Microtubule +TIPs at aglanceAnna Akhmanova1 and Michel O.Steinmetz2

1Department of Cell Biology, Erasmus MedicalCenter, PO Box 2040, 3000 CA Rotterdam, TheNetherlands2Biomolecular Research, Structural Biology, PaulScherrer Insititut, CH-5232 Villigen PSI, Switzerland(a.akhmanova@erasmusmc.nl;michel.steinmetz@psi.ch)

Journal of Cell Science 123, 3415-3419 © 2010. Published by The Company of Biologists Ltddoi:10.1242/jcs.062414

This article is part of a Minifocus on microtubuledynamics. For further reading, please see relatedarticles: ‘Kinesins at a glance’ by Sharyn A. Endowet al., (J. Cell Sci. 123, pp. 3420-3424), ‘Tubulindepolymerization may be an ancient biological motorprocess’ by J. Richard McIntosh et al. (J. Cell Sci.123, pp. 3425-3434), ‘Towards a quantitativeunderstanding of mitotic spindle assembly andmechanics’ by Alex Mogilner and Erin Craig (J. CellSci. 123, pp. 3435-3445) and ‘Post-translational

modifications of microtubules’ by Dorota Wloga andJacek Gaertig (J. Cell Sci. 123, pp. 3447-3455).

Microtubules are highly dynamic hollow tubesthat are involved in many vital cellularactivities, including maintenance of cell shape,division, migration and intracellular transport.They are assembled from heterodimers of -and -tubulin that align in a head-to-tail fashion.Microtubules are, thus, intrinsically polarbecause they contain two structurally distinctends: a slow-growing minus end, exposing -tubulin subunits; and a fast-growing plus end,exposing -tubulin subunits (for a review, seeNogales and Wang, 2006). In mammalian cells,microtubule minus ends are often stablyanchored, whereas the plus ends are highlydynamic and stochastically switch betweenphases of growth and shrinkage, a process that ispowered by GTP hydrolysis.

Microtubule plus-end tracking proteins(+TIPs) are a structurally and functionally

diverse group of proteins that are distinguishedby their specific accumulation at microtubuleplus ends (Mimori-Kiyosue et al., 2000; Perez etal., 1999; Schuyler and Pellman, 2001). +TIPstypically target growing but not shrinkingmicrotubule ends; however +TIP associationwith depolymerizing ends can occur and, insome organisms such as budding yeast, is evenquite common. In this Cell Science at a Glancearticle we review and illustrate the currentknowledge of these peculiar proteins,summarize their structural and functionalproperties, and discuss the proposed molecularmechanisms that they use to track microtubuleends.

Classification of +TIPsThe first reported +TIP was cytoplasmic linkerprotein of 170 kDa (CLIP-170, officially knownas CLIP1) (Perez et al., 1999). Since itsdiscovery, more than 20 different +TIP familieshave been identified. +TIPs are usually

3415Cell Science at a Glance

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© Journal of Cell Science 2010 (123, pp. 3414–3418)

Microtubule +TIPs at a GlanceAnna Akhmanova and Michel O. Steinmetz

Abbreviations: AAA, ATPase family associated with various cellular activities; APC, adenomateous polyposis coli protein; Arm, armadillo repeat; basic-S/P, sequence regions enriched in basic, serine and proline residues; CAP-Gly, cytoskeleton-associated protein glycine-rich; CDK5RAP2, CDK5 regulatory subunit- associated protein 2; CH, calponin homology; CLASP, CLIP-associated protein; CLIP-170, cytoplasmic linker protein of 170 kDa; Dam1, DUO1- and MPS1-interacting protein 1; EB, end-binding protein; EBH, EB homology; EEY/F, C-terminal Glu-Glu-Tyr/Phe tripeptide motif; EF-hand, Ca2+-binding motif; FOP, FGFR1 oncogene partner; Gas2, growth-arrest-specific protein 2; HC, heavy chain; Lis1, lissencephaly-1 protein; LisH, Lis1 homology; MACF, microtubule-actin

crosslinking factor; MCAK, mitotic centromere-associated kinesin; MT, microtubule; Ncd, non-claret disjunctional; p140Cap, p130Cas-associated protein of 140 kDa (also known as SRCIN1, SRC kinase signaling inhibitor 1, SNIP, SNAP-25-interacting protein); RhoGEF2, Rho-type guanine nucleotide-exchange factor 2; SAM, sterile α-motif domain; SAMP, Ser-Ala-Met-Pro repeat; STIM1, stromal interaction molecule 1; SxIP, Ser-x-Ile-Pro tetrapeptide motif, where x denotes any amino acid residue; +TIP, microtubule plus-end tracking protein; TIP150, +TIP of 150 kDa; TM, transmembrane domain; TOG, named after the discovery in human chTOG; WD40, ~40 amino acid motifs, often terminating in a Trp-Asp dipeptide; XMAP215, microtubule-associated protein of 215 kDa.

+TIP classification What is a +TIP?

+TIP localization

+TIP distribution in mammalian interphase cells

Monkeykidney cellstained forendogenousEB1

Dynein

SxIP proteins

EBH-SxIP

CAP-Gly-EEY/F

CAP-Gly proteins

Proteins withunknownEB-bindingmechanisms

Microtubule

+TIPs localize to and track dynamic MT plus ends

Growing MT

Catastrophe

GDP

GTP

Rescue

Shrinking MT

Plus end

Minus end

αβ

αβ

βα

βα

In vitro, some +TIPs canbind to growing but notshrinking MT minus ends

1D lattice diffusion(MCAK, XMAP215)

Kinesin-based transport(Tea2–Tip1, Kip2–Bik1)

Recognition of specificplus-end structure (EB1)

Recognitionof composite+TIP/tubulin-bindingsites (CLIP-170, MCAK)

3D diffusionin cytoplasm(RhoGEF2, Lis1)

Co-polymerization

Lattice-maturation-induced release

2D diffusionin membrane

(STIM1)

Hitchhiking

Processiveend tracking(XMAP215, Dam1)

Fastexchange(EB1,CLIP-170) Proteins in parentheses

indicate selected examplesof +TIPs using a particularmechanism

Microtubule dynamics

Interactions with cellular structures

Polymerization(XMAP215, EB1)

Depolymerization(MCAK)

Stabilization(CLASP, APC, MACF)

Cortex(CLASP, APC, MACF, CLIP-170, EB1,dynein, dynactin, LIS1)

Rescue(CLIP-170, CLASP)

Catastrophe(MCAK)

Kinetochores(Dam1, CLASP, CLIP-170, APC, EB1,dynein, dynactin, LIS1, MCAK)

F-actin(MACF, CLASP, APC, CLIP-170, Kar9,RhoGEF2, p140Cap)

Vesicles(dynein, dynactin,CLIP-170, Melanophilin)

Endoplasmic reticulum(EB1, STIM1)

Centrosome(XMAP215, EB1, CLASP, APC,dynein, dynactin, Lis1, FOP,CDK5RAP2)

Microtubules(Ncd, Klp2)

EEY/F

EB proteins

EBEBH

Coiledcoil

Coiledcoil

EEY/F

CAP–Gly proteins

CLIP170

p150glued

CAP–GlyCoiled coil ZnF

Basic-S/P

Motor proteins

Dynein HCHelical

Coiled coil

AAA

Tea2 NcdKinesin

TOG proteins

XMAP215

CLASP

HelicalBasic-S/P

TOG-like

SxIP

Other proteins

Lis1

Kar9

WD40LisH

Helical

Basic-S/P

15/20 aarepeats

SxIP proteins

APC

MACF

MCAK

STIM1 Further examples: CLASP, p140Cap, melanophilinRhoGEF2, CDK5RAP2, TIP150, navigator

Arm

Helical Basic-S/P

SAMPPlakin Spectrin EF

GAS2

TM

SAM

EF

SxIP

SxIP

SxIP

SxIP

+TIPs that belong to several classes: CLASP (SxIP, TOG), MCAK (SxIP, motor)

Dam1Multimericcomplex of10 proteins

CH

CH

Basic-S/P

TOG

Kar9

Tea2

Ncd

XMAP215

Lis1 CLIP-170

CH

EBH

EEY/F

CH

EB

Pro

x

Ile

Ser

CLASP

Melanophilin

APC

MCAK

MACF

RhoGEF2

CDK5RAP2

STIM1

p140Cap

TIP150

Live image of a human lungfibroblast expressing an MT marker(mCherry-α-tubulin, red) and a +TIP marker (EB3-GFP, green)

Autonomous+TIP

Non-autonomous+TIPs

GTP-tubulin

Kinesin

Lipid bilayer

GDP-tubulin

+TIP that tracks onlygrowing MT plus ends(e.g. EB1, CLIP-170)

+TIP that tracks growingand shrinking MT plusends (e.g. Dam1, Kar9)

GTP-tubulin

GDP-tubulin

+TIP functions

p150glued

Tyr

Glu

Glu

+TIP interactions Microtubule plus-end tracking mechanisms

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multidomain and/or multisubunit proteins thatrange in size from a few hundred up tothousands of residues. They can be cytoplasmicor membrane bound, and comprise motor andnon-motor proteins (for a review, seeAkhmanova and Steinmetz, 2008). +TIPs can beclassified on the basis of prominent structuralelements that enable them to interact with eachother and with microtubules; however, in somecases, +TIPs combine features characteristic ofseveral +TIP classes.

End-binding (EB) family proteins contain ahighly conserved N-terminal domain that adoptsa calponin homology (CH) fold (Korenbaumand Rivero, 2002) and is responsible formicrotubule binding (Hayashi and Ikura, 2003).In mammalian EB1 and EB3, a CH domain withthe adjacent linker sequence is sufficient forplus-end tracking (Komarova et al., 2009; Skubeet al., 2010); however dimerization is importantfor microtubule plus-end recognition by theiryeast homologue Bim1 (Zimniak et al., 2009).The C terminus of EB proteins harbors an-helical coiled-coil domain that mediatesparallel dimerization of EB monomers(Honnappa et al., 2005; Slep et al., 2005). Itfurther comprises the unique EB homology(EBH) domain and an acidic tail encompassinga C-terminal EEY/F motif, reminiscent of thoseof -tubulin and CLIP-170 (Komarova et al.,2005; Miller et al., 2006; Weisbrich et al., 2007).Notably, plant EB proteins lack the EEY/Fmotif, and some EB family members, such asEB1c in Arabidopsis thaliana, exhibit apositively charged C-terminus that isresponsible for nuclear localization (Komakiet al., 2010). Both the EBH domain and theEEY/F motif enable the EB proteins tophysically interact with an array of +TIPs torecruit them to microtubule ends.

The cytoskeleton-associated protein glycine-rich (CAP-Gly) domain is a small globularmodule that contains a unique conservedhydrophobic cavity and several characteristicglycine residues (Li et al., 2002; Saito et al.,2004). CAP-Gly domains use their hydrophobiccavity to confer interactions with microtubulesand EB proteins by specifically recognizingC-terminal EEY/F sequence motifs (Honnappaet al., 2006; Mishima et al., 2007; Weisbrich etal., 2007). Prominent examples are the CLIPproteins and the large subunit of the dynactincomplex p150glued. A single CAP-Gly domain ofCLIP-170, together with the adjacent serine-richregion, can track growing microtubule ends(Gupta et al., 2009).

The largest group of +TIPs comprises largeand complex, often multidomain, proteinscontaining low-complexity sequence regionsthat are rich in basic, serine and proline (basic-S/P) residues. They share the small four-residue

motif Ser-x-Ile-Pro (SxIP, where x denotes anyamino acid), which is specifically recognized bythe EBH domain of EB proteins (Honnappaet al., 2009). Prominent examples of this diverseclass of +TIPs are the adenomatous polyposiscoli (APC) tumour suppressor, the spectraplakinmicrotubule–actin crosslinking factor (MACF)and the mitotic centromere-associated kinesin(MCAK). Because SxIP motifs are very short,they can be easily acquired or lost duringevolution; for example, CDK5RAP2, a proteinimplicated in microcephaly, contains an EB1-binding SxIP motif in humans and dogs but notin rodents (Fong et al., 2009).

Proteins with TOG or TOG-like domains(named after their discovery in the protein ch-TOG) include members of the XMAP215/Dis1family and the CLASPs. Tandemly arrangedTOG domains mediate binding to tubulin andare probably responsible for microtubulegrowth-promoting activity of these proteins(Al-Bassam et al., 2006; Brouhard et al., 2008;Slep and Vale, 2007) (for a review, see Slep,2009a). Additional domains, such as SxIPmotifs in CLASPs, are required for targeting ofthese proteins to microtubule plus ends andother subcellular sites (Mimori-Kiyosue et al.,2005).

Both microtubule plus- and minus-end-directed motor proteins can track growingmicrotubule ends. Examples are the yeastkinesins Tea2 and Kip2, the microtubule-depolymerising kinesin 13 MCAK andcytoplasmic dynein (reviewed in Wu et al.,2006). Sequences outside the microtubule-binding motor domains, such as the SxIP motifof MCAK (Honnappa et al., 2009), might beneeded for the microtubule tip-trackingbehavior of these proteins.

Finally, there are other +TIPs that cannot begrouped in one of the five classes discussedabove. A prominent example is the Dam1complex – an assembly of ten subunits that formrings of 16-fold symmetry (Lampert et al., 2010;Wang et al., 2007) – and which is found in yeastbut not in higher organisms. Other examples arethe Saccharomyces cerevisiae protein Kar9(Liakopoulos et al., 2003; Moore and Miller,2007), and the highly conserved cytoplasmicdynein accessory factor lissencephaly-1protein (Lis1) (for a review, see Vallee and Tsai,2006).

Dynamic +TIP interaction networksOne hallmark of +TIPs is that they formdynamic interaction networks that rely on alimited number of protein modules and linearsequence motifs, such as the CH, EBH andCAP-Gly domains, and EEY/F and SxIP motifs.These elements mediate the interaction witheach other and microtubules, and typically

display affinities in the low micromolar range(Gupta et al., 2009; Mishima et al., 2007;Weisbrich et al., 2007).

EB proteins are now generally accepted torepresent core components of +TIP networksbecause they autonomously track growingmicrotubule plus ends independently of anybinding partners (Bieling et al., 2008; Bieling etal., 2007; Dixit et al., 2009; Komarova et al.,2009; Zimniak et al., 2009). Moreover, EBproteins directly associate with almost all otherknown +TIPs and, by doing so, target them togrowing microtubule plus ends (for reviews, seeAkhmanova and Steinmetz, 2008; Slep, 2009b).SxIP motifs act as a general ‘microtubule tiplocalization signal’ (MtLS) by interacting withthe EBH domain of EB proteins (Honnappaet al., 2009). Similarly, EEY/F motifs of EBproteins and -tubulin guide CAP-Gly proteinsto microtubule tips (Bieling et al., 2008; Dixitet al., 2009). Both the EBH-SxIP and theCAP-Gly-EEY/F interactions have beenanalyzed to high resolution (Hayashi et al.,2007; Honnappa et al., 2009; Honnappaet al., 2006; Mishima et al., 2007; Plevin et al.,2008; Weisbrich et al., 2007). The two distinctbinding modes were revealed through thesestructures and offer a molecular basis forunderstanding the majority of known interactionnodes in dynamic +TIP networks.

The EBH-SxIP and CAP-Gly-EEY/Finteractions can be regulated by post-translationalmodifications. Phosphorylation of Ser residues inthe vicinity of the SxIP motifs (Honnappa et al.,2009; Kumar et al., 2009; Watanabe et al., 2009)disrupts their interaction with EB proteins,whereas the removal of the C-terminal Tyr of -tubulin has a negative effect on the accumulationof CAP-Gly proteins at microtubule tips (Bielinget al., 2008; Peris et al., 2006).

+TIP tracking mechanismsBecause +TIPs form complex interactionnetworks, in-vitro reconstitution studies usingpurified components are required to determinewhether plus-end tracking behavior is anautonomous property of a particular protein.Using this approach, it was shown that some+TIPs can associate with growing microtubuleends in the absence of any additional factors.Autonomous processive microtubule tiptracking, whereby the protein stays bound to themicrotubule end during multiple rounds ofsubunit addition, has been described forXMAP215 (Brouhard et al., 2008). Anotherexample is the yeast Dam1 complex, whichcontinuously tracks both growing and shrinkingmicrotubule ends, possibly by using a form of adiffusion-based mechanism (Lampert et al.,2010). Finally, various EB family membersfrom different species bind to growing but not

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shortening plus- and minus ends in vitro(Bieling et al., 2008; Bieling et al., 2007; Dixit etal., 2009; Komarova et al., 2009; Zimniak et al.,2009). Unlike XMAP215, EB proteinsexchange rapidly at the microtubule end,undergoing several cycles of binding andunbinding events before the growingmicrotubule end converts into the maturelattice (Bieling et al., 2007; Dragestein et al.,2008).

It is currently unknown which structuralfeatures of the growing microtubule end arerecognized by autonomously tracking +TIPs;however, these might include the GTP cap at theend of the freshly polymerized microtubule(Lampert et al., 2010; Zanic et al., 2009) orsome specific protofilament arrangement (desGeorges et al., 2008; Sandblad et al., 2006) (for areview, see Coquelle et al., 2009). Anotherattractive idea is that autonomously tracking+TIPs co-polymerize with tubulin subunits andthen get released gradually from the maturelattice (Folker et al., 2005); this mechanism hasnot found support in the in-vitro reconstitutionstudies using EB and CLIP homologs of fissionyeast and vertebrates (Bieling et al., 2007;Bieling et al., 2008; Dixit et al., 2009), but mightstill apply to some other proteins.

Most +TIPs track the ends of growingmicrotubules in a non-autonomous manner.STIM1 and CDK5RAP2, for example, hitchhikeon microtubule tip-bound EB proteins (Fong etal., 2009; Grigoriev et al., 2008; Honnappa et al.,2009). Others, such as CLIP-170, recognizemore complex binding sites that encompassdomains of both EB proteins and tubulin (Bielinget al., 2008; Gupta et al., 2010). Because EBproteins rapidly exchange at microtubule tips,accumulation of their partners at microtubuleends is also dynamic, and mostly depends onthree-dimensional protein diffusion in thecytosol. However, one-dimensional diffusionalong the microtubule lattice might also occur, asis the case for MCAK (Helenius et al., 2006). Inthe case of STIM1, a transmembrane +TIP, two-dimensional diffusion in the membrane isrequired to enable accumulation at microtubuletips (Grigoriev et al., 2008).

For EB proteins and their partners thatdecorate the freshly polymerized microtubuletip, the specificity for microtubule plus ends – asopposed to minus ends – is explained by the factthat, in vivo, minus ends never grow in cells. Bycontrast, the exclusive accumulation atmicrotubule plus ends both in vitro and in vivo isobserved in systems in which plus-end-directedkinesins are involved. Among the best-studiedexamples are the yeast CLIP-170 orthologsBik1 and Tip1, which are concentrated atmicrotubule tips by the kinesins Kip2 and Tea2,respectively (Bieling et al., 2007; Busch et al.,

2004; Carvalho et al., 2004; Miller et al., 2006).It should be noted that kinesins, either alone ortogether with their binding partners, will trackmicrotubule ends only if they do not dissociateimmediately from microtubule ends but areretained on them, either because of interactionswith other +TIPs or through their intrinsicautonomous tip-tracking properties (Varga et al.,2009).

+TIP functionsLocalization at microtubule ends makes +TIPsideally suited to control different aspects ofmicrotubule dynamics; for example, bypromoting growth through catalyzing theaddition of tubulin to microtubule ends(XMAP215) (Brouhard et al., 2008), inducingcatastrophes (MCAK) (Kline-Smith andWalczak, 2002) or rescues (CLIP-170)(Komarova et al., 2002), or by stabilizingmicrotubules at the cell cortex (CLASPs, APC,MACF) (Kodama et al., 2003; Mimori-Kiyosueet al., 2005; Wen et al., 2004) (for reviews, seeHeald and Nogales, 2002; van der Vaart et al.,2009). For some +TIPs, the exact effect onmicrotubule dynamics varies depending on theassay conditions. EB proteins usually promotemicrotubule dynamics and growth, and suppresscatastrophes in cells (Busch and Brunner, 2004;Komarova et al., 2009; Tirnauer et al., 2002).However, the results of in-vitro experimentswith different EB family members have beencontroversial, because changes in growth andshrinkage rates, induction and suppression ofcatastrophes, or a complete lack of influence onsome or all microtubule dynamics parametershave been reported (Bieling et al., 2008; Bielinget al., 2007; Dixit et al., 2009; Katsuki et al.,2009; Komarova et al., 2009; Manna et al.,2007; Vitre et al., 2008). Taken together, thesestudies suggest that the regulation ofmicrotubule dynamics is an important +TIPfunction, but the underlying molecularmechanisms are still poorly understood.

In addition to regulating microtubuledynamics, +TIPs form links betweenmicrotubule ends and other cellular structures.For example, they can attach microtubule tips tothe cell cortex by binding to plasma-membrane-associated proteins – such as the CLASP–LL5complex (Lansbergen et al., 2006) – or byinteracting with actin fibers to which some+TIPs, such as spectraplakins, can bind directly(Applewhite et al., 2010; Kodama et al., 2003),whereas others (e.g. CLIP-170) might requireintermediary factors (Fukata et al., 2002). +TIPsalso participate in microtubule-actin crosstalk.The Tea1–Tea4 complex, for example, controlsactin organization through formins in buddingyeast (Martin et al., 2005), whereas CLIP-170 –which also acts in concert with a formin –

controls actin polymerization, a processessential for phagocytosis in mammalian cells(Lewkowicz et al., 2008). The EB1 partnerRhoGEF2 regulates contractility of epithelialcells in flies (Rogers et al., 2004), and p140Capacting together with EB3 affects F-actinorganization in dendritic spines of neurons(Jaworski et al., 2009). Furthermore, +TIPcomplexes are used for myosin-based transportof microtubule ends, e.g. Kar9-Myo2 in buddingyeast (Liakopoulos et al., 2003).

+TIPs also have an important role incoordinating microtubule attachment anddynamics at mitotic kinetochores – e.g. Dam1,CLIP-170, CLASPs, dynein (for a review, seeMaiato et al., 2004) – and participate in theextension of endoplasmic reticulum tubulestogether with growing microtubule ends(STIM1) (Grigoriev et al., 2008). +TIPs alsocontribute to loading cargo for minus-end-directed microtubule transport (dynactin, CLIP-170) (Lomakin et al., 2009; Vaughan et al.,2002) and in transporting microtubule endsalong other microtubules to promoteorganization of specialized microtubule arrays,such as mitotic spindles (Goshima et al., 2005)and bipolar microtubule bundles in fission yeast(Janson et al., 2007).

Finally, many +TIPs accumulate atcentrosomes and other microtubule organizingcenters where they might participate inmicrotubule nucleation and anchoring (for areview, see Bettencourt-Dias and Glover, 2007).The exact role +TIPs have at the centrosomesawaits to be explored.

PerspectivesGrowing microtubule ends have emerged asremarkably complex cellular sites wheremicrotubule dynamics can be coordinated withactin polymerization, cargo movement andremodeling of cell membranes. These processesare tightly regulated by a diverse set of proteinsthat form a dynamic and flexible interactionnetwork. In most cases, the exact role of themicrotubule plus-end tracking behavior for+TIP function has not been established and stillneeds to be examined. Remarkably, some of thekey microtubule tip-targeting motifs are veryshort and simple, and can be acquired easilyduring evolution. We thus expect that the list of+TIPs is incomplete and that many more proteinfamilies showing this peculiar localizationbehavior will be discovered in the near future.

A.A. is supported by the Netherlands Organization forScientific Research grants ALW-VICI and ZonMW-TOP. M.O.S. is supported by grants from the SwissNational Science Foundation.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/123/20/3415/DC1

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