In various studies, including this one, we have never observed an increase in axonal branch formation associated with experimental elevation of P60-katanin (Karabay et al., 2004
; Yu et al., 2005
; Qiang et al., 2006
). However, here we found a very dramatic and consistent elevation in axonal branch formation associated with overexpression of spastin. Thus we would conclude that, in a direct comparison of the two severing proteins, the properties of spastin are more conducive to the formation of branches. In support of this conclusion, depletion of spastin from neurons results in a fairly modest reduction in axonal length, but a far greater reduction in the formation of axonal branches. In addition, spastin has a far greater capacity to concentrate at sites of branch formation (and growth cones) than does P60-katanin. We envision a model wherein neurons contain high levels of P60-katanin that are absolutely essential for the ongoing severing of microtubules needed for axonal growth and contain much lower levels of spastin designed to participate in specialized duties such as axonal branch formation. Even so, it is noteworthy that branches can still form in the absence of spastin, and this is consistent with previous observations on spastin-compromised animals (Sherwood et al., 2004
; Trotta et al., 2004
; Orso et al., 2005
; Tarrade et al., 2006
; Wood et al., 2006
) and humans (McDermott et al., 2006
; Salinas et al., 2007
). Our data, presented here, suggest that there are two different modes by which axonal branches can form, one that utilizes spastin and the other than utilizes P60-katanin.
As noted earlier, the sequences of spastin and P60-katanin are very different in regions other than the AAA region responsible for severing microtubules (Errico et al., 2002
; Roll-Mecak and Vale, 2005
; White et al., 2007
). Thus, it may be that the other domains of spastin enable it to influence branch formation by means other than the severing of microtubules. For example, it is known that branch formation requires actin filaments as well as microtubules (Dent and Kalil, 2001
), and therefore it is conceivable that spastin influences the actin cytoskeleton in a way that P60-katanin does not. Indeed, the entire morphology of the neuron is markedly altered after spastin overexpression (i.e., greater numbers of filopodia and branches) in a manner not duplicated by P60-katanin overexpression. In the future, it will be of interest to ascertain whether spastin has domains that directly participate in pathways relevant to remodeling of the actin cytoskeleton.
For now, we are persuaded to believe that the dramatic morphological differences produced by the two severing proteins relate directly to their distinct patterns of microtubule severing. We have noticed in the past that overexpression of P60-katanin in neurons can often result in the appearance of very long microtubules that appear to be quite resistant to severing (Yu et al., 2005
), and the same observation was made here. By contrast, spastin overexpression appears to produce a far more consistent “even chopping” of the microtubules into small pieces. In other words, we have not detected any evidence for a population of microtubules that are strongly resistant to severing by spastin. Given these different severing patterns, the expectation would be that spastin is capable of producing high concentrations of short microtubules, whereas P60-katanin's properties are more suited for generating mixtures of long and short microtubules. Concentrations of plus ends of microtubules would be expected to recruit a variety of +tips which could also interact with structures within the actin-based cortical regions of the axon (Kornack and Giger, 2005
). Thus, the enhanced formation of filopodia and the remodeling of actin required for branches to form could relate directly to the manner by which spastin severs microtubules.
What accounts for the different microtubule-severing patterns of spastin and P60-katanin? Studies on P60-katanin suggest that its severing properties are regulated by phosphorylation, but probably not phosphorylation of P60-katanin itself (Vale, 1991
; McNally et al., 2002
; Baas and Qiang, 2005
). Rather, it appears that in nonneuronal cells, the phosphorylation state of MAP4 is crucial for regulating the degree to which P60-katanin can sever microtubules (McNally et al., 2002
). The binding of MAP4 to the microtubule apparently reduces the accessibility of the microtubule to P60-katanin. Phosphorylation of MAP4 causes it to dissociate from the microtubules, thereby permitting more robust severing by P60-katanin. MAP4 is not expressed in CNS neurons, but recent studies from our laboratory indicate that a comparable role is played by tau in the axon (Qiang et al., 2006
). The fact that tau and MAP4 are strong protectors against P60-katanin provides an appealing explanation for the severing pattern produced by P60-katanin in fibroblasts and neurons. P60-katanin would preferentially sever microtubules that are less rich in these protective proteins, which would then cause progressively more depolymerization of these microtubules. The molecules of tau or MAP4 that had been associated with these microtubules would then be available to bind to the microtubules that were already richer in these proteins, thereby rendering them even more resistant to severing and enabling them to achieve even greater lengths. In this manner, the properties of P60-katanin would promote a mixture of short and long microtubules.
Our current studies suggest that tau is a far less influential factor in the regulation of spastin's severing properties. The microtubules in the axon, rich in tau, show no greater resistance to spastin overexpression than the tau-deficient microtubules located in other compartments of the neuron. Moreover, axons depleted of tau undergo markedly more branching, and this effect is independent of whether or not spastin is present. These results are consistent with an alternative mechanism for axonal branching based on the properties of P60-katanin. In this mechanism, tau is locally detached from the microtubules at a site of impending branch formation. As a result, P60-katanin can produce the localized chopping of microtubules needed for the branch to form. This idea, which we have discussed in the past (Baas and Qiang, 2005
; Baas et al., 2006
), does not require a local concentration of P60-katanin, but rather a focal increase in the sensitivity of the microtubules to P60-katanin. This process would presumably be regulated by kinases and phosphatases that phosphorylate or dephosphorylate tau at motifs required for it to bind microtubules. Although our results suggest that tau is probably the main player in such a scenario, there may be other microtubule-associated proteins in the axon that conspire in regulating the sensitivity of the microtubules to P60-katanin.
In conclusion, we propose that there are two modes by which microtubule severing is orchestrated during axonal branch formation, one based on the local concentration of spastin at branch sites and the other based on local detachment from microtubules of molecules such as tau that regulate the severing properties of P60-katanin (see ). Each of these modes is presumably under the regulation of signaling cascades that regulate such events as the phosphorylation of tau, the attachment or detachment of other protective proteins, and the localization of spastin to sites of impending branch formation. The formation of a particular branch could utilize one mode or the other, or some combination of the two modes, working in tandem.
Figure 12. Schematic illustration of two modes by which microtubule severing may be regulated at sites of impending axonal branch formation. Both A and B show the axon undergoing the formation of branch. (A) In the “katanin mode,” microtubule severing (more ...)