hydrolysis (Moores and Milligan, 2008), and a recent muta- tional study indicates that tubulin release precedes ATP turn- over (Wagenbach et al., 2008). Although depolymerization activity is possible with the kinesin 13 motor core alone, the full-length (FL) protein is a more efficient depolymerizer, suggesting important roles for the other domains (Ovechkina et al., 2002; Ogawa et al., 2004; Hertzer et al., 2006). The N terminus of kinesin 13 is important for subcellular localization and contributes to dimerization, whereas the C terminus is required for dimerization (Maney et al., 1998, 2001; Wordeman et al., 1999; Walczak et al., 2002; Kline-Smith and Walczak, 2004). A charged sequence of 60 amino acids N terminal to the catalytic motor domain is a class- specific sequence known as the neck (Ovechkina et al., 2002; Ogawa et al., 2004). The neck is thought to be intimately in- volved in the depolymerization mechanism, as constructs con- taining only the neck in addition to the motor domain have depolymerization activity comparable to that seen for FL kine- sin 13 (Maney et al., 2001; Ovechkina et al., 2002; Hertzer et al., 2006). The role of the additional N- and C-terminal do- mains of the FL dimeric protein in the depolymerization mecha- nism, other than localization and regulation by phosphorylation, is not entirely clear and remains an important line of inquiry. Structural studies have provided important mechanistic in- sights into the function of conventional kinesin (e.g., Vale and Milligan, 2000; Endow, 2003), but similar attempts to character- ize the interactions of FL kinesin 13 with MT ends have been challenging as a result of their inherent heterogeneity. A recent study has shown that dolastatin-induced tubulin rings mimic the properties of MT ends (flexibility, shape, and stimulation of kinesin 13 ATPase) and that these rings can be decorated with the motor domain of kinesin 13 in various nucleotide states (Moores and Milligan, 2008). The size of these dolastatin tubulin rings and the suggestion that they mimic MT ends make them an ideal system for obtaining structural insights into the depolymeriza- tion mechanism of kinesin 13. It is important to note that these dolastatin tubulin rings are synthetic MT end mimics and should not be confused with the bracelets and spirals formed around MTs as products of active depolymerization by mitotic centromere- associated kinesin (MCAK) in the presence of 5-adenylyl-,- imidodiphosphate (AMPPNP; Moores et al., 2002, 2006; Tan et al., 2006, 2008). Such bracelets are complex and heterogeneous in structure, varying in thickness, connectivity, and symmetry. A beautiful example of careful structural experiments of such bracelets and spirals was recently published (Tan et al., 2008); we believe our experiments with synthetic MT end mimics to be complementary to these important studies. In this study, we have used EM and single particle image processing methods to visualize the molecular interactions by which kinesin 13 induces depolymerization at MT ends. We have visualized the interactions of kinesin 13 domain constructs of the motor core (M) alone, the neck plus motor core (NM), and the FL protein with MT end mimics in the AMPPNP state. Based on the binding patterns observed in our data, we propose a new model for MT depolymerization in which kinesin 13 achieves increased depolymerization efficiency by binding to adjacent protofilaments. 52 JCB • VOLUME 185 • NUMBER 1 • 2009 Results and discussion Decoration of tubulin rings As shown in previous studies, incubation of dolastatin-10 with tubulin resulted in the formation of single protofilament rings with 13, 14, and 15× heterodimer geometries, of which rings with the 14× geometry dominated the population (Bai et al., 1999; Boukari et al., 2007; Moores and Milligan, 2008). All kinesin 13 constructs investigated bound these rings along the inner periphery as can be seen by an inner ring of protein density in raw EM micrographs (Fig. 1 A, solid box); this corresponds to the outside MT surface (Bai et al., 1999; Boukari et al., 2007; Moores and Milligan, 2008). Raw EM images show that rings incubated with kinesin 13 constructs assume a less flexible and more rounded shape than undecorated rings (Fig. 1 A, solid vs. dashed box). A notable difference between ring construct complexes was the presence of stacks of rings in the NM and FL samples (Fig. 1 A, arrows). Such stacks contained tightly associated rings compared with the loosely associated rings occasionally observed for dolastatin rings alone or with the M construct (Fig. 1 A, NM and FL vs. ctrl and M). An inventory of 20 ran- domly selected micrographs from each of the ring-only, M, NM, and FL AMPPNP datasets confirmed that NM and FL data- sets contained more stacks per micrograph than the ring-only and M datasets (P < 0.001; Fig. 1 B and see Fig. S3 for equiva- lent behavior by a human M construct). Notably, stacks formed by FL were 2.41-fold longer than those formed by NM (P < 0.001; Fig. 1 C), indicating that dimerization of the motor sig- nificantly improved the ability of a single kinesin 13 molecule to interact with multiple rings. Stack formation in the presence of the monomeric NM construct is likely a result of nonspecific electrostatic inter- actions between the positively charged neck and the negatively charged tubulin C-terminal tails of nearby rings. Previous stud- ies have suggested multiple roles for such an electrostatic inter- action, including that of a motor to substrate tether during depolymerization (Ovechkina et al., 2002; Moores et al., 2006), a weak tether to aid 1D diffusion (Helenius et al., 2006), and an impediment to lateral MT interactions (Ogawa et al., 2004). Aside from providing further support for the existence of such an electrostatic interaction, we do not attribute physiological significance to ring stack formation in the presence of NM. However, because FL forms longer stacks than NM, we hypoth- esize that in addition to electrostatics, the extra domains present in FL contribute to longer stack formation. As such, these data suggest that the additional N- and C-terminal domains or the second head of dimeric FL is able to bind nearby rings, thereby resulting in the formation of longer stacks of rings. To further investigate the binding of M, NM, and FL to dolastatin rings, we performed cosedimentation assays of rings incubated with each construct in the presence of AMPPNP and analyzed the results by SDS-PAGE (Fig. 2 A). To compare ring- binding trends between constructs, the molar ratio of construct to heterodimer was determined for each pellet fraction and normal- ized for the concentration of construct used in the experiment. This analysis revealed that the molar amount of M bound to a