Interactions between CLIP-170, tubulin, and microtubules: Implications for the mechanism of CLIP-170 plus-end tracking behavior

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46565, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 12/2005; 16(11):5373-84. DOI: 10.1091/mbc.E04-12-1106
Source: PubMed


CLIP-170 belongs to a group of proteins (+TIPs) with the enigmatic ability to dynamically track growing microtubule plus-ends. CLIP-170 regulates microtubule dynamics in vivo and has been implicated in cargo-microtubule interactions in vivo and in vitro. Though plus-end tracking likely has intimate connections to +TIP function, little is known about the mechanism(s) by which this dynamic localization is achieved. Using a combination of biochemistry and live cell imaging, we provide evidence that CLIP-170 tracks microtubule plus-ends by a preassociation, copolymerization, and regulated release mechanism. As part of this analysis, we find that CLIP-170 has a stronger affinity for tubulin dimer than for polymer, and that CLIP-170 can distinguish between GTP- and GDP-like polymer. This work extends the previous analysis of CLIP-170 behavior in vivo and complements the existing fluorescence microscope characterization of CLIP-170 interactions with microtubules in vitro. In particular, these data explain observations that CLIP-170 localizes to newly polymerized microtubules in vitro but cannot track microtubule plus-ends in vitro. These observations have implications for the functions of CLIP-170 in regulating microtubule dynamics.

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    • "We note that several in vivo studies have indicated that EB1 can increase bundling of microtubules in cells [Bu and Su, 2003; Ligon et al., 2003], and in some cases, this bundling is increased by the presence of other MAPs [Tirnauer et al., 2002; Folker et al., 2005]. However, to our knowledge EB1-mediated bundling has not been observed in vitro and thus it is not clear whether EB1 is capable of bundling microtubules in isolation. "
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    ABSTRACT: Using the nonhydrolyzable GTP analog GMPCPP and the slowly hydrolyzable GTPγS, we polymerize microtubules that recapitulate the end binding behavior of the plus end interacting protein (+TIP) EB1 along their entire length, and use these to investigate the impact of EB1 binding on microtubule mechanics. To measure the stiffness of single filaments, we use a spectral analysis method to determine the ensemble of shapes adopted by a freely diffusing, fluorescently-labeled microtubule. We find that the presence of EB1 can stiffen microtubules in a manner that depends on the hydrolysis state of the tubulin-bound nucleotide, as well as the presence of the small-molecule stabilizer paclitaxel. We find that the magnitude of the EB1-induced stiffening is not proportional to the EB1-microtubule binding affinity, suggesting that the stiffening effect does not arise purely from an increase in the total amount of bound EB1. Additionally, we find that EB1 binds cooperatively to microtubules in manner that depends on tubulin-bound nucleotide state. © 2014 Wiley Periodicals, Inc.
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    • "It is currently unknown which structural features of the growing microtubule end are recognized by autonomously tracking +TIPs; however, these might include the GTP cap at the end of the freshly polymerized microtubule (Lampert et al., 2010; Zanic et al., 2009) or some specific protofilament arrangement (des Georges et al., 2008; Sandblad et al., 2006) (for a review, see Coquelle et al., 2009). Another attractive idea is that autonomously tracking +TIPs co-polymerize with tubulin subunits and then get released gradually from the mature lattice (Folker et al., 2005); this mechanism has not found support in the in-vitro reconstitution studies using EB and CLIP homologs of fission yeast and vertebrates (Bieling et al., 2007; Bieling et al., 2008; Dixit et al., 2009), but might still apply to some other proteins. Most +TIPs track the ends of growing microtubules in a non-autonomous manner. "

    Preview · Article · Oct 2010 · Journal of Cell Science
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    • "MT tip tracking can occur by three mechanisms: treadmilling , hitchhiking, and kinesin-mediated transport. In metazoa, the treadmilling proteins CLIP170 and p150 Glued contain Cap-Gly domains, and basic and serine-rich motifs that mediate interactions with the EB1 C terminus and tubulin, respectively, which facilitates treadmilling and potentially copolymerization at MT plus ends (Diamantopoulos et al., 1999; Ligon et al., 2003; Folker et al., 2005; Dixit et al., 2009). Other +TIPs, including APC and ACF-7, are instead thought to track MT ends by hitchhiking on EB1 (Mimori-Kiyosue et al., 2000a; Slep et al., 2005). "
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