Microtubules are dynamic filaments whose ends alternate between periods of slow growth and rapid shortening as they explore intracellular space and move organelles. A key question is how regulatory proteins modulate catastrophe, the conversion from growth to shortening. To study this process, we reconstituted microtubule dynamics in the absence and presence of the kinesin-8 Kip3 and the kinesin-13 MCAK. Surprisingly, we found that, even in the absence of the kinesins, the microtubule catastrophe frequency depends on the age of the microtubule, indicating that catastrophe is a multistep process. Kip3 slowed microtubule growth in a length-dependent manner and increased the rate of aging. In contrast, MCAK eliminated the aging process. Thus, both kinesins are catastrophe factors; Kip3 mediates fine control of microtubule length by narrowing the distribution of maximum lengths prior to catastrophe, whereas MCAK promotes rapid restructuring of the microtubule cytoskeleton by making catastrophe a first-order random process.

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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...

In cells, a complex network of proteins regulates the dynamic growth of microtubules that is essential for division and migration. In vitro approaches with purified components have so far been unable to reconstitute fast microtubule growth observed in vivo . Here we show that two well-studied plus-end-binding proteins-end-tracking protein EB1 and microtubule polymerase XMAP215-act together to strongly promote microtubule growth to cellular rates. Unexpectedly, the combined effects of XMAP215 and EB1 are highly synergistic, with acceleration of growth well beyond the product of the individual effects of either protein. The synergistic growth promotion does not rely on any of the canonical EB1 interactions, suggesting an allosteric interaction through the microtubule end. This hypothesis is supported by the finding that taxol and XMAP215, which have non-overlapping binding sites on tubulin, also act synergistically on growth. The increase in growth rates is accompanied by a strong enhancement of microtubule catastrophe by EB1, thereby rendering the fast and dynamic microtubule behaviour typically observed in cells.