XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2011; 108(7):2741-6. DOI: 10.1073/pnas.1016498108
Source: PubMed


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.


Available from: Andrei I. Pozniakovsky
  • Source
    • "Recently, the importance of the 1st and 2nd N-terminal TOG domains has been the subject of focus. Inactivation of the 3rd and 4th TOG domains of full-length XMAP215 increased the growth rate similar to normal XMAP215 in in vitro reconstitution experiments (Widlund et al., 2011). Interestingly, truncated proteins with the 1st and 2nd N-terminal TOG domains (TOG12) of XMAP215 or MOR1 also increased the growth rate or amount of microtubules in vitro, respectively (Widlund et al., 2011; Lechner et al., 2012), suggesting that TOG12 acts as a microtubule polymerase at least. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, many microtubule-associated proteins (MAPs) govern microtubule dynamics in cells. In plants, major MAPs are identified as microtubule stabilizers (CLASP and MAP65 etc.), microtubule destabilizers (kinesin-13, katanin, MAP18 and MDP25), and microtubule dynamics promoters (EB1, MAP215, MOR1, MAP200, SPR2). Mutant analyses with forward and reverse genetics have shown the importance of microtubules and individual MAPs in plants. However, it is difficult to understand how each MAP regulates microtubule dynamics, such as growth and shortening, through mutant analyses. In vitro reconstitution analyses with individual purified MAPs and tubulin are powerful tools to reveal how each MAP regulates microtubule dynamics at the molecular level. In this review, I summarize the results of in vitro reconstitution analyses and introduce current models of how each MAP regulates microtubule dynamic instability.
    Frontiers in Plant Science 08/2014; 5:409. DOI:10.3389/fpls.2014.00409 · 3.95 Impact Factor
  • Source
    • "Stu2p(TOG2-TOG2)-Δcc showed rescue efficiency very similar to that of Stu2p(TOG1-TOG2)-Δcc (Figure 4B). Dimerization impaired variants of Stu2p in which TOG1 or TOG2 was defective for αβ-tubulin binding did not show any rescue activity (Figure 4B), consistent with a prior in vitro study of XMAP215 that demonstrated a requirement for at least two TOG domains (Widlund et al., 2011). More importantly, the ability of TOG2 to substitute for TOG1 in this more stringent, dimerization-impaired background strengthens the conclusion that the polymerase does not require different TOG domains for its function. "
    [Show abstract] [Hide abstract]
    ABSTRACT: eLife digest Dynamic filaments of proteins, called microtubules, have several important roles inside cells. Microtubules provide structural support for the cell; they help to pull chromosomes apart during cell division; and they guide the trafficking of proteins and molecules across the cell. The building blocks of microtubules are proteins called αβ-tubulin, which are continually added to and removed from the ends of a microtubule, causing it to grow and shrink. Other proteins that interact with the microtubules can help to speed up these construction and deconstruction processes. Ayaz et al. took a closer look at the structure of one particular family of proteins that make it easier for the microtubules to grow, using a technique called X-ray crystallography. The resulting images show two sites—called TOG1 and TOG2—on the enzymes that attach to the αβ-tubulin proteins. Ayaz et al. found that this binding can only occur when αβ-tubulin has a curved shape, which only happens when the tubulins are not included in, or are only bound weakly to the end of, a microtubule. Previous research suggested that the two binding sites might work together to provide ‘scaffolding’ that stabilizes the microtubule. However, genetic experiments by Ayaz et al. show that microtubules will grow even if one of the binding sites is missing. Both TOG1 and TOG2 bind to αβ-tubulin in the same way, and by using computer simulations Ayaz et al. found that this helps to speed up the growth of microtubules. This is because the enzyme's two sites concentrate the individual tubulin building blocks at the ends of the filament. For example, TOG2 could bind to the end of the microtubule, while TOG1 holds an αβ-tubulin protein nearby and ready to bind to the filament's end. This tethering allows the microtubules to be assembled more efficiently. DOI: http://dx.doi.org/10.7554/eLife.03069.002
    eLife Sciences 08/2014; 3:e03069. DOI:10.7554/eLife.03069 · 9.32 Impact Factor
  • Source
    • "The founding member, XMAP215, was originally identified as a MT-associated protein from Xenopus laevis egg extracts that promotes MT assembly in vitro[9]. More recently, reconstitution assays and single-molecule imaging combined with structure-function analyses have provided useful insights into the mechanism by which XMAP215 catalyzes MT polymerization in vitro[10,11]. However, there have been few studies of XMAP215 and its family members in vivo[12-14], and none have examined its role(s) specifically within the neuronal growth cone. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Microtubule (MT) regulators play essential roles in multiple aspects of neural development. In vitro reconstitution assays have established that the XMAP215/Dis1/TOG family of MT regulators function as MT 'plus-end-tracking proteins' (+TIPs) that act as processive polymerases to drive MT growth in all eukaryotes, but few studies have examined their functions in vivo. In this study, we use quantitative analysis of high-resolution live imaging to examine the function of XMAP215 in embryonic Xenopus laevis neurons. Here, we show that XMAP215 is required for persistent axon outgrowth in vivo and ex vivo by preventing actomyosin-mediated axon retraction. Moreover, we discover that the effect of XMAP215 function on MT behavior depends on cell type and context. While partial knockdown leads to slower MT plus-end velocities in most cell types, it results in a surprising increase in MT plus-end velocities selective to growth cones. We investigate this further by using MT speckle microscopy to determine that differences in overall MT translocation are a major contributor of the velocity change within the growth cone. We also find that growth cone MT trajectories in the XMAP215 knockdown (KD) lack the constrained co-linearity that normally results from MT-F-actin interactions. Collectively, our findings reveal unexpected functions for XMAP215 in axon outgrowth and growth cone MT dynamics. Not only does XMAP215 balance actomyosin-mediated axon retraction, but it also affects growth cone MT translocation rates and MT trajectory colinearity, all of which depend on regulated linkages to F-actin. Thus, our analysis suggests that XMAP215 functions as more than a simple MT polymerase, and that in both axon and growth cone, XMAP215 contributes to the coupling between MTs and F-actin. This indicates that the function and regulation of XMAP215 may be significantly more complicated than previously appreciated, and points to the importance of future investigations of XMAP215 function during MT and F-actin interactions.
    Neural Development 12/2013; 8(1):22. DOI:10.1186/1749-8104-8-22 · 3.45 Impact Factor
Show more