Members of the MAP65 family organize microtubules by preferentially crosslinking filaments with an anti-parallel orientation. In this study, we provide a structural framework for how three structurally distinct domains in human MAP65 (PRC1) are combined to achieve selective and efficient crosslinking of anti-parallel microtubules, as would be needed during the self-organization of dynamic cytoskeletal networks.
The conserved microtubule binding domain in PRC1 has a spectrin fold. First identified in spectrin, a constituent of the membrane skeleton, this fold has since been seen in several actin crosslinking proteins such as alpha-actinin and dystrophin (Djinovic-Carugo et al., 2002
). In these proteins, spectrin-domains appear as repeated units that connect other actin-binding domains and regulate properties of the cytoskeletal structures. In PRC1, this domain appears to have evolved to mediate microtubule binding, an entirely different role for this protein fold. Further, the spectrin fold is unrelated to the other protein folds (e.g Cap/Gly motifs and calponin-homology domains) that are known to mediate interactions with the microtubule lattice. It has been proposed that some protein surfaces are evolutionarily selected to act as a versatile protein interaction platform. This is best exemplified in the Fc domain of IgG, which is structurally adapted to interact with several different protein scaffolds (DeLano et al., 2000
). Similarly, it appears that the microtubule surface has evolved to bind a large variety of unrelated structural motifs and thereby accommodate diverse MAPs that can carry out a wide range of functions (Amos and Schlieper, 2005
Microtubule binding by the spectrin domain is augmented by a Lys/Arg rich unstructured domain in PRC1. Synergistic microtubule binding by structured and unstructured domains have also been seen in other MAPs such as Ndc80 (Guimaraes et al., 2008
; Miller et al., 2008
), suggesting that this feature may be a frequent adaptation in MAPs. There are also at least two other functions ascribed to these unstructured microtubule binding domains. First, these unstructured domains are often sites of phospho-regulation (Holt et al., 2009
). Interestingly, Cdk1 phosphorylation sites in PRC1 map to the unstructured Lys/Arg domain (Zhu et al., 2006
). Our data suggest that these phosphorylations would directly attenuate microtubule affinity by reducing the net positive charge of the domain. Dephosphorylation of these residues would activate PRC1's microtubule binding at anaphase, as would be needed for PRC1's functions during the final stages of cell division. Second, as has been suggested for Ndc80, these domains are proposed to allow a mode of attachment which does not significantly resist microtubule movement. We find that PRC1 does not strongly oppose microtubule sliding by kinesin-5, indicating that moderate binding affinities and diffusive microtubule interactions of PRC1 can permit microtubule movement while maintaining attachment. This result is also consistent with the reported diffusion constant of Ase1 which indicates that >100 crosslinker molecules would be needed to generate a resistive force greater than 1.5 pN, which is in the range of the force required to inhibit kinesin-5 movement (Kapitein et al., 2008a
; Korneev et al., 2007
; Valentine and Block, 2009
). This would be relevant in vivo, when microtubule bundling by Ase1 and sliding by kinesin-5 are both required for spindle elongation in anaphase B (Khmelinskii et al., 2009
Crossbridges between two PRC1-crosslinked microtubules are formed by PRC1's dimerization domain. These are seen to project at an angle of 70° relative to the microtubule lattice. Binding at a fixed angle relative to the microtubule lattice has also been reported for Ndc80 (Wilson-Kubalek et al., 2008
) but the implications of defined projection angles for any microtubule binding protein is thus far unknown. The crossbridge also determines the inter-microtubule spacing between two microtubules. This inter-microtubule distance could also affect the ability of motor proteins to bind and slide PRC1 crosslinked microtubules, providing an additional mechanism for activating or deactivating specific motors at these structures. For example, the reported length of kinesin-5 motor is approximately 95 nm, which is ~ 2-fold greater than the 37 nm inter-microtubule spacing of PRC1 crosslinked microtubules (Kashina et al., 1996
). Hence, the reduction in the velocity of microtubule sliding by kinesin-5 seen in our experiments at high PRC1 concentrations could result from the inability of kinesin-5 to bind properly and efficiently walk along both microtubules with dense PRC1 crosslinks. Such modulation of motor activity through control of inter-filament spacing has been proposed to affect the magnitude of active forces generated in striated muscles during muscle contraction (Millman, 1998
Based on our results, we propose a structural model for how polarity-specific crosslinking is mediated by PRC1 (). The ability of a microtubule associated protein to distinguish parallel and anti-parallel filaments relies on two factors. First, PRC1 molecules must decode filament polarity when bound to one microtubule. Our results show that the spectrin domain is the most ordered region in a PRC1-microtubule complex and uses a well-defined surface for microtubule binding. This suggests that this domain makes contacts with the microtubule lattice that decode filament orientation. Second, the microtubule polarity needs to be transmitted across the linker to the second spectrin domain in the PRC1 homodimer. Our data show that the dimerization domain in PRC1 has a single conformation when crosslinking two microtubules. The structural rigidity of this domain is likely to be responsible for PRC1's selectivity for anti-parallel microtubules. Though specificity can be achieved by oriented binding of the spectrin domain and rigidity in the linker domain, the weak microtubule binding affinity of the spectrin domain alone does not explain the extensive filament bundling induced by PRC1. To increase binding and consequently crosslinking efficiency, PRC1 uses an unstructured positively charged domain for maintaining long-lived associations with microtubules. Additionally, the flexibility in the linker domain of PRC1, which appears to have more than one conformation when bound to one microtubule, may allow for an initial contact with a second microtubule that could have a wide-range of orientations. Relative to a highly rigid structure, such flexibility could increase the crosslinking efficiency of PRC1 molecules. These features would enable PRC1 to stay associated with the first microtubule encountered, explore its length by 1-D diffusion, and thereby increase the probability of capturing a second filament for establishing anti-parallel linkages between two microtubules.
FIGURE 7 PRC1 is a compliant, microtubule-overlap tracking protein that tunes structural rigidity to specifically crosslink two anti-parallel microtubules. (A) A model for how PRC1 can align microtubules into anti-parallel arrays. The spectrin domain in PRC1 can (more ...)
Many cellular processes require recognition of a “mark” at precise locations. Post-translational modifications such as ubiquitination and methylation are some of the common marking mechanisms for proteins and DNA in cells. In the microtubule cytoskeleton, the +TIP-proteins track and identify microtubule growing microtubule plus-ends (Akhmanova and Steinmetz, 2008
). Similarly, by recognizing specific microtubule geometries and forming compliant crosslinks, the MAP65 proteins can mark anti-parallel microtubule overlap during the self-organization of dynamic cytoskeletal networks.