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.

No abstract

XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215's overall affinity for free tubulin. The mutants' overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a "bonsai" protein, with two TOG domains and a basic region, that has almost full polymerase activity.C ells assemble and disassemble actin filaments and microtubules to carry out a vast array of functions, such as defining cell shape, directing cellular movement, and mediating chromosome segregation and cell division. Although these polymeric filaments have different structures and display different dynamics, the cell regulates their assembly and disassembly in related ways. Polymer growth is polar in both cases and occurs at the plus ends of microtubules and the barbed ends of actin filaments. Both polymers have specific nucleating proteins, assemble with the help of polymerases, and disassemble with the aid of severing proteins and depolymerases (1-4). How these various activities coordinate to create the cytoskeleton is a central question in cell biology (5). This work focuses on assembly.The main promoters of polymer growth are the XMAP215/Dis family for microtubules and the formins for actin (4,(6)(7)(8)(9). The function of formins in actin polymerization is well characterized. Formins have two key domains that are important for their activity, FH1 and FH2 (8,10). While the FH2 domain is necessary for binding to the barbed end of actin, repeats of polyproline in the FH1 domain are required to interact with actin/profilin complexes and recruit them to the barbed end (4,11,12).Much less is known about how the regions of XMAP215 coordinate in promoting microtubule growth (13). Recent work has shown that XMAP215 acts as a classic catalyst (14). At physiological tubulin concentrations, XMAP215 is a tubulin polymerase that promotes incorporation of tubulin into the growing plus end. However, in the absence of free tubulin, XMAP215 accelerates depolymerization of GMPCPP-stabili...

Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1.

Previous studies of mitosis show that capture of single kinetochores by microtubules from both centrosomes (merotelic orientation) is a major cause of aneuploidy. We have characterized hcp-6, a temperature-sensitive chromosome segregation mutant in C. elegans that exhibits chromosomes attached to both poles via a single sister kinetochore. We demonstrate that the primary defect in this mutant is a failure to fully condense chromosomes during prophase. Although centromere formation and sister centromere resolution remain unaffected in hcp-6, the chromosomes lack the rigidity of wild-type chromosomes and twist around the long axis of the chromosome. As such, they are unable to establish a proper orientation at prometaphase, allowing individual kinetochores to be captured by microtubules from both poles. We therefore propose that chromosome rigidity plays an essential role in maintaining chromosome orientation to prevent merotelic capture.