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.     

CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms

CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.     

CLIP-170 facilitates the formation of kinetochore-microtubule attachments

CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochore-microtubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochore-microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore-microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore-microtubule attachments, possibly through direct capture of microtubules at the kinetochore.     

Purification and analysis of authentic CLIP-170 and recombinant fragments.

We have purified authentic CLIP-170 (cytoplasmic linker protein of 170 kDa) and fragments comprising functional domains of the protein to characterize the structural basis of the function of CLIP-170. Analysis of authentic CLIP-170 and the recombinant fragments by electron microscopy after glycerol spraying/low angle rotary metal shadowing reveals CLIP-170 as a thin, 135-nm-long molecule with two kinks in its central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is alpha-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced approximately 37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated molecule. The N-terminal domain of CLIP-170 binds to microtubules in vitro with a stoichiometry of one dimeric head domain per four tubulin heterodimers. Authentic CLIP-170 binds to microtubules with lower stoichiometry, indicating that the rod and tail domains affect microtubule binding of CLIP-170. These results document that CLIP-170 is a highly elongated polar molecule with the microtubule-binding domain and the organelle-interacting domains at opposite ends of the homodimer, thus providing a structural basis for the function of CLIP-170 as a microtubule-organelle linker protein.     

CLIPR-59 is a lipid raft-associated protein containing a cytoskeleton-associated protein glycine-rich domain (CAP-Gly) that perturbs microtubule dynamics

We recently have identified a new cytoplasmic linker protein (CLIP), CLIPR-59, which is involved in the regulation of early endosome/trans-Golgi network dynamics. In contrast with CLIP-170, CLIPR-59 is not localized to microtubules at steady state but is associated with the trans-Golgi network and the plasma membrane. Here we show that the last 30 amino acids (C30) are sufficient for membrane targeting and that two cysteines in the C30 domain are palmitoylated. We demonstrate that CLIPR-59 is associated with lipid rafts via its C-terminal palmitoylated domain. In vitro experiments suggest that CLIPR-59 and its microtubule-binding domain alone have a better affinity for unpolymerized tubulin or small oligomers than for microtubules. In contrast with the CLIP-170 microtubule-binding domain, the CLIPR-59 microtubule-binding domain diminishes microtubule regrowth after nocodazole washout in vivo, showing that this domain can prevent microtubule polymerization. In contrast with the role of linker between membranes and microtubules that was proposed for CLIP function, CLIPR-59 thus may have an "anti-CLIP" function by preventing microtubule-raft interactions.     

Functionally distinct isoforms of dynactin are expressed in human neurons.

P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.     

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.     

CLIP-170 links endocytic vesicles to microtubules.

Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A. The structure of CLIP-170 has been analyzed by cloning its cDNA. The predicted non-helical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain. We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules. This motif is also found in the Drosophila Glued and yeast BIK1 proteins. These features, together with its very elongated structure, suggest that CLIP-170 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPs), mediating interactions of organelles with microtubules.     

The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150(Glued) subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150(Glued) in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150(Glued) on microtubule polymerization, and they show that p150(Glued) has a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.     

Microtubule binding proteins CLIP-170, EB1, and p150Glued form distinct plus-end complexes

Microtubule plus-end proteins CLIP-170 and EB1 dynamically track the tips of growing microtubules in vivo. Here we examine the association of these proteins with microtubules in vitro. CLIP-170 binds tubulin dimers and co-assembles into growing microtubules. EB1 binds tubulin dimers more weakly, so no co-assembly is observed. However, EB1 binds to CLIP-170, and forms a co-complex with CLIP-170 and tubulin that is recruited to growing microtubule plus ends. The interaction between CLIP-170 and EB1 is competitively inhibited by the related CAP-Gly protein p150Glued, which also localizes to microtubule plus ends in vivo. Based on these observations, we propose a model in which the formation of distinct plus-end complexes may differentially affect microtubule dynamics in vivo.     

A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150(Glued) by live-cell imaging. Time-lapse analysis of p150(Glued) revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein-glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150(Glued) phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150(Glued) phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150(Glued) phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150(Glued) during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150(Glued)-labeled microtubules just prior to transport, implicating microtubules and dynactin in a search-capture mechanism for minus-end-directed organelles.     

CLIPR-59, a new trans-Golgi/TGN cytoplasmic linker protein belonging to the CLIP-170 family

The microtubule cytoskeleton plays a fundamental role in cell organization and membrane traffic in higher eukaryotes. It is well established that molecular motors are involved in membrane-microtubule interactions, but it has also been proposed that nonmotor microtubule-binding (MTB) proteins known as CLIPs (cytoplasmic linker proteins) have basic roles in these processes. We report here the characterization of CLIPR-59, a CLIP-170-related protein localized to the trans-most part of the Golgi apparatus. CLIPR-59 contains an acidic region followed by three ankyrin-like repeats and two CLIP-170-related MTB motifs. We show that the 60-amino acid-long carboxy-terminal domain of CLIPR-59 is necessary and sufficient to achieve Golgi targeting, which represents the first identification of a membrane targeting domain in a CLIP-170-related protein. The MTB domain of CLIPR-59 is functional because it localizes to microtubules when expressed as a fragment in HeLa cells. However, our results suggest that this domain is normally inhibited by the presence of adjacent domains, because neither full-length CLIPR-59 nor a CLIPR-59 mutant missing its membrane-targeting region localize to microtubules. Consistent with this observation, overexpression of CLIPR-59 does not affect the microtubule network. However, CLIPR-59 overexpression strongly perturbs early/recycling endosome-TGN dynamics, implicating CLIPR-59 in the regulation of this pathway.     

A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.     

Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.     

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

CLIP-170, the founding member of microtubule “plus ends tracking” proteins, is involved in many critical microtubule-related functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr²⁸⁷ in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.Microtubule dynamics consist of alternating phases of growth and shortening, a pattern of behavior known as dynamic instability (1). This process is tightly regulated by a group of proteins that bind specifically to the plus ends of the growing microtubules (2). Cytoplasmic linker protein (CLIP)³ -170, the founding member of the microtubule plus end family (3), is composed of three separate regions: N terminus, central coiled-coil region, and C terminus. In addition to two conserved cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, the N terminus has three serine-rich regions. The N-terminal domain plays an essential role in microtubule targeting (4), the long central coiled-coil domain is responsible for dimerization of the protein, and the C-terminal region, which contains two zinc-finger domains interferes with microtubule binding by interacting with the N terminus (5). Experiments in a variety of organisms have demonstrated that CLIP-170 plays an important role in microtubule dynamics (6, 7). In addition to its positive role in regulating microtubule growth in both yeast and humans (8, 9), CLIP-170 is involved in recruitment of dynactin to the microtubule plus ends and in linking microtubules to the cortex through Cdc42 and IQGAP (10, 11). The role of CLIP-170 during mitosis was recently examined by loss-of-function approaches. It was shown that CLIP-170 localizes to unattached kinetochores in prometaphase and that such localization is essential for the formation of kinetochore-microtubule attachments (12, 13).It was previously reported that CLIP-170 is a phosphoprotein and that overall phosphorylation of CLIP-170 affects its microtubule binding ability (14). More recently, metabolic labeling experiments indicated that CLIP-170 is phosphorylated at multiple sites (15). However, individual phosphorylation sites have not been identified. Moreover, the FKBP12-rapamycin-associated protein (FRAP) is the only kinase identified to date for CLIP-170 (15). Therefore, to fully understand the regulation of CLIP-170, it is important to identify individual phosphorylation sites and the responsible kinases. In this communication, we describe a novel kinase/substrate partnership between Cdc2 and CLIP-170. We provide evidence that Cdc2 phosphorylates CLIP-170 at Thr²⁸⁷, and the Cdc2-mediated phosphorylation of CLIP-170 is essential for its localization at microtubule plus ends in the G2 phase and the G2/M transition.     

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.     

CLIP-170 family members: a motor-driven ride to microtubule plus ends

CLIP-170 family proteins regulate microtubule plus end dynamics. Two reports published in this issue of Developmental Cell show that Bik1 and tip1p, the CLIP-170-like proteins of budding and fission yeast, are carried to microtubule plus ends by kinesin motor proteins. These findings indicate a complex interplay between microtubule-associated proteins and suggest a novel mechanism by which kinesin proteins stabilize microtubules.     

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.     

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.     

The microtubule plus end-tracking proteins mal3p and tip1p cooperate for cell-end targeting of interphase microtubules

BACKGROUND: CLIP-170 and EB1 protein family members localize to growing microtubule tips and link spatial information with the control of microtubule dynamics. It is unknown whether these proteins operate independently or whether their actions are coordinated. In fission yeast the CLIP-170 homolog tip1p is required for targeting of microtubules to cell ends, whereas the role of the EB1 homolog mal3p in microtubule organization has not been investigated. RESULTS: We show that mal3p promotes the initiation of microtubule growth and inhibits catastrophes. Premature catastrophes occur randomly throughout the cell in the absence of mal3p. mal3p decorates the entire microtubule lattice and localizes to particles along the microtubules and at their growing tips. Particles move in two directions, outbound toward the cell ends or inbound toward the cell center. At cell ends, the microtubule tip-associated mal3p particles disappear followed by a catastrophe. mal3p localizes normally in tip1-deleted cells and disappears from microtubule tips preceding the premature catastrophes. In contrast, tip1p requires mal3p to localize at microtubule tips. mal3p and tip1p directly interact in vitro. CONCLUSIONS: mal3p and tip1p form a system allowing microtubules to target cell ends. We propose that mal3p stimulates growth initiation and maintains growth by suppressing catastrophes. At cell ends, mal3p disappears from microtubule tips followed by a catastrophe. mal3p is involved in recruiting tip1p to microtubule tips. This becomes important when microtubules contact the cell cortex outside the cell ends because mal3p dissociates prematurely without tip1p, which is followed by a premature catastrophe.