CLIP170 autoinhibition mimics intermolecular interactions with p150Glued or EB1

CLIP170 and p150(Glued) localize to the plus ends of growing microtubules. Using crystallography and NMR, we show that autoinhibitory interactions within CLIP170 use the same binding determinants as CLIP170's intermolecular interactions with p150(Glued). These interactions have both similar and distinct features when compared with the p150(Glued)-EB1 complex. Our data thus demonstrate that regulation of microtubule dynamics by plus end-tracking proteins (+TIPs) occurs through direct competition between homologous binding interfaces.     

EB1 and EB3 control CLIP dissociation from the ends of growing microtubules

EBs and CLIPs are evolutionarily conserved proteins, which associate with the tips of growing microtubules, and regulate microtubule dynamics and their interactions with intracellular structures. In this study we investigated the functional relationship of CLIP-170 and CLIP-115 with the three EB family members, EB1, EB2(RP1), and EB3 in mammalian cells. We showed that both CLIPs bind to EB proteins directly. The C-terminal tyrosine residue of EB proteins is important for this interaction. When EB1 and EB3 or all three EBs were significantly depleted using RNA interference, CLIPs accumulated at the MT tips at a reduced level, because CLIP dissociation from the tips was accelerated. Normal CLIP localization was restored by expression of EB1 but not of EB2. An EB1 mutant lacking the C-terminal tail could also fully rescue CLIP dissociation kinetics, but could only partially restore CLIP accumulation at the tips, suggesting that the interaction of CLIPs with the EB tails contributes to CLIP localization. When EB1 was distributed evenly along the microtubules because of overexpression, it slowed down CLIP dissociation but did not abolish its preferential plus-end localization, indicating that CLIPs possess an intrinsic affinity for growing microtubule ends, which is enhanced by an interaction with the EBs.     

Cooperative stabilization of microtubule dynamics by EB1 and CLIP-170 involves displacement of stably bound P(i) at microtubule ends

End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 μM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ(32)P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound (32)P(i). Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap.     

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.     

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.     

CLIP170-like tip1p spatially organizes microtubular dynamics in fission yeast

Rod-shaped fission yeast cells grow in a polarized manner, and unlike budding yeast, the correct positioning of the growth sites at cell ends requires interphase microtubules. Here we describe a microtubule guidance mechanism that orients microtubules in the intracellular space along the long axis of the cell, guiding them to their target region at the cell ends. This mechanism involves tip1p, a CLIP170-like protein that localizes to distal tips of cytoplasmic microtubules. In the absence of tip1p, microtubular catastrophe is no longer restricted to cell ends but occurs when microtubules reach any region of the cellular cortex. Thus, tip1p enables microtubules to discriminate different cortical regions and regulates their dynamics accordingly.     

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.     

The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules

Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein [1] [2] [3]. We previously showed that APC tagged with green fluorescent protein (GFP) in Xenopus A6 epithelial cells moves along a subset of microtubules and accumulates at their growing plus ends in cell extensions [4]. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis [5], was also reported to be associated with microtubules [6] [7] [8]. To examine the interaction between APC and EB1 within cells, we compared the dynamic behavior of EB1-GFP with that of APC-GFP in A6 transfectants. Time-lapse microscopy of live cells at interphase revealed that EB1-GFP was concentrated at all of the growing microtubule ends throughout the cytoplasm and abruptly disappeared from the ends when microtubules began to shorten. Therefore, EB1 appeared to be co-localized and interact with APC on the growing ends of a subset of microtubules. When APC-GFP was overexpressed, endogenous EB1 was recruited to APC-GFP, which accumulated in large amounts on microtubules. On the other hand, when microtubules were disassembled by nocodazole, EB1 was not co-localized with APC-GFP, which was concentrated along the basal plasma membrane. During mitosis, APC appeared to be dissociated from microtubules, whereas EB1-GFP continued to concentrate at microtubule growing ends. These findings showed that the APC-EB1 interaction is regulated within cells and is allowed near the ends of microtubules only under restricted conditions.     

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.     

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.     

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.     

Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules

The positioning of growth sites in fission yeast cells is mediated by spatially controlled microtubule dynamics brought about by tip1p, a CLIP-170-like protein, which is localized at the microtubule tips and guides them to the cell ends. The kinesin tea2p is also located at microtubule tips and affects microtubule dynamics. Here we show that tea2p interacts with tip1p and that the two proteins move with high velocity along the microtubules toward their growing tips. There, tea2p and tip1p accumulate in larger particles. Particle formation requires the EB1 homolog, mal3p. Our results suggest a model in which kinesins regulate microtubule growth by transporting regulatory factors such as tip1p to the growing microtubule tips.     

NudEL targets dynein to microtubule ends through LIS1

Dynein is a minus-end-directed microtubule motor with critical roles in mitosis, membrane transport and intracellular transport. Several proteins regulate dynein activity, including dynactin, LIS1 (refs 2, 3) and NudEL (NudE-like). Here, we identify a NUDEL homologue in budding yeast and name it Ndl1. The ndl1delta null mutant shows decreased targeting of dynein to microtubule plus ends, an essential element of the model for dynein function. We find that Ndl1 regulates dynein targeting through LIS1, with which it interacts biochemically, but not through CLIP170, another plus-end protein involved in dynein targeting. Ndl1 is found at far fewer microtubule ends than are LIS1 and dynein. However, when Ndl1 is present at a plus end, the molar amount of Ndl1 approaches that of LIS1 and dynein. We propose a model in which Ndl1 binds transiently to the plus end to promote targeting of LIS1, which cooperatively recruits dynein.     

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.     

Phosphorylation of CLIP-170 by Plk1 and CK2 promotes timely formation of kinetochore�Cmicrotubule attachments

CLIP‐170 is implicated in the formation of kinetochore–microtubule attachments through direct interaction with the dynein/dynactin complex. However, whether this important function of CLIP‐170 is regulated by phosphorylation is unknown. Herein, we have identified polo‐like kinase 1 (Plk1) and casein kinase 2 (CK2) as two kinases of CLIP‐170 and mapped S195 and S1318 as their respective phosphorylation sites. We showed that a CK2 unphosphorylatable mutant lost its ability to bind to dynactin and to localize to kinetochores during prometaphase, indicating that the CK2 phosphorylation of CLIP‐170 is involved in its dynactin‐mediated kinetochore localization. Furthermore, we provide evidence that Plk1 phosphorylation of CLIP‐170 at S195 enhances its association with CK2. Finally, we detected defects in the formation of kinetochore fibres in cells expressing the CLIP‐S195A and ‐S1318A, but not the CLIP‐S195E and ‐S1318D, confirming that Plk1‐ and CK2‐associated phosphorylations of CLIP‐170 are involved in the timely formation of kinetochore–microtubule attachments in mitosis.     

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.     

CLIP‐170 spatially modulates receptor tyrosine kinase recycling to coordinate cell migration

Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location and surface complement of RTKs. The microtubule (MT) plus‐end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalization in response to ligand remain poorly understood. We report that net outward‐directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP‐170, as well as the association of CLIP‐170 to MT plus‐ends. In response to HGF, entry of Met into Rab4‐positive endosomes results in Golgi‐localized γ‐ear‐containing Arf‐binding protein 3 (GGA3) and CLIP‐170 recruitment to an activated Met RTK complex. We conclude that CLIP‐170 co‐ordinates the recycling and the transport of Met‐positive endocytic vesicles to plus‐ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration.      

Dynein,Lis1 and CLIP‐170 counteract Eg5‐dependent centrosome separation during bipolar spindle assembly

Bipolar spindle assembly critically depends on the microtubule plus‐end‐directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus‐end‐directed motor dynein, or the dynein‐binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5‐dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus‐end‐tracking protein CLIP‐170. This function of CLIP‐170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP‐115, a highly homologous protein that lacks the dynein–dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP‐170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.     

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.     

Dynamics of interphase microtubules in Schizosaccharomyces pombe

BACKGROUND: Microtubules in interphase Schizosaccharomyces pombe are essential for maintaining the linear growth habit of these cells. The dynamics of assembly and disassembly of these microtubules are so far uncharacterised. RESULTS: Live cell confocal imaging of alpha1 tubulin tagged with enhanced green fluorescent protein revealed longitudinally oriented, dynamically unstable interphase microtubule assemblies (IMAs). The IMAs were uniformly bright along their length apart from a zone of approximately doubly intense fluorescence commonly present close to their centres. The ends of each IMA switched from growth ( approximately 3.0 microm/min) to shrinkage ( approximately 4.5 microm/min) at 1.0 events per minute and from shrinkage to growth at 1.9 events per minute, and the two ends were equivalently dynamic, suggesting equivalent structure. We accordingly propose a symmetrical model for microtubule packing within the IMAs, in which microtubules are plus ends out and overlap close to the equator of the cell. IMAs may contain multiple copies of this motif; if so, then within each IMA end, the microtubule ends must synchronise catastrophe and rescue. When both ends of an IMA lodge in the hemispherical cell ends, the IMAs start to bend under compression and their overall growth rate is inhibited about twofold. Similar microtubule dynamics were observed in cells ranging in size from half to twice normal length. Patterned photobleaching indicated no detectable treadmilling or microtubule sliding during interphase. CONCLUSIONS: The consequence of the mechanisms described is continuous recruitment of microtubule ends to the ends of growing cells, supporting microtubule-based transport into the cell ends and qualitatively accounting for the essential role for microtubules in directing linear cell growth in S. pombe.