Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers.

Kinetochores are multicomponent assemblies that connect chromosomal centromeres to mitotic-spindle microtubules. The Ndc80 complex is an essential core element of kinetochores, conserved from yeast to humans. It is a rod-like assembly of four proteins- Ndc80p (HEC1 in humans), Nuf2p, Spc24p and Spc25p. We describe here the crystal structure of the most conserved region of HEC1, which lies at one end of the rod and near the N terminus of the polypeptide chain. It folds into a calponin-homology domain, resembling the microtubule-binding domain of the plus-end-associated protein EB1. We show that an Ndc80p-Nuf2p heterodimer binds microtubules in vitro. The less conserved, N-terminal segment of Ndc80p contributes to the interaction and may be a crucial regulatory element. We propose that the Ndc80 complex forms a direct link between kinetochore core components and spindle microtubules.

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either α or β tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.

Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an αβ tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer.

The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. Here, we review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, while CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behaviors of MT plus ends.

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

Alp14, a XMAP215 orthologue in fission yeast, is a microtubule (MT) polymerase. It tracks growing MT plus ends and regulates the polymerization state of tubulin by cycling between a tubulin dimer–bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly.

Members of the XMAP215/Dis1 family of microtubule-associated proteins (MAPs) are essential for microtubule growth. MAPs in this family contain several 250 residue repeats, called TOG domains, which are thought to bind tubulin dimers and promote microtubule polymerization. We have determined the crystal structure of a single TOG domain from the Caenorhabditis elegans homolog, Zyg9, to 1.9 A resolution, and from it we describe a structural blueprint for TOG domains. These domains are flat, paddle-like structures, composed of six HEAT-repeat elements stacked side by side. The two wide faces of the paddle contain the HEAT-repeat helices, and the two narrow faces, the intra- and inter-HEAT repeat turns. Solvent-exposed residues in the intrarepeat turns are conserved, both within a particular protein and across the XMAP215/Dis1 family. Mutation of some of these residues in the TOG1 domain from the budding yeast homolog, Stu2p, shows that this face indeed participates in the tubulin contact.