the state of dynamic interactions between XMAP215 and other MAPs in solution. It remains to be determined how this is achieved by CDK1: directly or indirectly. It is tempting to think that CDK1 alters the balance between kinases and phosphatases that modulate the dynamics of interactions between XMAP215 and the other MAPs.