Regulation of end-binding protein EB1 in the control of microtubule dynamics

Abstract

The regulation of microtubule dynamics is critical to ensure essential cell functions, such as proper segregation of chromosomes during mitosis or cell polarity and migration. End-binding protein 1 (EB1) is a plus-end-tracking protein (+TIP) that accumulates at growing microtubule ends and plays a pivotal role in the regulation of microtubule dynamics. EB1 autonomously binds an extended tubulin-GTP/GDP-Pi structure at growing microtubule ends and acts as a molecular scaffold that recruits a large number of regulatory +TIPs through interaction with CAP-Gly or SxIP motifs. While extensive studies have focused on the structure of EB1-interacting site at microtubule ends and its role as a molecular platform, the mechanisms involved in the negative regulation of EB1 have only started to emerge and remain poorly understood. In this review, we summarize recent studies showing that EB1 association with MT ends is regulated by post-translational modifications and affected by microtubule-targeting agents. We also present recent findings that structural MAPs, that have no tip-tracking activity, physically interact with EB1 to prevent its accumulation at microtubule plus ends. These observations point out a novel concept of “endogenous EB1 antagonists” and emphasize the importance of finely regulating EB1 function at growing microtubule ends.

Introduction

The microtubule (MT) cytoskeleton is involved in essential cellular functions such as intracellular transport, maintenance of cell shape, polarity, cell signaling, and mitosis. MTs are assembled by polymerization of α-/β-tubulin heterodimers that are organized head-to-tail to form 13 protofilaments. Each tubulin dimer has two GTP binding sites. The α-tubulin monomer contains a non-exchangeable site always filled with GTP, whereas the β-tubulin monomer has an exchangeable site which is exposed at the dimer surface and can bind GTP, its hydrolyzed form GDP-Pi, or GDP alone when the Pi is released [1]. MT filaments are polarized with a minus end that exposes α-tubulin and a plus end that exposes β-tubulin. MT-plus ends have been extensively characterized over the past few years, whereas the organization and regulation of MT minus ends have only recently started to be understood [2].

MT ends undergo continuous cycles of polymerization (growth) and depolymerization (shrinkage), with periods of pauses, a process referred to as “dynamic instability” [3]. The transition between MT growth and shrinkage is defined as catastrophe, and a rescue defines the switch from shortening to growth [4, 5]. MT dynamic instability is intrinsically driven by hydrolysis of β-tubulin-bound GTP, which occurs with a delay after a tubulin dimer has been incorporated into the sheet-like structure of growing microtubule ends [6]. Due to the delay in GTP hydrolysis, a cap of GTP-tubulin (GTP-cap) is formed at the end of growing MTs and is believed to protect the MT from depolymerization. A catastrophe occurs once the GTP-cap disappears and GDP-tubulin is exposed at the end of the MT [3]. This so-called “GTP-cap model” is a standard to explain MT dynamic instability [4, 5], and has been challenged by several groups over the past few years [7–10].

Dynamic instability is essential to MT functions, including exploration of the cytosol, connection with various cellular components, and segregation of chromosomes towards cell poles during mitosis. This process is finely controlled by a large number of regulatory MT-associated proteins (MAPs) that interact with tubulin or MTs. Among these, MT-plus-end-tracking proteins (+TIPs) constitute a family of structurally and functionally diverse MT regulators that accumulate at growing plus ends and are in a privileged position to control the fate of MT tips [6]. The end-binding protein EB1 is a +TIP that has the intrinsic ability to bind growing MT ends and recruit networks of interacting +TIP partners. Due to its pivotal role in regulating MT dynamics at the plus ends, EB1 has been the subject of extensive studies.

This review summarizes recent insights into the structural basis of EB1 binding at growing MT ends and interaction with other +TIPs, as well as its regulation by post-translational modifications and microtubule-targeting agents (MTAs). We also present recent findings that EB1 localization at growing MT ends is negatively regulated by direct interaction with different structural MAPs that do not localize at MT ends. Relevance of these studies in human disease is discussed.

A family of end-binding (EB) proteins

EB1 was first discovered in 1995 as an Adenomatous Polyposis Coli (APC)-interacting partner [11] and is now recognized as the leading member of a family of end-binding (EB) proteins, namely, EB1, EB2/RP1 and EB3, encoded by evolutionarily conserved genes designated MAPRE1, MAPRE2 and MAPRE3, respectively [12]. EBs are proteins of approximately 300 amino-acid residues organized into an N-terminal calponin homology (CH) domain involved in MT binding, a flexible and unstructured linker region, and a C-terminal coiled-coil domain responsible for dimerization (Fig. 1). The coiled-coil domain overlaps with a unique motif, referred to as end-binding homology (EBH) domain implicated in self-inhibition and interaction with binding partners, that ends with a flexible acidic tail containing the C-terminal sequence EEY/F [6].

Fig. 1.

Structural organization and post-translational modifications of EB orthologs. Schematic representation of the organization of human EB1, EB2, EB3, and yeast Bim1 and Mal3 polypeptides into functional domains, showing post-translational modifications. Two stars indicate phosphorylation sites that have been validated and for which the kinase is identified, and one star indicates those that have been validated but with no identified kinase. Other potential phosphorylation sites were identified by a phosphoproteomic approach and are not validated. Note that residues K220(A) and Y268(Tyr) in EB1 are sites of acetylation and detyrosination, respectively. CH calponin-homology, EBH EB-homology, C-ter Carboxy-terminal region. Amino-acid numbering is indicated below each EB polypeptide

EB1, EB2, and EB3 show high amino-acid sequence conservation and are ubiquitous, EB1 being highly expressed in most tissues and EB3 being particularly abundant in the brain and skeletal muscle [13]. EB1 and EB3 are generally considered equivalent in their ability to track MT ends and they form homo-and heterodimers in the cytosol [14–16]. In contrast, EB2 is less potent in binding MT ends and +TIPs, and does not heterodimerize with EB1 and EB3 [14, 15, 17]. Most analyses of EBs structure and function have focused on EB1 and its yeast homologs (Mal3 and Bim1), whereas EB3 has been mainly studied in neurons. EB2 remains poorly characterized.

A high-affinity, GTP-dependent EB-binding site at growing MT ends

During MT growth episodes, EB1 and EB3 accumulate at the tip of growing MTs and form “comet-like” structures that disappear during pauses and MT shrinkage. This particular feature has been largely exploited for studying MT dynamics using GFP-tagged EB1 and EB3 as surrogate markers of MT growth [18–20]. The intriguing property of EBs to track MT ends has launched a number of studies that aimed at investigating the structural basis of EB-binding sites at growing ends. In vitro reconstitution experiments using purified proteins indicated that EBs bind autonomously an extended region at the end of dynamic MTs [21, 22] with more than tenfold higher affinity compared to the MT lattice [23]. EB1 turns over rapidly at its binding region on the MT end both in vitro and in vivo [22, 24] and this rapid exchange implies that free EB1 diffusion in the cytosol is a requisite and a rate-limiting step for binding to MT-plus ends. Importantly, EB proteins recognize a transient, GTP/GDP-Pi-dependent conformation at the MT end [25] (Fig. 2). High-resolution analyses revealed that the CH domain of EB1 binds at the corner of four tubulin dimers present in two adjacent protofilaments [26, 27], which makes it ideally positioned to sense MT conformational changes induced by GTP hydrolysis. EBs do not bind to the extreme end of MT ends at which GTP is loaded, but rather bind an extended region corresponding to transient intermediate states generated during GTP hydrolysis. Recent studies further revealed that EB1 interacts with the outer surface of curved and straight tubulin sheets as well as with closed regions of the microtubule lattice [28]. The initial observation that EB binding is lost a few seconds before catastrophe suggested that the EB protein binding site may protect the MT from depolymerization [26]. Further studies [10] indicated that the EB-binding region may indeed correspond to the stabilizing cap, whose length depends on MT growth rate and GTP hydrolysis, and revealed that catastrophe occurs when the number of EB-binding sites at the MT end are reduced to 15–30% of their initial level [10].

Fig. 2.

Nucleotide-dependent EB-binding region at the MT-plus end. MT polymerization is managed by incorporation of GTP-loaded α-/β-tubulin dimers at growing ends. The different nucleotide states of tubulin are presented with the β-tubulin subunit in blue (GTP), grey (GDP-Pi), and black (GDP). Microtubule plus ends undergo two maturation steps: GTP hydrolysis and Pi release from GDP-Pi. The EB1-binding site comprises two adjacent tubulin dimers loaded with either GTP-, GDP-Pi, or other intermediate states of GTP hydrolysis

Notably, EB1 not only binds the nucleotide-sensitive cap at the MT end, but also contributes to its maturation. Paradoxically, EB1 accelerates a conformational transition leading to MT catastrophe by shortening the lifetime of its own protective binding site at the MT-plus end [29], a finding that corroborates previous observations that EB1 increases catastrophe frequency in vitro [14, 21, 29–32]. Together these studies point to EB1 both as a surrogate marker of MT growth and as a crucial regulator of the stabilizing cap, and thus of MT dynamic instability. In living cells, however, EB1 more likely induces persistent MT growth [14] suggesting that the pro-catastrophe effect of EB1 is inhibited. This highlights the complex regulatory effects of EBs on MT dynamics in a cellular context. Post-translational modifications of EBs, as well as local modifications of EBs expression levels, stability, conformation, and/or free diffusion in the cytosol, are thus expected to promote important changes in MT-plus end dynamics.

EB1 at the core of +TIP-interacting networks

Through its C-terminal part, EB1 interacts with numerous partners and recruits them at growing MT ends to form +TIP molecular networks that contribute to the regulation of MT dynamic instability [33–35]. EB1 partners at the MT-plus ends include two major families of proteins, the CAP-Gly, and the SxIP-containing proteins. Cytoskeleton-Associated Protein Glycine-rich (CAP-Gly) proteins, including CLIP-115, CLIP-170, and the dynactin complex p150^glued, contain CAP-Gly domains that mediate their interaction with microtubules and EB proteins. CAP-Gly domains contain a unique hydrophobic cavity that encompasses the highly conserved GKNDG sequence motif and several characteristic Glycine residues [6, 33]. SxIP-containing proteins constitute a large family of EB1 partners characterized by the presence of one or several copies of a Ser/Thr-X-Ile-Pro (serine/threonine-any aminoacid–isoleucine–proline) motif embedded in an unstructured polypeptide region enriched in serine, proline, and basic residues [36, 37]. EB1-interacting SxIP-containing proteins are structurally heterogeneous and control different aspects of MT dynamics and function [17, 38], including the regulation of catastrophes or rescues. They also provide a link between MT ends and other cellular components, such as the plasma membrane, the endoplasmic reticulum, actin fibers, or the kinetochores. Different sets of SxIP-containing proteins were found to interact with EB1 in interphase and at different stages of mitosis, highlighting the importance of temporal changes in EB1 interactome to ensure normal cell division [38].

Global quantification of protein expression conducted in human cell lines [39–41] revealed that EB1 is significantly more abundant than any of its +TIP partners, which suggests that multiple proteins may be recruited to MT ends simultaneously through binding to EB1 [42]. The organization of complex +TIP networks involves both hierarchical and non-hierarchical interactions with EB1 and depends on cooperation [43, 44] or competition [45] between different +TIPs for EB1 binding. The organization of dynamic +TIP networks is also orchestrated by phosphorylation of SxIP-containing proteins. Indeed, regions flanking the SxIP sequence contain positively charged residues that contribute to stabilizing the interaction with surface-exposed acidic residues of the EBH domain [36, 37], which explains why the addition of local negative charges by phosphorylation of serine/threonine residues at the vicinity of the SxIP motif leads to loss of EB1-binding and tip-tracking properties. Phosphorylation by various kinases, including GSK3β and mitotic kinases, appears as a major mechanism for spatiotemporal regulation of SxIP proteins at growing MT ends, with subsequent consequences on MT dynamics [33–37].

EB1 regulation by post-translational modifications

A growing body of evidence indicates that EBs themselves are the targets of post-translational modifications, including phosphorylation, acetylation, and detyrosination (Fig. 1; Table 1).

Table 1.

Post-translational modifications of human and yeast EB orthologs

Protein	PTM site	Domain	Kinase	Effect	References
EB1 (human)	S155, T166	linker	Akt/GSK3β	MT dynamics	[52, 58]
	S40, T154, T206	CH, linker, CC	ASK1	Mitotic spindle	[53, 54]
	Y247, Y268	EBH, C-ter	Src	MT dynamics	[55]
	Y71, T206, Y217	CH, CC	nd	MT dynamics	[58]
	S27, T33, T154, S156, S165, S157	CH, linker, CC	nd	nd	[58]
	K220 (A)	CC	PCAF acetyltransferase	Cell migration, MT dynamics, Mitosis	[59–61]
	Y268 (Tyr)	C-ter	nd	Cell migration	[62]
EB2 (human)	S9, S208, S209, S216, T217, S222, S223	CH, linker	Aurora B, CDK1	MT dynamics, Mitosis	[50]
	S236	CC	CK2	Cell adhesion	[56]
EB3 (human)	S176	linker	Aurora A/B	Proteasome, MT dynamics	[48, 49]
	S162	linker	nd	MT dynamics	[57]
Bim1 (yeast)	S139, S148, S149, S165, S166, S176	linker	Aurora (Ipl1)	MT dynamics, Mitosis	[46]
Mal3 (yeast)	S147, S149, S151	linker	nd	MT dynamics	[47]

Major sites of post-translational modification of EB1 family orthologs are indicated, together with the kinase involved and known associated effects. All residues correspond to phosphorylation sites except for K220 (A) and Y268 (Tyr) in EB1 that are sites of acetylation and detyrosination/tyrosination, respectively

PTM post-translational modification, CC coiled-coil domain, nd not determined

Serine and threonine residues in the flexible linker region of EBs are major sites of phosphorylation during mitosis. In yeast, EB1 homologues Bim1 and Mal3 are phosphorylated on a cluster of six serine residues in the linker region (Fig. 1) and this promotes their dissociation from MTs [46, 47]. The Aurora kinase homolog lpl1 was shown to phosphorylate Bim1 on these sites during anaphase to regulate spindle MT dynamics and spindle midzone disassembly [46]. In humans, Aurora A and Aurora B phosphorylate EB3 on S176 during the early and late stages of mitosis, respectively [48]. Aurora B-mediated phosphorylation of EB3 on S176 both stabilizes EB3 during mitosis by protecting it from degradation by the proteasome [48] and allows coordination of cell adhesion with cytokinesis [49]. Recent studies indicate that EB2 is also phosphorylated on six clustered serine/threonine residues in the linker region by mitotic kinases Aurora B and cdk1 during mitosis, which results in loss of EB2 association with MTs and ensures proper kinetochore MT dynamics and chromosome segregation [50]. Of note, EB1 interacts with Auroras both in cells and in vitro but is not a substrate for these kinases [48, 51]. EB1 rather enhances Aurora B kinase activity by blocking its interaction with protein phosphatase PP2A, thereby preventing subsequent Aurora kinase inactivation [51].

The Akt/GSK3 pathway is another major signaling pathway that induces EB1 phosphorylation on residues S155 and T166 located in the linker region [52]. Interestingly, phosphorylation at these two sites has opposite effects on EB1. Phosphorylation on S155 contributes to EB1 accumulation at growing MT ends by increasing the lifetime or number of EB1 binding sites, resulting in increased cell migration and proliferation [52]. Such post-translational modification may potentially contribute to inhibiting the effect of EB1 on GTP/GDP-Pi maturation. In contrast, T166 phosphorylation leads to a decrease in the lifetime of EB1 binding sites at MT-plus ends, thus reducing EB1 comet length. As will be discussed in the following, the cytotoxic and anti-migratory effects of MT-targeting agents (MTAs) are sensitive to EB1 phosphorylation on residue T166 [52].

Recent studies [53, 54] have identified residues S40, T154, and T206, located in the CH, linker, and coiled-coil region of EB1, respectively, as being phosphorylated by apoptosis signal-regulated kinase (ASK1). Phosphorylation at these sites increases EB1 binding at the plus ends of astral microtubules, thereby regulating mitotic spindle orientation and positioning [53], and ASK1-mediated EB1 phosphorylation at S40 contributes to the recruitment of CLIP-170 and p150^Glued at the plus ends of astral microtubules [54]. Another recent study indicated that tyrosine phosphorylation also contributes to the regulation of EB1 functions [55]. Src kinase was shown to interact with EB1 and to phosphorylate residues Y247 and Y268 in the EBH region and the EEY end of the protein, respectively. EB1 phosphorylation by src at Y247, which is prominent at the centrosome and focal adhesions, reduces EB1 binding to SxIP-containing proteins APC and MCAK, leading to the promotion of cell migration through an increase in MT dynamics [55]. Of note, in endothelial cells, EB2 phosphorylation at residue S236 by the CK2 kinase has been shown to reduce cell adhesion under shear stress [56], whereas VE-cadherin-mediated phosphorylation of EB3 at S162 [57] regulates junctional integrity.

Large-scale phosphoproteomic studies [58] have identified a total of 11 putative phosphorylation sites on human EB1 (Fig. 1; Table 1), most of which correspond to residues already mentioned above [52–55]. Functional studies indicated that overexpression of EB1 phospho-deficient mutants at positions Y71 and T206 leads to decreased MT growth rate and growth length compared to wild-type EB1 [58], suggesting that phosphorylation at these sites may be required for EB1 function on MT dynamics at growing ends. Interestingly, a phospho-mimic mutation at position T206 abrogated EB1 interaction with CLIP-170 and p150^Glued but not with APC nor MCAK, whereas residue Y217 was found essential for EB1 tip-tracking activity, dimerization and interaction with both SxIP-containing (APC, MCAK) and CAP-Gly (CLIP-170, p150^Glued) partners [58]. Further studies should be conducted to validate additional potential EB1 phosphorylation sites (S27, T33, S156, S157, and S165) identified by mass spectrometry.

Other post-translational modifications, such as ubiquitination and acetylation, regulate EBs stability or functions during mitosis. EB3 interacts with the SIAH-1 E3 ligase and gets ubiquitinated and degraded by the proteasome at the end of mitosis, in an Aurora B-dependent manner [48]. Other studies have shown that EB1 is acetylated at residue K220 by P300/CBP-associated factor (PCAF acetyltransferase) in mitosis [59] (Table 1). Acetylation does not affect EB1 dimerization but impairs its interaction with several SxIP-containing +TIPs, including TIP150, MCAK, and DDA3 [59]. Persistent EB1 acetylation leads to aberrant metaphase alignment [59, 60] and impaired directional migration [61]. Finally, the C-terminal tail of EB1 (residues EEY) is the target of detyrosination/tyrosination cycles, similar to what was shown for tubulin. EB1 detyrosination may increase EB1 decoration time at MT-plus ends and decrease MT catastrophe, thereby contributing to increased cell migration [62].

Reciprocal effects of MTAs and EB1

Microtubule-targeting agents (MTAs) are a broad class of compounds that bind to tubulin and/or MTs and interfere with MT polymerization and dynamics. Due to their potent anti-cancer effects, a large number of studies have been dedicated to evaluating their mechanisms of action [63, 64]. Different MTAs bind MTs at different sites. Vinblastine binds the β-subunit of tubulin in a region called “vinca-binding domain” and has been shown to bind with high affinity at the extreme ends of purified MTs in vitro [63]. Eribulin binds another high-affinity binding site on β-tubulin that is exposed at the plus ends of MTs [65, 66], whereas paclitaxel binds to β-tubulin on the inside surface of the MT, at the “taxane-binding site” [67].

MTAs are usually classified as MT polymerizing and depolymerizing compounds according to the effects they have on the MT polymer mass when used at relatively high concentrations. Taxanes (paclitaxel, docetaxel) and epothilones promote MT assembly and stabilization, whereas vinca-alkaloids (vincristine, vinflunine) and eribulin depolymerize MTs. At lower concentrations that do not affect polymer mass but are still cytotoxic, MTAs of both classes suppress MT dynamic instability and behave as mitotic poisons that target the mitotic spindle, inducing mitotic arrest and apoptosis. At very low doses of the nanomolar range showing no cytotoxicity, these compounds impair cell migration and differentiation, and increase rather than suppress dynamic instability. Indeed, ultra-low doses of MTAs often increase growth and shortening rates of interphase MTs and consistently increase catastrophe frequency (Fig. 3), with a concomitant reduction [68–70] and splitting [65] of EB comets.

Fig. 3.

Reciprocal effects of EB1 and MTAs in regulating MT dynamics. Microtubule-targeting agents (MTAs) regulate MT dynamics in two different ways. (1) They accelerate MT maturation steps, thereby shortening the EB-stabilizing cap at MT-plus ends and leading to MT catastrophes. Note that EB1 and MTAs act synergistically to shorten the EB1 binding region. (2) MTAs also trigger Akt/GSK3β-dependent phosphorylation of EB1 on residue T166, which contributes to MTA effects on MT-plus-end maturation. Those two effects of MTAs are not mutually exclusive

Recent studies revealed that in addition to their direct effect through binding to MTs, different classes of MTAs including vincristine, paclitaxel, and patupilone also regulate MT dynamics by promoting GSK3β-mediated phosphorylation of EB1 [52] (Fig. 3). These agents were shown to increase mitochondrial ROS, leading to a decrease in Akt phosphorylation and a subsequent increase in GSK3β activity that in turn induces EB1 phosphorylation on S155 and T166 residues in the linker region. As mentioned above, S155 phosphorylation in cancer cells promotes EB1 accumulation at MT ends by increasing the lifetime of EB1 binding sites, whereas T166 phosphorylation reduces EB1 comet length by decreasing MT growth rate. Of interest, a phospho-defective mutant (T166A) of EB1 induces resistance to MTA actions [52]. In astrocytes, the PI3K-GSK3β signaling pathway also leads to a decrease in EB1 comet length together with decreased cell migration following exposure to HYS-32, a derivative of MT depolymerizing agent combrestatin–A4 [71]. Finally, the vinca-alkaloid compound vinflunine was shown to interfere with the EB1 tyrosination/detyrosination cycle in endothelial and glioblastoma cells by decreasing the level of the detyrosinated form and increasing the native tyrosinated form of EB1, thereby reducing cell migration [62].

Not surprisingly, the presence of EB1 or EB3 was shown to modify the effects of MTAs on MT dynamics in vitro, illustrating the existence of an intrinsic mechanism of cross-regulation between MTs, EBs, and drugs [32, 69]. Indeed, in the absence of EBs, MTAs more likely suppress dynamic instability parameters, such as MT growth and shortening rates in vitro, whereas in the presence of EBs, both polymerizing and depolymerizing agents strongly induce MT catastrophes even at ultra-low drug concentration [32]. Thus, EBs synergize with MTAs to increase MT catastrophes [32] (Fig. 3), which is consistent with the above-mentioned contribution of EB1 to the maturation of MT ends in vitro, but may seem counterintuitive given the opposite effects of EB1 and MTAs on cell proliferation and migration. In glioblastoma cells, the effects of vinflunine on MT dynamics, cell proliferation, and migration as well as tumor growth were also sensitized by overexpression of EB1 [72]. Furthermore, EB1-overexpressing glioblastoma cancer stem cells were highly sensitive to sub-cytotoxic doses of BAL27862, a colchicine site binder, leading to inhibition of cell migration and self-renewal and promoting astrocytic differentiation, whereas EB1-silenced cancer stem cells remained unaffected [73]. In contrast, results remain controversial in breast cancer, as EB1 has been shown either to sensitize [74] or counteract [75] the effects of paclitaxel. The discrepancy between the latter studies may relate to the pharmacological complexity of MTAs and/or to different drug concentrations used in the experiments. Indeed, the sensitizing effect of EB1 on MTAs was found to be more prevalent at low concentrations of drugs [72].

Negative regulation of EB1 at growing ends by structural MAPs

While the structural basis and functional relevance of EB1 interaction with numerous +TIPs have been extensively studied in the past few years, the notion has only recently emerged that EB1 and EB3 accumulation at growing MT ends is negatively regulated by direct interaction with structural MAPs, which bind to the MT lattice and not to the end.

MAP1B, a classical MAP highly expressed in developing neurons, is known to regulate neurite extension and axon growth by promoting MT polymerization and stabilization [76]. An additional layer of MT regulation by MAP1B through functional crosstalk with EBs has been described by Tortosa et al. [77] who showed that MAP1B co-localizes with EB1 and EB3 in growth cones where it locally impairs EB1 and EB3 accumulation at the MT-plus ends. Direct interaction between MAP1B and EBs takes place in the cytosol in a MT-independent manner, and is sensitive to phosphorylation by GSK3 and cdk5 kinases. Physical EB3-MAP1B interaction lowers EB3 mobility in the cytosol, thereby reducing the « effective » concentration of EB3 available for free exchange at MT ends [77]. This in turn leads to local changes in MT dynamics, illustrated by an increase in MT growth rate, and a decrease in time-based catastrophe frequency and MT pauses, in extending neurites of MAP1B-deficient neurons. Thus, MAP1B indirectly regulates MT dynamics at growing plus ends in developing neurons by a mechanism involving cytosolic sequestration of EB proteins (Fig. 4).

Fig. 4.

Negative regulation of EB1 at growing ends by structural MAPs. MAP1B negatively regulates EB1 by sequestration in the cytosol. Highly expressed tau and NMDA-activated MAP2 proteins redistribute EB1 to MT bundles. ATIP3 reduces EB1 turnover on its binding site, probably through post-translational modifications. These three mechanisms result in decreased EB1 accumulation at its binding site on growing MT ends and subsequent changes in MT dynamics

Tau is another major neuronal MAP that promotes MT polymerization and stabilization. Recent studies showed that tau and EBs are enriched in differentiating neuroblastoma cells and that they partially co-localize in extending neurites [78]. When expressed at high levels, tau induces MT bundling and promotes redistribution of EB1 and EB3 along MT bundles [78, 79] by a mechanism that depends on tau phosphorylation on S262 [79]. Direct interaction between tau and EBs in the cytosol leads to reduced binding of EBs at growing MT ends and antagonizes EB1 and EB3 functions [78–81]. A similar mechanism of EB3 trapping on MT bundles has been reported for MAP2-EB3 interaction upon prolonged stimulation of NMDA receptors in hippocampal neurons [82]. Direct interaction between MAP2 and EB3, which is concomitant with NMDA-mediated MAP2 dephosphorylation, gradually recruits EB3 along MAP2-positive microtubule bundles in the dendritic shaft [82] resulting in MT stabilization, allowing synaptic plasticity.

A third mechanism of EB1 negative regulation by a structural MAP has been depicted through the studies of MT-associated tumor suppressor ATIP3 [83]. ATIP3 is a stabilizing MAP [84–87], whose silencing regulates MT dynamics by increasing MT growth rate and reducing catastrophe, which results in an increase of EB1 comet length [87]. Recent studies have shown that ATIP3 interacts directly with EB1 in a MT-independent manner, via a proline-rich motif that differs from the consensus SxIP sequence. ATIP3 does not co-localize with EB1 at growing MT ends and EB1-ATIP3 molecular complexes are mainly detected in the cytosol and along the MT lattice [83]. In contrast to other classical MAPs, ATIP3 does not modify EB1 mobility in the cytosol, and therefore, its effects are unlikely to involve EB1 sequestration. Interestingly, EB1-GFP fluorescence recovery measured by FRAP analyses at growing MT ends was significantly accelerated upon ATIP3 silencing, indicating that ATIP3 reduces the rate of EB1 binding/unbinding to its high-affinity binding site [83]. In vitro studies indicated that purified EB1-interacting domain of ATIP3 has no significant effect on EB1 binding to growing MT ends in a cell-free system, suggesting that negative regulation of EB1 by ATIP3 may involve other cellular components and/or post-translational modifications.

Together, these studies illustrate three major mechanisms by which structural MAPs directly interact with EBs in the cytosol to regulate EBs localization and function at MT-plus ends (Fig. 4), which does not exclude that overexpression of classical MAPs may also alter EB1 comet length through changes in MT stability and dynamics.

These novel findings lead to the interesting concept that a subset of MAPs may represent « endogenous EB1 antagonists », that are immediately available to regulate EB1 and/or EB3 in the cytosol or at the vicinity of MTs. EBs’ regulation by classical MAPs, either by cytosolic sequestration (MAP1B), trapping at MT bundles (tau/MAP2), or reduced turnover at MT ends (ATIP3), results in decreased accumulation of EBs and +TIPs molecular complexes at growing ends with subsequent changes in MT dynamics. Interestingly, EBs interactions with MAP1B and MAP2 are sensitive to phosphorylation, as are +TIPs/EBs complexes, raising the possibility that a functional interplay between classical MAPs and +TIPs may be orchestrated by phosphorylation.

The discovery of novel regulatory mechanisms, by which direct interaction between classical MAPs and EB1 in the cytosol modifies EB1 binding properties at the plus ends, brings an additional layer of complexity to the regulation of MT dynamics, which may take place either locally (in neurite extensions) or upon external stimulation. Given the large number of structural MAPs that are known to promote MT polymerization and stabilization in different physiological situations, it is likely that a growing number of EB-interacting MAPs will be identified in the coming years.

Relevance in human pathology

These observations are of particular relevance in pathological situations, such as cancer and neurodegenerative diseases, in which MT dynamics plays an essential role and is often dysregulated. EB1 has been found overexpressed in various cancer types, including hepatocellular carcinoma [88] and glioblastoma [72] as well as breast [89], colon [90, 91] and oral cancers [92], and stands as a prognostic biomarker whose high expression level correlates with poor survival of the patients. Given its effects on cell sensitivity to MTAs, EB1 may also represent a predictive biomarker for the response of tumors to MT-targeting chemotherapy [72, 74]. Indeed, it was recently demonstrated that high levels of EB1 expression in glioblastoma are associated with a better response to vincristine both in cells and in nude mice [72] and that breast cancer patients with high EB1 levels are better responders to paclitaxel-based chemotherapy [74]. Moreover, functional studies have uncovered mitotic and pro-migratory effects of EB1 overexpression, suggesting that EB1 may also represent a valuable therapeutic target against cancer. Deeper understanding of endogenous and drug-induced regulation of EB1 at growing MT ends, and the identification of molecular alterations of complex +TIP networks in tumor cells, will be an important step towards the design of novel efficient +TIP-targeted anti-cancer therapies. The demonstration that ATIP3, a potent MT-associated tumor suppressor in breast, colon, liver and oral cancers, interacts with EB1 to negatively regulate its turnover at MT growing ends [83], further illustrates the importance of targeting EB1 and its interactome in cancer. In this context, peptide aptamers that bind either EB1 or EB3, and interfere with MT dynamics [93] may represent an interesting option.

Fine regulation of MT dynamics is also essential to neuronal development and homeostasis, which rely on cell polarity and intracellular transport. A wide range of SxIP-containing or CAP-Gly proteins are involved in neuronal functions and were reported to be altered in neurological diseases (reviewed in [94]), suggesting that EB-interacting molecular networks may also be modified in the central nervous system in pathological situations. In addition to +TIPs, well-characterized neuronal MAPs, such as MAP1B, MAP2, and tau, that play essential roles in neuronal polarity and synaptic plasticity, also directly bind EB1 and EB3 to antagonize their functions, as discussed above. Interestingly, EB1 indirectly contributes to the phosphorylation of KIF2A depolymerizing kinesin by tau-tubulin kinases TTBK2 [95], an observation that adds additional complexity to the effects of EB1 in MT dynamics regulation. Together, these findings suggest that not only +TIPs or MAPs, but also protein kinases that control EBs molecular interactions, are likely to locally contribute to spatiotemporal regulation of MT dynamics and may be targeted in human diseases.

Concluding remarks

Since the discovery of the EB1 protein family, extensive studies have highlighted its pivotal role both as a marker and a regulator of MT dynamic instability at growing ends. The EB1 binding site at the MT-plus end has been described at high resolution, and the molecular basis for EB1 recruitment of a wide family of proteins at the plus ends has been largely documented. The apparent contradiction between EB1 effects in vitro (inducing catastrophe) and in vivo (promoting persistent growth), together with the impact of post-translational modifications of EBs and intracellular partners on MT dynamics remain to be fully addressed. Recent studies have evidenced an additional level of indirect regulation of EBs at the plus ends by structural MAPs that bind EBs in the cytosol and antagonize their functions. Given the key role of EBs molecular complexes in the control of essential MT functions, further understanding the exquisite regulation of EBs interactions may pave the way to attractive therapeutic strategies targeting the MT cytoskeleton against human diseases.

Abbreviations

APC

Adenomatous polyposis coli

ATIP

AT2 receptor-interacting protein

CAP-Gly

Cytoskeletal-associated protein glycine-rich

Cdk

Cyclin-dependent kinase

CH

Calponin homology

CLIP

Cytoplasmic linker protein

DDA3

Differential display activated by p53

EB

End-binding protein

EBH

End-binding homology

GDP

Guanosine diphosphate

GSK3β

Glycogen synthase kinase 3 beta

GTP

Guanosine triphosphate

KIF2A

Kinesin heavy chain member 2A

MAP

Microtubule-associated protein

MAPRE

Microtubule-associated protein RP/EB

MCAK

Mitotic centromere-associated kinesin

MT

Microtubule

MTA

Microtubule-targeting agent

NMDA

N-Methyl-d-aspartate

PCAF

P300/CBP-associated factor

Pi

Inorganic phosphate

PI3K

Phosphoinositide 3-kinase

PP2A

Protein phosphatase 2

ROS

Reactive oxygen species

SxIP

Serine-any amino acid–isoleucine–proline

+TIP

Microtubule plus end-tracking protein

TTBK

Tau-tubulin kinase

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.