Mammalian end binding proteins control persistent microtubule growth

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.     

Interaction of mammalian end binding proteins with CAP-Gly domains of CLIP-170 and p150(glued)

End binding proteins (EBs) track growing microtubule ends and play a master role in organizing dynamic protein networks. Mammalian cells express up to three different EBs (EB1, EB2, and EB3). Besides forming homodimers, EB1 and EB3 also assemble into heterodimers. One group of EB-binding partners encompasses proteins that harbor CAP-Gly domains. The binding properties of the different EBs towards CAP-Gly proteins have not been systematically investigated. This information is, however, important to compare and contrast functional differences. Here we analyzed the interactions between CLIP-170 and p150(glued) CAP-Gly domains with the three EB homodimers and the EB1-EB3 heterodimer. Using isothermal titration calorimetry we observed that some EBs bind to the individual CAP-Gly domains with similar affinities while others interact with their targets with pronounced differences. We further found that the two types of CAP-Gly domains use alternative mechanisms to target the C-terminal domains of EBs. We succeeded to solve the crystal structure of a complex composed of a heterodimer of EB1 and EB3 C-termini together with the CAP-Gly domain of p150(glued). Together, our results provide mechanistic insights into the interaction properties of EBs and offer a molecular framework for the systematic investigation of their functional differences in cells.     

Sequence Determinants of a Microtubule Tip Localization Signal (MtLS)

Microtubule plus-end-tracking proteins (+TIPs) specifically localize to the growing plus-ends of microtubules to regulate microtubule dynamics and functions. A large group of +TIPs contain a short linear motif, SXIP, which is essential for them to bind to end-binding proteins (EBs) and target microtubule ends. The SXIP sequence site thus acts as a widespread microtubule tip localization signal (MtLS). Here we have analyzed the sequence-function relationship of a canonical MtLS. Using synthetic peptide arrays on membrane supports, we identified the residue preferences at each amino acid position of the SXIP motif and its surrounding sequence with respect to EB binding. We further developed an assay based on fluorescence polarization to assess the mechanism of the EB-SXIP interaction and to correlate EB binding and microtubule tip tracking of MtLS sequences from different +TIPs. Finally, we investigated the role of phosphorylation in regulating the EB-SXIP interaction. Together, our results define the sequence determinants of a canonical MtLS and provide the experimental data for bioinformatics approaches to carry out genome-wide predictions of novel +TIPs in multiple organisms.     

Tyrosine-dependent capture of CAP-Gly domain-containing proteins in complex mixture by EB1 C-terminal peptidic probes

Microtubule dynamics is regulated by an array of microtubule associated proteins of which the microtubule plus-end tracking proteins (+TIPs) are prominent examples. +TIPs form dynamic interaction networks at growing microtubule ends in an EB1-dependent manner. The interaction between the C-terminal domain of EB1 and the CAP-Gly domains of the +TIP CLIP-170 depends on the last tyrosine residue of EB1. In the present study, we generated peptidic probes corresponding to the C-terminal tail of EB1 to affinity-capture binding partners from cell lysates. Using an MS-based approach, we showed that the last 15 amino-acid residues of EB1, either free or immobilized on beads, bound recombinant CAP-Gly domains of CLIP-170. We further demonstrate that this binding was prevented when the C-terminal tyrosine of EB1 was absent in the peptidic probes. Western blotting in combination with a label-free quantitative proteomic analysis revealed that the peptidic probe harboring the C-terminal tyrosine of EB1 effectively pulled-down proteins with CAP-Gly domains from endothelial cell extracts. Additional proteins known to interact directly or indirectly with EB1 and the microtubule cytoskeleton were also identified. Our peptidic probes represent valuable tools to detect changes induced in EB1-dependent +TIP networks by external cues such as growth factors and small molecules.     

Adenomatous Polyposis Coli as a Scaffold for Microtubule End-Binding Proteins

End-binding proteins (EBs), referred to as the core components of the microtubule plus-end tracking protein network, interact with the C-terminus of the adenomatous polyposis coli (APC) tumor suppressor. This interaction is disrupted in colon cancers expressing truncated APC. APC and EBs act in synergy to regulate microtubule dynamics during spindle formation, chromosome segregation and cell migration. Since EBs autonomously end-track microtubules and partially co-localize with APC at microtubule tips in cells, EBs have been proposed to direct APC to microtubule ends. However, the interdependency of EB and APC localization on microtubules remains elusive. Here, using in vitro reconstitution and single-molecule imaging, we have investigated the interplay between EBs and the C-terminal domain of APC (APC-C) on dynamic microtubules. Our results show that APC-C binds along the microtubule wall but does not accumulate at microtubule tips, even when EB proteins are present. APC-C was also found to enhance EB binding at the extremity of growing microtubules and on the microtubule lattice: APC-C promotes EB end-tracking properties by increasing the time EBs spend at microtubule growing ends, whereas a pool of EBs with a fast turnover accumulates along the microtubule surface. Overall, our results suggest that APC is a promoter of EB interaction with microtubules, providing molecular determinants to reassess the relationship between APC and EBs.     

Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1

Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtubules, where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1, and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we report crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. We propose that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end.     

Multisite phosphorylation disrupts arginine-glutamate salt bridge networks required for binding of cytoplasmic linker-associated protein 2 (CLASP2) to end-binding protein 1 (EB1)

A group of diverse proteins reversibly binds to growing microtubule plus ends through interactions with end-binding proteins (EBs). These +TIPs control microtubule dynamics and microtubule interactions with other intracellular structures. Here, we use cytoplasmic linker-associated protein 2 (CLASP2) binding to EB1 to determine how multisite phosphorylation regulates interactions with EB1. The central, intrinsically disordered region of vertebrate CLASP proteins contains two SXIP EB1 binding motifs that are required for EB1-mediated plus-end-tracking in vitro. In cells, both EB1 binding motifs can be functional, but most of the binding free energy results from nearby electrostatic interactions. By employing molecular dynamics simulations of the EB1 interaction with a minimal CLASP2 plus-end-tracking module, we find that conserved arginine residues in CLASP2 form extensive hydrogen-bond networks with glutamate residues predominantly in the unstructured, acidic C-terminal tail of EB1. Multisite phosphorylation of glycogen synthase kinase 3 (GSK3) sites near the EB1 binding motifs disrupts this electrostatic "molecular Velcro." Molecular dynamics simulations and (31)P NMR spectroscopy indicate that phosphorylated serines participate in intramolecular interactions with and sequester arginine residues required for EB1 binding. Multisite phosphorylation of these GSK3 motifs requires priming phosphorylation by interphase or mitotic cyclin-dependent kinases (CDKs), and we find that CDK- and GSK3-dependent phosphorylation completely disrupts CLASP2 microtubule plus-end-tracking in mitosis.     

Amer2 Protein Interacts with EB1 Protein and Adenomatous Polyposis Coli (APC) and Controls Microtubule Stability and Cell Migration

EB1 is key factor in the organization of the microtubule cytoskeleton by binding to the plus-ends of microtubules and serving as a platform for a number of interacting proteins (termed +TIPs) that control microtubule dynamics. Together with its direct binding partner adenomatous polyposis coli (APC), EB1 can stabilize microtubules. Here, we show that Amer2 (APC membrane recruitment 2), a previously identified membrane-associated APC-binding protein, is a direct interaction partner of EB1 and acts as regulator of microtubule stability together with EB1. Amer2 binds to EB1 via specific (S/T)xIP motifs and recruits it to the plasma membrane. Coexpression of Amer2 and EB1 generates stabilized microtubules at the plasma membrane, whereas knockdown of Amer2 leads to destabilization of microtubules. Knockdown of Amer2, APC, or EB1 reduces cell migration, and morpholino-mediated down-regulation of Xenopus Amer2 blocks convergent extension cell movements, suggesting that the Amer2-EB1-APC complex regulates cell migration by altering microtubule stability.     

EB1 Recognizes the Nucleotide State of Tubulin in the Microtubule Lattice

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.     

Insights into EB1 structure and the role of its C-terminal domain for discriminating microtubule tips from the lattice

End-binding proteins (EBs) comprise a conserved family of microtubule plus end-tracking proteins. The concerted action of calponin homology (CH), linker, and C-terminal domains of EBs is important for their autonomous microtubule tip tracking, regulation of microtubule dynamics, and recruitment of numerous partners to microtubule ends. Here we report the detailed structural and biochemical analysis of mammalian EBs. Small-angle X-ray scattering, electron microscopy, and chemical cross-linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains, an arrangement reminiscent of that seen in other microtubule- and actin-binding proteins. Removal of the negatively charged C-terminal tail did not affect the overall conformation of EBs; however, it increased the dwell times of EBs on the microtubule lattice in microtubule tip-tracking reconstitution experiments. An even more stable association with the microtubule lattice was observed when the entire negatively charged C-terminal domain of EBs was replaced by a neutral coiled-coil motif. In contrast, the interaction of EBs with growing microtubule tips was not significantly affected by these C-terminal domain mutations. Our data indicate that long-range electrostatic repulsive interactions between the C-terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends.     

MAP1B regulates microtubule dynamics by sequestering EB1/3 in the cytosol of developing neuronal cells

MAP1B, a structural microtubule (MT)‐associated protein highly expressed in developing neurons, plays a key role in neurite and axon extension. However, not all molecular mechanisms by which MAP1B controls MT dynamics during these processes have been revealed. Here, we show that MAP1B interacts directly with EB1 and EB3 (EBs), two core ‘microtubule plus‐end tracking proteins’ (+TIPs), and sequesters them in the cytosol of developing neuronal cells. MAP1B overexpression reduces EBs binding to plus‐ends, whereas MAP1B downregulation increases binding of EBs to MTs. These alterations in EBs behaviour lead to changes in MT dynamics, in particular overstabilization and looping, in growth cones of MAP1B‐deficient neurons. This contributes to growth cone remodelling and a delay in axon outgrowth. Together, our findings define a new and crucial role of MAP1B as a direct regulator of EBs function and MT dynamics during neurite and axon extension. Our data provide a new layer of MT regulation: a classical MAP, which binds to the MT lattice and not to the end, controls effective concentration of core +TIPs thereby regulating MTs at their plus‐ends.     

A novel localization pattern for an EB1-like protein links microtubule dynamics to endomembrane organization

A group of microtubule-associated proteins called +TIPs (plus end tracking proteins), including EB1 family proteins, label growing microtubule ends specifically in diverse organisms and are implicated in spindle dynamics, chromosome segregation, and directing microtubules toward cortical sites. Here, we report three new EB1-like proteins from Arabidopsis and provide the intracellular localization for AtEB1, which differs from all known EB1 proteins in having a very long acidic C-terminal tail. In marked contrast to other EB1 proteins, the GFP-AtEB1 fusion protein localizes not only to microtubule plus ends but also to motile, pleiomorphic tubulovesicular membrane networks that surround other organelles and frequently merge with the endoplasmic reticulum. AtEB1 behavior thus resembles that of +TIPs, such as the cytoplasmic linker protein CLIP-170, that are known to associate with and pull along membrane tubules in animal systems but for which homologs have not been identified in plants. In addition, though EB1 proteins are believed to stabilize microtubules, a different behavior is observed for AtEB1 where instead of stabilizing a microtubule it localizes to already stabilized regions on a microtubule. The dual localization pattern of AtEB1 suggests links between microtubule plus end dynamics and endomembrane organization during polarized growth of plant cells.     

SxIP binding disrupts the constitutive homodimer interface of EB1 and stabilizes EB1 monomer

SxIP is a microtubule tip localizing signal found in many +TIP proteins that bind to the hydrophobic cavity of the C-terminal domain of end binding protein 1 (EB1) and then positively regulate the microtubule plus-end tracking of EBs. However, the exact mechanism of microtubule activation of EBs in the presence of SxIP signaling motif is not known. Here, we studied the effect of SxIP peptide on the native conformation of EB1 in solution. Using various NMR experiments, we found that SxIP peptide promoted the dissociation of natively formed EB1 dimer. We also discovered that I224A mutation of EB1 resulted in an unfolded C-terminal domain, which upon binding with the SxIP motif folded to its native structure. Molecular dynamics simulations also confirmed the relative structural stability of EB1 monomer in the SxIP bound state. Residual dipolar couplings and heteronuclear NOE analysis suggested that the binding of SxIP peptide at the C-terminal domain of EB1 decreased the dynamics and conformational flexibility of the N-terminal domain involved in EB1-microtubule interaction. The SxIP-induced disruption of the dimeric interactions in EB1, coupled with the reduction in conformational flexibility of the N-terminal domain of EB1, might facilitate the microtubule association of EB1.     

The Microtubule Plus-End Tracking Proteins SPR1 and EB1b Interact to Maintain Polar Cell Elongation and Directional Organ Growth in Arabidopsis

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.     

Interactions between EB1 and Microtubules: DRAMATIC EFFECT OF AFFINITY TAGS AND EVIDENCE FOR COOPERATIVE BEHAVIOR*

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.     

CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end–binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated α-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.     

Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization

Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca²⁺-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca²⁺ signaling since Ca²⁺- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.     

EB1 and EB3 control CLIP dissociation from the ends of growing microtubules

EBs and CLIPs are evolutionarily conserved proteins, which associate with the tips of growing microtubules, and regulate microtubule dynamics and their interactions with intracellular structures. In this study we investigated the functional relationship of CLIP-170 and CLIP-115 with the three EB family members, EB1, EB2(RP1), and EB3 in mammalian cells. We showed that both CLIPs bind to EB proteins directly. The C-terminal tyrosine residue of EB proteins is important for this interaction. When EB1 and EB3 or all three EBs were significantly depleted using RNA interference, CLIPs accumulated at the MT tips at a reduced level, because CLIP dissociation from the tips was accelerated. Normal CLIP localization was restored by expression of EB1 but not of EB2. An EB1 mutant lacking the C-terminal tail could also fully rescue CLIP dissociation kinetics, but could only partially restore CLIP accumulation at the tips, suggesting that the interaction of CLIPs with the EB tails contributes to CLIP localization. When EB1 was distributed evenly along the microtubules because of overexpression, it slowed down CLIP dissociation but did not abolish its preferential plus-end localization, indicating that CLIPs possess an intrinsic affinity for growing microtubule ends, which is enhanced by an interaction with the EBs.     

SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase

The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.     

Key interaction modes of dynamic +TIP networks

Dynamic microtubule plus-end tracking protein (+TIP) networks are implicated in all functions of microtubules, but the molecular determinants of their interactions are largely unknown. Here, we have explored key binding modes of +TIPs by analyzing the interactions between selected CAP-Gly, EB-like, and carboxy-terminal EEY/F-COO(-) sequence motifs. X-ray crystallography and biophysical binding studies demonstrate that the beta2-beta3 loop of CAP-Gly domains determines EB-like motif binding specificity. They further show how CAP-Gly domains serve as recognition domains for EEY/F-COO(-) motifs, which represent characteristic and functionally important sequence elements in EB, CLIP-170, and alpha-tubulin. Our findings provide a molecular basis for understanding the modular interaction modes between alpha-tubulin, CLIPs, EB proteins, and the dynactin-dynein motor complex and suggest that multiple low-affinity binding sites in different combinations control dynamic +TIP networks at microtubule ends. They further offer insights into the structural consequences of genetic CAP-Gly domain defects found in severe human disorders.