Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends

Microtubule (MT) plus end-tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.     

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.     

EBs clip CLIPs to growing microtubule ends

Proteins that track growing microtubule (MT) ends are important for many aspects of intracellular MT function, but the mechanism by which these +TIPs accumulate at MT ends has been the subject of a long-standing controversy. In this issue, Bieling et al. (Bieling, P., S. Kandels-Lewis, I.A. Telley, J. van Dijk, C. Janke, and T. Surrey. 2008. J. Cell Biol. 183:1223–1233) reconstitute plus end tracking of EB1 and CLIP-170 in vitro, which demonstrates that CLIP-170 plus end tracking is EB1-dependent and that both +TIPs rapidly exchange between a soluble and a plus end–associated pool. This strongly supports the hypothesis that plus end tracking depends on a biochemical property of growing MT ends, and that the characteristic +TIP comets result from the generation of new +TIP binding sites through MT polymerization in combination with the exponential decay of these binding sites.     

Stimulation of the CLIP-170--dependent capture of membrane organelles by microtubules through fine tuning of microtubule assembly dynamics

Cytoplasmic microtubules (MTs) continuously grow and shorten at their free plus ends, a behavior that allows them to capture membrane organelles destined for MT minus end-directed transport. In Xenopus melanophores, the capture of pigment granules (melanosomes) involves the +TIP CLIP-170, which is enriched at growing MT plus ends. Here we used Xenopus melanophores to test whether signals that stimulate minus end MT transport also enhance CLIP-170-dependent binding of melanosomes to MT tips. We found that these signals significantly (>twofold) increased the number of growing MT plus ends and their density at the cell periphery, thereby enhancing the likelihood of interaction with dispersed melanosomes. Computational simulations showed that local and global increases in the density of CLIP-170-decorated MT plus ends could reduce the half-time of melanosome aggregation by ~50%. We conclude that pigment granule aggregation signals in melanophores stimulate MT minus end-directed transport by the increasing number of growing MT plus ends decorated with CLIP-170 and redistributing these ends to more efficiently capture melanosomes throughout the cytoplasm.     

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.     

TIP150 interacts with and targets MCAK at the microtubule plus ends

The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins—called plus-end tracking proteins (+TIPs)—bind to and control the plus-end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus-end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end-binding protein 1 (EB1) in vitro and co-localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus-end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere-associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus-end localization of MCAK. Importantly, aurora B-mediated phosphorylation disrupts the TIP150–MCAK association in vitro. We reason that TIP150 facilitates the EB1-dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.     

Regulation of Microtubule Dynamics by Bim1 and Bik1, the Budding Yeast Members of the EB1 and CLIP-170 Families of Plus-End Tracking Proteins

Microtubule dynamics are regulated by plus-end tracking proteins (+TIPs), which bind microtubule ends and influence their polymerization properties. In addition to binding microtubules, most +TIPs physically associate with other +TIPs, creating a complex web of interactions. To fully understand how +TIPs regulate microtubule dynamics, it is essential to know the intrinsic biochemical activities of each +TIP and how +TIP interactions affect these activities. Here, we describe the activities of Bim1 and Bik1, two +TIP proteins from budding yeast and members of the EB1 and CLIP-170 families, respectively. We find that purified Bim1 and Bik1 form homodimers that interact with each other to form a tetramer. Bim1 binds along the microtubule lattice but with highest affinity for the microtubule end; however, Bik1 requires Bim1 for localization to the microtubule lattice and end. In vitro microtubule polymerization assays show that Bim1 promotes microtubule assembly, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. Interestingly, the Bim1-Bik1 complex affects microtubule dynamics in much the same way as Bim1 alone. These studies reveal new activities for EB1 and CLIP-170 family members and demonstrate how interactions between two +TIP proteins influence their activities.     

Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150(Glued) are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH(2) terminus of p150(Glued) binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150(Glued) and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH(2)-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH(2) and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.     

The FKBP12-rapamycin-associated protein (FRAP) is a CLIP-170 kinase

CLIP-170/Restin belongs to a family of conserved microtubule (MT)-associated proteins, which are important for MT organization and functions. CLIP-170 is a phosphoprotein and phosphorylation is thought to regulate the binding of CLIP-170 to MTs. However, little is known about the kinase(s) involved. In this study, we show that FKBP12-rapamycin-associated protein (FRAP, also called mTOR/RAFT) interacts with CLIP-170. CLIP-170 is phosphorylated in vivo at multiple sites, including rapamycin-sensitive and -insensitive sites, and is phosphorylated by FRAP in vitro at the rapamycin-sensitive sites. In addition, rapamycin inhibited the ability of CLIP-170 to bind to MTs. Our observations suggest that multiple CLIP-170 kinases are involved in positive and negative control of CLIP-170, and FRAP is a CLIP-170 kinase positively regulating the MT-binding behavior of CLIP-170.     

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.     

The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis

Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by αMβ2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during αMβ2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of αMβ2 integrins.     

LIS1, CLIP-170's key to the dynein/dynactin pathway

CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.     

TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation

Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end–tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.     

Microtubule plus end: a hub of cellular activities

Microtubules (MTs) are highly dynamic polymers, which control many aspects of cellular architecture. Growing MT plus ends accumulate a specific set of evolutionary conserved factors, the so-called MT plus-end-tracking proteins (+TIPs). +TIPs regulate MT dynamics and the reciprocal interactions of MTs with the cell cortex, mitotic kinetochores or different cellular organelles. Most +TIPs can directly bind to MTs, but the molecular mechanisms of their specific targeting to the growing plus ends remain poorly understood. Recent studies suggest that the members of one particular +TIP family, EB1 and its homologues, are present in all eucaryotic kingdoms, interact directly with the majority of other known plus-end-associated proteins and may be responsible for their specific accumulation at the MT tips.     

Phosphorylation Controls Autoinhibition of Cytoplasmic Linker Protein-170

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150^Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an “open” conformation and a higher binding affinity for growing MT ends and p150^Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the “folded back” conformation shows decreased MT association and does not interact with p150^Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.     

Interactions between CLIP-170, tubulin, and microtubules: implications for the mechanism of Clip-170 plus-end tracking behavior

CLIP-170 belongs to a group of proteins (+TIPs) with the enigmatic ability to dynamically track growing microtubule plus-ends. CLIP-170 regulates microtubule dynamics in vivo and has been implicated in cargo-microtubule interactions in vivo and in vitro. Though plus-end tracking likely has intimate connections to +TIP function, little is known about the mechanism(s) by which this dynamic localization is achieved. Using a combination of biochemistry and live cell imaging, we provide evidence that CLIP-170 tracks microtubule plus-ends by a preassociation, copolymerization, and regulated release mechanism. As part of this analysis, we find that CLIP-170 has a stronger affinity for tubulin dimer than for polymer, and that CLIP-170 can distinguish between GTP- and GDP-like polymer. This work extends the previous analysis of CLIP-170 behavior in vivo and complements the existing fluorescence microscope characterization of CLIP-170 interactions with microtubules in vitro. In particular, these data explain observations that CLIP-170 localizes to newly polymerized microtubules in vitro but cannot track microtubule plus-ends in vitro. These observations have implications for the functions of CLIP-170 in regulating microtubule dynamics.     

Microtubule plus end‐tracking proteins play critical roles in directional growth of hyphae by regulating the dynamics of cytoplasmic microtubules in Aspergillus nidulans

Cytoplasmic microtubules (MTs) serve as a rate‐limiting factor for hyphal tip growth in the filamentous fungus Aspergillus nidulans. We hypothesized that this function depended on the MT plus end‐tracking proteins (+TIPs) including the EB1 family protein EBA that decorated the MT plus ends undergoing polymerization. The ebAΔ mutation reduced colony growth and the mutant hyphae appeared in an undulating pattern instead of exhibiting unidirectional growth in the control. These phenotypes were enhanced by a mutation in another +TIP gene clipA. EBA was required for plus end‐tracking of CLIPA, the Kinesin‐7 motor KipA, and the XMAP215 homologue AlpA. In addition, cytoplasmic dynein also depended on EBA to track on most polymerizing MT plus ends, but not for its conspicuous appearance at the MT ends near the hyphal apex. The loss of EBA reduced the number of cytoplasmic MTs and prolonged dwelling times for MTs after reaching the hyphal apex. Finally, we found that colonies were formed in the absence of EBA, CLIPA, and NUDA together, suggesting that they were dispensable for fundamental functions of MTs. This study provided a comprehensive delineation of the relationship among different +TIPs and their contributions to MT dynamics and unidirectional hyphal expansion in filamentous fungi.     

CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.     

Cytoplasmic linker protein-170 enhances spreading and phagocytosis in activated macrophages by stabilizing microtubules

Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.