EB1 regulates microtubule dynamics and tubulin sheet closure in vitro

Vitre, Benjamin; Coquelle, Frédéric M.; Heichette, Claire; Garnier, Cyrille; Chrétien, Denis; Arnal, Isabelle

doi:10.1038/ncb1703

Cited by 252 publications

(256 citation statements)

References 35 publications

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“…The sum of our observations indicates that native EB1 FL and even native EB1 have intrinsically weak activity, contrary to the strong MT binding and polymerization activities previously reported with tagged proteins (22,26). Our results also suggest that EB1 FL and EB1 can bind to MTs cooperatively, thus increasing their activity.…”

Section: Microtubules (Mts)contrasting

confidence: 54%

“…For example, Tirnauer et al (22) reported that the K d of the EB1-MT interaction is 0.44 M, whereas the behavior of EB1 (or its relatives) in other experiments has implied a much weaker interaction (23)(24)(25). Some studies have reported that EB1 can induce the polymerization of low concentrations of tubulin (26), whereas others report that EB1 by itself has no effect on MT polymerization; EB1 activity requires the removal of its autoinhibitory tail (aa 249 -268) or activation by proteins that bind this tail (19,(27)(28)(29). Although one might predict that the greater activity of the activated EB1 fragment lacking the autoinhibitory tail (EB1 ) is due to its having a higher affinity for MTs, comparative affinities of full-length EB1 and EB1 have not yet been measured.…”

Section: Microtubules (Mts)mentioning

confidence: 99%

“…As mentioned above, it has been suggested that full-length EB1 can promote MT growth in vitro (26), but this conclusion remains controversial (19,28,29). To clarify whether EB1 has the ability to promote MT polymerization and whether EB1 polymerization promoting activity is also influenced by the presence of His tags, we performed tubulin polymerization (light scattering) assays with all three full-length EB1 proteins (His EB1 FL , His-cut EB1 FL , and untagged EB1 FL ).…”

Section: Preparation Of His-tagged His-tag-cleaved and Untaggedmentioning

confidence: 99%

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Interactions between EB1 and Microtubules

Zhu

Gupta

Slabbekoorn

et al. 2009

Journal of Biological Chemistry

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Plus end tracking proteins (؉TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved ؉TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip. Microtubules (MTs)2 are a major component of the cytoskeleton, the network of proteinacious fibers that endow the cell with structural integrity, motile properties, and internal organization (1-4). MTs play a particularly important role in cell organization: they help to pull the chromosomes apart at mitosis, act as a "railway" for intracellular transport, and define the localization and structure of internal membrane systems (5-11).The cellular functions of MTs are highly dependent on their dynamic nature, which is regulated by a number of microtubule associated proteins (MAPs) (3, 9, 12, 13). The plus end tracking proteins (ϩTIPs) are an unusual group of MAPs that preferentially localize to growing MT plus ends (13)(14)(15). A large number of proteins have now been identified as ϩTIPs, but one of the most important is EB1 (end binding protein-1) (4,13,16,17). EB1 was initially discovered as a binding partner of the adenomatous polyposis coli protein, but it is becoming apparent that EB1 binds to an astonishing array of other proteins including other ϩTIPs (CLIP-170, p150, and CLASPs), molecular motors (Tea2 and kinesin), signal transduction proteins (Rho-GEF2), and cytoskeletal scaffolding proteins (spectraplakins and formins) (4, 13, 15). As might be expected from a protein with so many interactions, EB1 is strikingly well conserved across eukaryotes (18 -21). These observations suggest that EB1 is the structural and evolutionary core of a complex of proteins that regulates the behavior of MT plus ends (4, 13-15). However, its activities and the mechanisms of i...

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Section: Microtubules (Mts)contrasting

confidence: 54%

Section: Microtubules (Mts)mentioning

confidence: 99%

Section: Preparation Of His-tagged His-tag-cleaved and Untaggedmentioning

confidence: 99%

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Interactions between EB1 and Microtubules

Zhu

Gupta

Slabbekoorn

et al. 2009

Journal of Biological Chemistry

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“…EB1 has been proposed to promote tubulin sheet closure during microtubule polymerization (35), potentially through preferential binding along the seam (36). In vitro, EB1 has been reported to suppress the shortening rate and the frequency of catastrophe (37) or, alternatively, to promote both catastrophes and rescues (35).…”

Section: Discussionmentioning

confidence: 99%

Microtubule plus-end tracking by CLIP-170 requires EB1

Dixit

Barnett

Lazarus

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

182

180

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Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (؉TIPs) localize to and track with growing microtubule plus-ends in the cell. ؉TIPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the ؉TIP tracking activity are not well understood, however. We reconstituted the ؉TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our singlemolecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170 -EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for ؉TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5-[␣,␤-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian ؉TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end.ϩTIP ͉ single molecule ͉ total internal reflection fluorescence (TIRF) microscopy ͉ dynamic instability

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“…End-binding proteins recognize a tubulin conformation unique to the growing ends of MTs and can affect the dynamics of plusends by intrinsically altering the structure of the MT end (6)(7)(8) as well as recruiting other interacting proteins (9). In contrast, TOG domain-containing proteins, such as XMAP215, promote MT growth and have been suggested to act as MT "polymerases" (10,11).…”

mentioning

confidence: 99%

Regulation of microtubule minus-end dynamics by CAMSAPs and Patronin

Hendershott

Vale

2014

Proc. Natl. Acad. Sci. U.S.A.

158

184

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The microtubule (MT) cytoskeleton plays an essential role in mitosis, intracellular transport, cell shape, and cell migration. The assembly and disassembly of MTs, which can occur through the addition or loss of subunits at the plus-or minus-ends of the polymer, is essential for MTs to carry out their biological functions. A variety of proteins act on MT ends to regulate their dynamics, including a recently described family of MT minus-end binding proteins called calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin/Nezha. Patronin, the single member of this family in Drosophila, was previously shown to stabilize MT minusends against depolymerization in vitro and in vivo. Here, we show that all three mammalian CAMSAP family members also bind specifically to MT minus-ends and protect them against kinesin-13-induced depolymerization. However, these proteins differ in their abilities to suppress tubulin addition at minus-ends and to dissociate from MTs. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends. By using truncation analysis, we find that somewhat different minimal domains of CAMSAP and Patronin are involved in minus-end localization. However, we find that, in both cases, a highly conserved C-terminal domain and a more variable central domain cooperate to suppress minus-end dynamics in vitro and that both regions are required to stabilize minus-ends in Drosophila S2 cells. These results show that members of the CAMSAP/Patronin family all localize to and protect minus-ends but have evolved distinct effects on MT dynamics.tubulin polymerization | cytoskeletal regulation | TIRF microscopy M icrotubules (MTs) are cellular polymers that are important for a variety of functions, including cargo transport and mitotic spindle formation. MTs are composed of dimers of α-and β-tubulin that assemble head-to-tail, creating a polar protofilament. Protofilaments then assemble laterally to form the canonical 13-protofilament MT structure, with β-tubulin exposed at fast-growing plus-ends and α-tubulin exposed at slow-growing minus-ends (1). MTs exhibit an intriguing property termed "dynamic instability," whereby the polymer can abruptly switch between episodes of net growth and shrinkage (2). The rates of growth and shrinkage as well as the frequency of transitions between these two states are regulated by numerous MT-associated proteins, many of which bind to the ends of the polymer (3, 4).The dynamics of MT plus-ends are regulated by a well-characterized network of plus-end tracking proteins (+TIPs) (5). End-binding proteins recognize a tubulin conformation unique to the growing ends of MTs and can affect the dynamics of plusends by intrinsically altering the structure of the MT end (6-8) as well as recruiting other interacting proteins (9). In contrast, TOG domain-containing proteins, such as XMAP215, promote MT growth and have been suggested ...

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EB1 regulates microtubule dynamics and tubulin sheet closure in vitro

Cited by 252 publications

References 35 publications

Interactions between EB1 and Microtubules

Interactions between EB1 and Microtubules

Microtubule plus-end tracking by CLIP-170 requires EB1

Regulation of microtubule minus-end dynamics by CAMSAPs and Patronin

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