Cell Science at a Glance 3415

Microtubule +TIPs at a modifications of ’ by Dorota Wloga and diverse group of proteins that are distinguished Jacek Gaertig (J. Cell Sci. 123, pp. 3447-3455). by their specific accumulation at glance plus ends (Mimori-Kiyosue et al., 2000; Perez et Microtubules are highly dynamic hollow tubes al., 1999; Schuyler and Pellman, 2001). +TIPs Anna Akhmanova1 and Michel O. that are involved in many vital cellular typically target growing but not shrinking Steinmetz2 activities, including maintenance of cell shape, microtubule ends; however +TIP association 1 Department of , Erasmus Medical division, migration and intracellular transport. with depolymerizing ends can occur and, in Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands They are assembled from heterodimers of - some organisms such as budding yeast, is even 2Biomolecular Research, Structural Biology, Paul and -tubulin that align in a head-to-tail fashion. quite common. In this Cell Science at a Glance Scherrer Insititut, CH-5232 Villigen PSI, Switzerland Microtubules are, thus, intrinsically polar article we review and illustrate the current ([email protected]; [email protected]) because they contain two structurally distinct knowledge of these peculiar proteins, ends: a slow-growing minus end, exposing - summarize their structural and functional Journal of Cell Science 123, 3415-3419 © 2010. Published by The Company of Biologists Ltd tubulin subunits; and a fast-growing plus end, properties, and discuss the proposed molecular doi:10.1242/jcs.062414 exposing -tubulin subunits (for a review, see mechanisms that they use to track microtubule Nogales and Wang, 2006). In mammalian cells, ends. This article is part of a Minifocus on microtubule dynamics. For further reading, please see related microtubule minus ends are often stably articles: ‘ at a glance’ by Sharyn A. Endow anchored, whereas the plus ends are highly Classification of +TIPs et al., (J. Cell Sci. 123, pp. 3420-3424), ‘Tubulin dynamic and stochastically switch between The first reported +TIP was cytoplasmic linker depolymerization may be an ancient biological motor process’ by J. Richard McIntosh et al. (J. Cell Sci. phases of growth and shrinkage, a process that is protein of 170 kDa (CLIP-170, officially known 123, pp. 3425-3434), ‘Towards a quantitative powered by GTP hydrolysis. as CLIP1) (Perez et al., 1999). Since its understanding of mitotic spindle assembly and Microtubule plus-end tracking proteins discovery, more than 20 different +TIP families mechanics’ by Alex Mogilner and Erin Craig (J. Cell Sci. 123, pp. 3435-3445) and ‘Post-translational (+TIPs) are a structurally and functionally have been identified. +TIPs are usually

Microtubule +TIPs at a Glance Anna Akhmanova and Michel O. Steinmetz

+TIP classification What is a +TIP? +TIP functions

EB proteins CAP–Gly proteins +TIPs localize to and track dynamic MT plus ends Microtubule dynamics CAP–Gly EBH Coiled coil ZnF +TIP that tracks only Growing MT GTP Shrinking MT Polymerization Depolymerization EB CH CLIP170 growing MT plus ends β β (XMAP215, EB1) (MCAK) Rescue Coiled EEY/F Basic-S/P EEY/F (e.g. EB1, CLIP-170) Plus end α α (CLIP-170, CLASP) coil p150glued +TIP that tracks growing GDP SxIP proteins and shrinking MT plus Coiled 15/20 aa ends (e.g. Dam1, Kar9) Catastrophe SxIP Rescue coil Helical repeats β (MCAK) Basic-S/P Stabilization APC GTP-tubulin α (CLASP, APC, MACF) Arm SAMP SxIP β Catastrophe Journal of Cell Science Plakin Spectrin EF MACF CH α GDP-tubulin GAS2 SAM SxIP Further examples: CLASP, p140Cap, melanophilin In vitro, some +TIPs can STIM1 Interactions with cellular structures RhoGEF2, CDK5RAP2, TIP150, navigator bind to growing but not EF TM Minus end shrinking MT minus ends Cortex Endoplasmic reticulum (CLASP, APC, MACF, CLIP-170, EB1, (EB1, STIM1) TOG proteins Other proteins , dynactin, LIS1) TOG Basic-S/P Helical Multimeric XMAP215 Dam1 complex of 10 proteins +TIP localization SxIP CLASP LisH WD40 Lis1 +TIP distribution in mammalian interphase cells TOG-like Live image of a Helical Monkey Kar9 kidney cell human lung Motor proteins Basic-S/P stained for fibroblast expressing endogenous an MT marker Basic-S/P SxIP EB1 (mCherry-α-tubulin, F-actin Vesicles Tea2 Ncd MCAK red) and a +TIP (MACF, CLASP, APC, CLIP-170, Kar9, (dynein, dynactin, RhoGEF2, p140Cap) CLIP-170, Melanophilin) Helical AAA marker (EB3-GFP, Dynein HC green) Coiled coil

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

+TIP interactions Microtubule plus-end tracking mechanisms Kinetochores (Dam1, CLASP, CLIP-170, APC, EB1, SxIP proteins CAP-Gly-EEY/F 3D diffusion dynein, dynactin, LIS1, MCAK) Proteins with Autonomous (STIM1) Microtubules APC in cytoplasm 2D diffusion unknown Microtubule +TIP (Ncd, Klp2) Tyr Lattice- (RhoGEF2, Lis1) in membrane EB-binding MACF Glu maturation- mechanisms CH CH Melanophilin Non-autonomous induced release Hitchhiking XMAP215 EB MCAK +TIPs Kar9 STIM1 Kinesin-based transport EBH Tea2 TIP150 Glu (Tea2–Tip1, Kip2–Bik1) Kinesin Ncd EEY/F RhoGEF2 Co-polymerization EBH-SxIP CDK5RAP2 (XMAP215, EB1, CLASP, APC, p140Cap GTP-tubulin dynein, dynactin, Lis1, FOP, CDK5RAP2) CLASP Pro CAP-Gly GDP-tubulin 1D lattice diffusion proteins (MCAK, XMAP215) Processive Ile Fast end tracking Lipid bilayer Dynein p150glued x Recognition exchange (XMAP215, Dam1) Ser of composite (EB1, Proteins in parentheses +TIP/tubulin-binding CLIP-170) indicate selected examples sites (CLIP-170, MCAK) of +TIPs using a particular Recognition of specific Lis1 CLIP-170 mechanism plus-end structure (EB1)

Abbreviations: AAA, ATPase family associated with various cellular activities; APC, adenomateous polyposis crosslinking factor; MCAK, mitotic centromere-associated kinesin; MT, microtubule; Ncd, non-claret disjunctional; coli protein; Arm, armadillo repeat; basic-S/P, sequence regions enriched in basic, serine and proline residues; p140Cap, p130Cas-associated protein of 140 kDa (also known as SRCIN1, SRC kinase signaling inhibitor 1, SNIP, CAP-Gly, -associated protein glycine-rich; CDK5RAP2, CDK5 regulatory subunit- associated protein 2; SNAP-25-interacting protein); RhoGEF2, Rho-type guanine nucleotide-exchange factor 2; SAM, sterile α-motif CH, calponin homology; CLASP, CLIP-associated protein; CLIP-170, cytoplasmic linker protein of 170 kDa; Dam1, domain; SAMP, Ser-Ala-Met-Pro repeat; STIM1, stromal interaction molecule 1; SxIP, Ser-x-Ile-Pro tetrapeptide DUO1- and MPS1-interacting protein 1; EB, end-binding protein; EBH, EB homology; EEY/F, C-terminal Glu-Glu- motif, where x denotes any amino acid residue; +TIP, microtubule plus-end tracking protein; TIP150, +TIP of 150 Tyr/Phe tripeptide motif; EF-hand, Ca2+-binding motif; FOP, FGFR1 oncogene partner; Gas2, growth-arrest-specific kDa; TM, transmembrane domain; TOG, named after the discovery in human chTOG; WD40, ~40 amino acid protein 2; HC, heavy chain; Lis1, lissencephaly-1 protein; LisH, Lis1 homology; MACF, microtubule-actin motifs, often terminating in a Trp-Asp dipeptide; XMAP215, microtubule-associated protein of 215 kDa. © Journal of Cell Science 2010 (123, pp. 3414–3418)

(See poster insert) 3416 Journal of Cell Science 123 (20)

multidomain and/or multisubunit proteins that motif Ser-x-Ile-Pro (SxIP, where x denotes any display affinities in the low micromolar range range in size from a few hundred up to amino acid), which is specifically recognized by (Gupta et al., 2009; Mishima et al., 2007; thousands of residues. They can be cytoplasmic the EBH domain of EB proteins (Honnappa Weisbrich et al., 2007). or membrane bound, and comprise motor and et al., 2009). Prominent examples of this diverse EB proteins are now generally accepted to non-motor proteins (for a review, see class of +TIPs are the adenomatous polyposis represent core components of +TIP networks Akhmanova and Steinmetz, 2008). +TIPs can be coli (APC) tumour suppressor, the spectraplakin because they autonomously track growing classified on the basis of prominent structural microtubule–actin crosslinking factor (MACF) microtubule plus ends independently of any elements that enable them to interact with each and the mitotic centromere-associated kinesin binding partners (Bieling et al., 2008; Bieling et other and with microtubules; however, in some (MCAK). Because SxIP motifs are very short, al., 2007; Dixit et al., 2009; Komarova et al., cases, +TIPs combine features characteristic of they can be easily acquired or lost during 2009; Zimniak et al., 2009). Moreover, EB several +TIP classes. evolution; for example, CDK5RAP2, a protein proteins directly associate with almost all other End-binding (EB) family proteins contain a implicated in microcephaly, contains an EB1- known +TIPs and, by doing so, target them to highly conserved N-terminal domain that adopts binding SxIP motif in humans and dogs but not growing microtubule plus ends (for reviews, see a calponin homology (CH) fold (Korenbaum in rodents (Fong et al., 2009). Akhmanova and Steinmetz, 2008; Slep, 2009b). and Rivero, 2002) and is responsible for Proteins with TOG or TOG-like domains SxIP motifs act as a general ‘microtubule tip microtubule binding (Hayashi and Ikura, 2003). (named after their discovery in the protein ch- localization signal’ (MtLS) by interacting with In mammalian EB1 and EB3, a CH domain with TOG) include members of the XMAP215/Dis1 the EBH domain of EB proteins (Honnappa the adjacent linker sequence is sufficient for family and the CLASPs. Tandemly arranged et al., 2009). Similarly, EEY/F motifs of EB plus-end tracking (Komarova et al., 2009; Skube TOG domains mediate binding to tubulin and proteins and -tubulin guide CAP-Gly proteins et al., 2010); however dimerization is important are probably responsible for microtubule to microtubule tips (Bieling et al., 2008; Dixit for microtubule plus-end recognition by their growth-promoting activity of these proteins et al., 2009). Both the EBH-SxIP and the yeast homologue Bim1 (Zimniak et al., 2009). (Al-Bassam et al., 2006; Brouhard et al., 2008; CAP-Gly-EEY/F interactions have been The C terminus of EB proteins harbors an Slep and Vale, 2007) (for a review, see Slep, analyzed to high resolution (Hayashi et al., -helical coiled-coil domain that mediates 2009a). Additional domains, such as SxIP 2007; Honnappa et al., 2009; Honnappa parallel dimerization of EB monomers motifs in CLASPs, are required for targeting of et al., 2006; Mishima et al., 2007; Plevin et al., (Honnappa et al., 2005; Slep et al., 2005). It these proteins to microtubule plus ends and 2008; Weisbrich et al., 2007). The two distinct further comprises the unique EB homology other subcellular sites (Mimori-Kiyosue et al., binding modes were revealed through these (EBH) domain and an acidic tail encompassing 2005). structures and offer a molecular basis for a C-terminal EEY/F motif, reminiscent of those Both microtubule plus- and minus-end- understanding the majority of known interaction of -tubulin and CLIP-170 (Komarova et al., directed motor proteins can track growing nodes in dynamic +TIP networks. 2005; Miller et al., 2006; Weisbrich et al., 2007). microtubule ends. Examples are the yeast The EBH-SxIP and CAP-Gly-EEY/F Notably, plant EB proteins lack the EEY/F kinesins Tea2 and Kip2, the microtubule- interactions can be regulated by post-translational motif, and some EB family members, such as depolymerising kinesin 13 MCAK and modifications. Phosphorylation of Ser residues in EB1c in Arabidopsis thaliana, exhibit a cytoplasmic dynein (reviewed in Wu et al., the vicinity of the SxIP motifs (Honnappa et al.,

Journal of Cell Science positively charged C-terminus that is 2006). Sequences outside the microtubule- 2009; Kumar et al., 2009; Watanabe et al., 2009) responsible for nuclear localization (Komaki binding motor domains, such as the SxIP motif disrupts their interaction with EB proteins, et al., 2010). Both the EBH domain and the of MCAK (Honnappa et al., 2009), might be whereas the removal of the C-terminal Tyr of - EEY/F motif enable the EB proteins to needed for the microtubule tip-tracking tubulin has a negative effect on the accumulation physically interact with an array of +TIPs to behavior of these proteins. of CAP-Gly proteins at microtubule tips (Bieling recruit them to microtubule ends. Finally, there are other +TIPs that cannot be et al., 2008; Peris et al., 2006). The cytoskeleton-associated protein glycine- grouped in one of the five classes discussed rich (CAP-Gly) domain is a small globular above. A prominent example is the Dam1 +TIP tracking mechanisms module that contains a unique conserved complex – an assembly of ten subunits that form Because +TIPs form complex interaction hydrophobic cavity and several characteristic rings of 16-fold symmetry (Lampert et al., 2010; networks, in-vitro reconstitution studies using glycine residues (Li et al., 2002; Saito et al., Wang et al., 2007) – and which is found in yeast purified components are required to determine 2004). CAP-Gly domains use their hydrophobic but not in higher organisms. Other examples are whether plus-end tracking behavior is an cavity to confer interactions with microtubules the Saccharomyces cerevisiae protein Kar9 autonomous property of a particular protein. and EB proteins by specifically recognizing (Liakopoulos et al., 2003; Moore and Miller, Using this approach, it was shown that some C-terminal EEY/F sequence motifs (Honnappa 2007), and the highly conserved cytoplasmic +TIPs can associate with growing microtubule et al., 2006; Mishima et al., 2007; Weisbrich et dynein accessory factor lissencephaly-1 ends in the absence of any additional factors. al., 2007). Prominent examples are the CLIP protein (Lis1) (for a review, see Vallee and Tsai, Autonomous processive microtubule tip proteins and the large subunit of the dynactin 2006). tracking, whereby the protein stays bound to the complex p150glued. A single CAP-Gly domain of microtubule end during multiple rounds of CLIP-170, together with the adjacent serine-rich Dynamic +TIP interaction networks subunit addition, has been described for region, can track growing microtubule ends One hallmark of +TIPs is that they form XMAP215 (Brouhard et al., 2008). Another (Gupta et al., 2009). dynamic interaction networks that rely on a example is the yeast Dam1 complex, which The largest group of +TIPs comprises large limited number of protein modules and linear continuously tracks both growing and shrinking and complex, often multidomain, proteins sequence motifs, such as the CH, EBH and microtubule ends, possibly by using a form of a containing low-complexity sequence regions CAP-Gly domains, and EEY/F and SxIP motifs. diffusion-based mechanism (Lampert et al., that are rich in basic, serine and proline (basic- These elements mediate the interaction with 2010). Finally, various EB family members S/P) residues. They share the small four-residue each other and microtubules, and typically from different species bind to growing but not Journal of Cell Science 123 (20) 3417

shortening plus- and minus ends in vitro 2004; Carvalho et al., 2004; Miller et al., 2006). controls actin polymerization, a process (Bieling et al., 2008; Bieling et al., 2007; Dixit et It should be noted that kinesins, either alone or essential for phagocytosis in mammalian cells al., 2009; Komarova et al., 2009; Zimniak et al., together with their binding partners, will track (Lewkowicz et al., 2008). The EB1 partner 2009). Unlike XMAP215, EB proteins microtubule ends only if they do not dissociate RhoGEF2 regulates contractility of epithelial exchange rapidly at the microtubule end, immediately from microtubule ends but are cells in flies (Rogers et al., 2004), and p140Cap undergoing several cycles of binding and retained on them, either because of interactions acting together with EB3 affects F-actin unbinding events before the growing with other +TIPs or through their intrinsic organization in dendritic spines of neurons microtubule end converts into the mature autonomous tip-tracking properties (Varga et al., (Jaworski et al., 2009). Furthermore, +TIP lattice (Bieling et al., 2007; Dragestein et al., 2009). complexes are used for myosin-based transport 2008). of microtubule ends, e.g. Kar9-Myo2 in budding It is currently unknown which structural +TIP functions yeast (Liakopoulos et al., 2003). features of the growing microtubule end are Localization at microtubule ends makes +TIPs +TIPs also have an important role in recognized by autonomously tracking +TIPs; ideally suited to control different aspects of coordinating microtubule attachment and however, these might include the GTP cap at the microtubule dynamics; for example, by dynamics at mitotic kinetochores – e.g. Dam1, end of the freshly polymerized microtubule promoting growth through catalyzing the CLIP-170, CLASPs, dynein (for a review, see (Lampert et al., 2010; Zanic et al., 2009) or addition of tubulin to microtubule ends Maiato et al., 2004) – and participate in the some specific protofilament arrangement (des (XMAP215) (Brouhard et al., 2008), inducing extension of endoplasmic reticulum tubules Georges et al., 2008; Sandblad et al., 2006) (for a catastrophes (MCAK) (Kline-Smith and together with growing microtubule ends review, see Coquelle et al., 2009). Another Walczak, 2002) or rescues (CLIP-170) (STIM1) (Grigoriev et al., 2008). +TIPs also attractive idea is that autonomously tracking (Komarova et al., 2002), or by stabilizing contribute to loading cargo for minus-end- +TIPs co-polymerize with tubulin subunits and microtubules at the cell cortex (CLASPs, APC, directed microtubule transport (dynactin, CLIP- then get released gradually from the mature MACF) (Kodama et al., 2003; Mimori-Kiyosue 170) (Lomakin et al., 2009; Vaughan et al., lattice (Folker et al., 2005); this mechanism has et al., 2005; Wen et al., 2004) (for reviews, see 2002) and in transporting microtubule ends not found support in the in-vitro reconstitution Heald and Nogales, 2002; van der Vaart et al., along other microtubules to promote studies using EB and CLIP homologs of fission 2009). For some +TIPs, the exact effect on organization of specialized microtubule arrays, yeast and vertebrates (Bieling et al., 2007; microtubule dynamics varies depending on the such as mitotic spindles (Goshima et al., 2005) Bieling et al., 2008; Dixit et al., 2009), but might assay conditions. EB proteins usually promote and bipolar microtubule bundles in fission yeast still apply to some other proteins. microtubule dynamics and growth, and suppress (Janson et al., 2007). Most +TIPs track the ends of growing catastrophes in cells (Busch and Brunner, 2004; Finally, many +TIPs accumulate at microtubules in a non-autonomous manner. Komarova et al., 2009; Tirnauer et al., 2002). and other microtubule organizing STIM1 and CDK5RAP2, for example, hitchhike However, the results of in-vitro experiments centers where they might participate in on microtubule tip-bound EB proteins (Fong et with different EB family members have been microtubule nucleation and anchoring (for a al., 2009; Grigoriev et al., 2008; Honnappa et al., controversial, because changes in growth and review, see Bettencourt-Dias and Glover, 2007). 2009). Others, such as CLIP-170, recognize shrinkage rates, induction and suppression of The exact role +TIPs have at the centrosomes

Journal of Cell Science more complex binding sites that encompass catastrophes, or a complete lack of influence on awaits to be explored. domains of both EB proteins and tubulin (Bieling some or all microtubule dynamics parameters et al., 2008; Gupta et al., 2010). Because EB have been reported (Bieling et al., 2008; Bieling Perspectives proteins rapidly exchange at microtubule tips, et al., 2007; Dixit et al., 2009; Katsuki et al., Growing microtubule ends have emerged as accumulation of their partners at microtubule 2009; Komarova et al., 2009; Manna et al., remarkably complex cellular sites where ends is also dynamic, and mostly depends on 2007; Vitre et al., 2008). Taken together, these microtubule dynamics can be coordinated with three-dimensional protein diffusion in the studies suggest that the regulation of actin polymerization, cargo movement and cytosol. However, one-dimensional diffusion microtubule dynamics is an important +TIP remodeling of cell membranes. These processes along the microtubule lattice might also occur, as function, but the underlying molecular are tightly regulated by a diverse set of proteins is the case for MCAK (Helenius et al., 2006). In mechanisms are still poorly understood. that form a dynamic and flexible interaction the case of STIM1, a transmembrane +TIP, two- In addition to regulating microtubule network. In most cases, the exact role of the dimensional diffusion in the membrane is dynamics, +TIPs form links between microtubule plus-end tracking behavior for required to enable accumulation at microtubule microtubule ends and other cellular structures. +TIP function has not been established and still tips (Grigoriev et al., 2008). For example, they can attach microtubule tips to needs to be examined. Remarkably, some of the For EB proteins and their partners that the cell cortex by binding to plasma-membrane- key microtubule tip-targeting motifs are very decorate the freshly polymerized microtubule associated proteins – such as the CLASP–LL5 short and simple, and can be acquired easily tip, the specificity for microtubule plus ends – as complex (Lansbergen et al., 2006) – or by during evolution. We thus expect that the list of opposed to minus ends – is explained by the fact interacting with actin fibers to which some +TIPs is incomplete and that many more protein that, in vivo, minus ends never grow in cells. By +TIPs, such as spectraplakins, can bind directly families showing this peculiar localization contrast, the exclusive accumulation at (Applewhite et al., 2010; Kodama et al., 2003), behavior will be discovered in the near future. microtubule plus ends both in vitro and in vivo is whereas others (e.g. CLIP-170) might require A.A. is supported by the Netherlands Organization for observed in systems in which plus-end-directed intermediary factors (Fukata et al., 2002). +TIPs Scientific Research grants ALW-VICI and ZonMW- kinesins are involved. Among the best-studied also participate in microtubule-actin crosstalk. TOP. M.O.S. is supported by grants from the Swiss examples are the yeast CLIP-170 orthologs The Tea1–Tea4 complex, for example, controls National Science Foundation. Bik1 and Tip1, which are concentrated at actin organization through formins in budding Supplementary material available online at microtubule tips by the kinesins Kip2 and Tea2, yeast (Martin et al., 2005), whereas CLIP-170 – http://jcs.biologists.org/cgi/content/full/123/20/ respectively (Bieling et al., 2007; Busch et al., which also acts in concert with a formin – 3415/DC1 3418 Journal of Cell Science 123 (20)

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