Arl4D-EB1 interaction promotes centrosomal recruitment of EB1 and microtubule growth

ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. End-binding 1 (EB1) is a microtubule (MT) plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing MTs and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates MT nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EB homology region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal MT nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased MT nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and the EB1, which is important for MT stabilization. Together, our results indicate that Arl4D modulates MT nucleation through regulation of the EB1–p150 association at the centrosome.     

Kif18B interacts with EB1 and controls astral microtubule length during mitosis

Regulation of microtubule (MT) dynamics is essential for proper spindle assembly and organization. Kinesin-8 family members are plus-end-directed motors that modulate plus-end MT dynamics by acting as MT depolymerases or as MT plus-end capping proteins. In this paper, we show that the human kinesin-8 Kif18B functions during mitosis to control astral MT organization. Kif18B is a MT plus-tip-tracking protein that localizes to the nucleus in interphase and is enriched at astral MT plus ends during early mitosis. Knockdown of Kif18B caused spindle defects, resulting in an increased number and length of MTs. A yeast two-hybrid screen identified an interaction of the C-terminal domain of Kif18B with the plus-end MT-binding protein EB1. EB1 knockdown disrupted Kif18B targeting to MT plus ends, indicating that EB1/Kif18B interaction is physiologically important. This interaction is direct, as the far C-terminal end of Kif18B is sufficient for binding to EB1 in vitro. Overexpression of this domain is sufficient for plus-end MT targeting in cells; however, targeting is enhanced by the motor domain, which cooperates with the tail to achieve proper Kif18B localization at MT plus ends. Our results suggest that Kif18B is a new MT dynamics regulatory protein that interacts with EB1 to control astral MT length.     

Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase

XMAP215/Dis1 proteins are conserved tubulin-binding TOG-domain proteins that regulate microtubule (MT) plus-end dynamics. Here we show that Alp14, a XMAP215 orthologue in fission yeast, Schizosaccharomyces pombe, has properties of a MT polymerase. In vivo, Alp14 localizes to growing MT plus ends in a manner independent of Mal3 (EB1). alp14-null mutants display short interphase MTs with twofold slower assembly rate and frequent pauses. Alp14 is a homodimer that binds a single tubulin dimer. In vitro, purified Alp14 molecules track growing MT plus ends and accelerate MT assembly threefold. TOG-domain mutants demonstrate that tubulin binding is critical for function and plus end localization. Overexpression of Alp14 or only its TOG domains causes complete MT loss in vivo, and high Alp14 concentration inhibits MT assembly in vitro. These inhibitory effects may arise from Alp14 sequestration of tubulin and effects on the MT. Our studies suggest that Alp14 regulates the polymerization state of tubulin by cycling between a tubulin dimer-bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly.     

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.     

Centriolar Association of ALMS1 and Likely Centrosomal Functions of the ALMS Motif–containing Proteins C10orf90 and KIAA1731

Mutations in the human gene ALMS1 cause Alström syndrome, a rare progressive condition characterized by neurosensory degeneration and metabolic defects. ALMS1 protein localizes to the centrosome and has been implicated in the assembly and/or maintenance of primary cilia; however its precise function, distribution within the centrosome, and mechanism of centrosomal recruitment are unknown. The C-terminus of ALMS1 contains a region with similarity to the uncharacterized human protein C10orf90, termed the ALMS motif. Here, we show that a third human protein, the candidate centrosomal protein KIAA1731, contains an ALMS motif and that exogenously expressed KIAA1731 and C10orf90 localize to the centrosome. However, based on deletion analysis of ALMS1, the ALMS motif appears unlikely to be critical for centrosomal targeting. RNAi analyses suggest that C10orf90 and KIAA1731 have roles in primary cilium assembly and centriole formation/stability, respectively. We also show that ALMS1 localizes specifically to the proximal ends of centrioles and basal bodies, where it colocalizes with the centrosome cohesion protein C-Nap1. RNAi analysis reveals markedly diminished centrosomal levels of C-Nap1 and compromised cohesion of parental centrioles in ALMS1-depleted cells. In summary, these data suggest centrosomal functions for C10orf90 and KIAA1731 and new centriole-related functions for ALMS1.     

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.     

The Microtubule plus end-tracking protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii

Flagellar axonemes assemble and continuously turn over at the flagellar tip. The supply and removal of axonemal subunits at the tip are mediated by intraflagellar transport (IFT), a motility process essential for the assembly and maintenance of all eukaryotic flagella and cilia. IFT is characterized by the movement of large protein complexes (IFT particles) from the basal bodies to the flagellar tip by kinesin-II and from the tip back to the basal bodies by cytoplasmic dynein 1b. The IFT particles consist of approximately 16 polypeptides partitioned into two complexes, A and B, and associate with axonemal precursors/turn over products. The mechanisms by which IFT motor regulation and cargo loading/unloading occur at the flagellar tip are unknown. We identified a Chlamydomonas reinhardtii ortholog of the microtubule (MT) plus end-tracking protein EB1 [4] (CrEB1) and show here that CrEB1 localizes to the tip of flagella and to the proximal part of the basal bodies. Furthermore, we found that CrEB1 is depleted from flagella of the temperature-sensitive (ts) flagellar assembly-defective (fla) mutant fla11(ts) at the restrictive temperature. This depletion of CrEB1 is accompanied by a dramatic accumulation of IFT particle polypeptides near the flagellar tip.     

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.     

The LIM protein Ajuba is required for ciliogenesis and left-right axis determination in medaka

Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left-right organ asymmetries and altered expression of early genes responsible for left-right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer’s vesicle; it is these cells that induce the asymmetric fluid flow required for left-right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left-right axis determination.     

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 complex of two centrosomal proteins, CAP350 and FOP, cooperates with EB1 in microtubule anchoring

The anchoring of microtubules (MTs) to subcellular structures is critical for cell shape, polarity, and motility. In mammalian cells, the centrosome is a prominent MT anchoring structure. A number of proteins, including ninein, p150Glued, and EB1, have been implicated in centrosomal MT anchoring, but the process is far from understood. Here we show that CAP350 and FOP (FGFR1 oncogene partner) form a centrosomal complex required for MT anchoring. We show that the C-terminal domain of CAP350 interacts directly with FOP and that both proteins localize to the centrosome throughout the cell cycle. FOP also binds to EB1 and is required for localizing EB1 to the centrosome. Depletion of either CAP350, FOP, or EB1 by siRNA causes loss of MT anchoring and profound disorganization of the MT network. These results have implications for the mechanisms underlying MT anchoring at the centrosome and they attribute a key MT anchoring function to two novel centrosomal proteins, CAP350 and FOP.     

TIP maker and TIP marker; EB1 as a master controller of microtubule plus ends

The EB1 protein is a member of the exciting and enigmatic family of microtubule (MT) tip-tracking proteins. EB1 acts as an exquisite marker of dynamic MT plus ends in some cases, whereas in others EB1 is thought to directly dictate the behavior of the plus ends. How EB1 differentiates between these two roles remains unclear; however, a growing list of interactions between EB1 and other MT binding proteins suggests there may be a single mechanism. Adding another layer of complexity to these interactions, two studies published in this issue implicate EB1 in cross-talk between mitotic MTs and between MTs and actin filaments (Goshima et al., p. 229; Wu et al., p. 201). These results raise the possibility that EB1 is a central player in MT-based transport, and that the activity of MT-binding proteins depends on their ability or inability to interact with EB1.     

Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1

In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-green fluorescent protein and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 small interfering RNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remained bound to MTs stabilized with low nocodazole, whereas EB1 did not. Thus, we demonstrate that the association of endogenous APC with MT ends correlates directly with their increased growth stability, that this can occur independently of its association with EB1, and that APC and EB1 can associate with MT plus ends by distinct mechanisms.     

A novel localization pattern for an EB1-like protein links microtubule dynamics to endomembrane organization

A group of microtubule-associated proteins called +TIPs (plus end tracking proteins), including EB1 family proteins, label growing microtubule ends specifically in diverse organisms and are implicated in spindle dynamics, chromosome segregation, and directing microtubules toward cortical sites. Here, we report three new EB1-like proteins from Arabidopsis and provide the intracellular localization for AtEB1, which differs from all known EB1 proteins in having a very long acidic C-terminal tail. In marked contrast to other EB1 proteins, the GFP-AtEB1 fusion protein localizes not only to microtubule plus ends but also to motile, pleiomorphic tubulovesicular membrane networks that surround other organelles and frequently merge with the endoplasmic reticulum. AtEB1 behavior thus resembles that of +TIPs, such as the cytoplasmic linker protein CLIP-170, that are known to associate with and pull along membrane tubules in animal systems but for which homologs have not been identified in plants. In addition, though EB1 proteins are believed to stabilize microtubules, a different behavior is observed for AtEB1 where instead of stabilizing a microtubule it localizes to already stabilized regions on a microtubule. The dual localization pattern of AtEB1 suggests links between microtubule plus end dynamics and endomembrane organization during polarized growth of plant cells.     

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.     

Axoneme-specific beta-tubulin specialization: a conserved C-terminal motif specifies the central pair.

Axonemes are ancient organelles that mediate motility of cilia and flagella in animals, plants, and protists. The long evolutionary conservation of axoneme architecture, a cylinder of nine doublet microtubules surrounding a central pair of singlet microtubules, suggests all motile axonemes may share common assembly mechanisms. Consistent with this, alpha- and beta-tubulins utilized in motile axonemes fall among the most conserved tubulin sequences [1, 2], and the beta-tubulins contain a sequence motif at the same position in the carboxyl terminus [3]. Axoneme doublet microtubules are initiated from the corresponding triplet microtubules of the basal body [4], but the large macromolecular "central apparatus" that includes the central pair microtubules and associated structures [5] is a specialization unique to motile axonemes. In Drosophila spermatogenesis, basal bodies and axonemes utilize the same alpha-tubulin but different beta-tubulins [6--13]. beta 1 is utilized for the centriole/basal body, and beta 2 is utilized for the motile sperm tail axoneme. beta 2 contains the motile axoneme-specific sequence motif, but beta 1 does not [3]. Here, we show that the "axoneme motif" specifies the central pair. beta 1 can provide partial function for axoneme assembly but cannot make the central microtubules [14]. Introducing the axoneme motif into the beta 1 carboxyl terminus, a two amino acid change, conferred upon beta 1 the ability to assemble 9 + 2 axonemes. This finding explains the conservation of the axoneme-specific sequence motif through 1.5 billion years of evolution.     

Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly

Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.     

EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.     

Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase

The central spindle forms between segregating chromosomes during anaphase and is required for cytokinesis. Although anaphase-specific bundling and stabilization of interpolar microtubules (MTs) contribute to formation of the central spindle, it remains largely unknown how these MTs are prepared. Using live imaging of MT plus ends and an MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle occur independently of preanaphase MTs or centrosomal MT nucleation but require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during preanaphase. MTs generated in a hepatoma up-regulated protein (HURP)-dependent manner during anaphase also contribute to central spindle formation redundantly with preanaphase MTs. Based on these results, a new model for central spindle assembly is proposed.     

Centriolar kinesin Kif24 interacts with CP110 to remodel microtubules and regulate ciliogenesis

We have identified a protein, Kif24, that shares homology with the kinesin-13 subfamily of motor proteins and specifically interacts with CP110 and Cep97, centrosomal proteins that play a role in regulating centriolar length and ciliogenesis. Kif24 preferentially localizes to mother centrioles. Loss of Kif24 from cycling cells resulted in aberrant cilia assembly but did not promote growth of abnormally long centrioles, unlike CP110 and Cep97 depletion. We found that loss of Kif24 leads to the disappearance of CP110 from mother centrioles, specifically in cycling cells able to form cilia. Kif24 is able to bind and depolymerize microtubules in vitro. Remarkably, ectopically expressed Kif24 specifically remodels centriolar microtubules without significantly altering cytoplasmic microtubules. Thus, our studies have identified a centriolar kinesin that specifically remodels a subset of microtubules, thereby regulating cilia assembly. These studies also suggest mechanistic differences between the regulation of microtubule elongation associated with centrioles and cilia.