Yeast Bim1p promotes the G1-specific dynamics of microtubules

Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells. This study reports that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1. During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state. Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein. Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules. This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end. These in vivo data provide evidence of a cell cycle-specific microtubule-binding protein that promotes microtubule dynamicity (Tirnauer, 1999; full text of article).

Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables

Microtubules and actin filaments interact and cooperate in many processes in eukaryotic cells, but the functional implications of such interactions are not well understood. In the yeast Saccharomyces cerevisiae, both cytoplasmic microtubules and actin filaments are needed for spindle orientation. In addition, this process requires the type V myosin protein Myo2, the microtubule end-binding protein Bim1 (homolog of EB1), and Kar9. Fusing Bim1 to the tail of the Myo2 is sufficient to orient spindles in the absence of Kar9, suggesting that the role of Kar9 is to link Myo2 to Bim1. In addition, Myo2 localizes to the plus ends of cytoplasmic microtubules, and the rate of movement of these cytoplasmic microtubules to the bud neck depends on the intrinsic velocity of Myo2 along actin filaments. These results support a model for spindle orientation in which a Myo2-Kar9-Bim1 complex transports microtubule ends along polarized actin cables. Data is presented suggesting that a similar process plays a role in orienting cytoplasmic microtubules in mating yeast cells (Hwang, 2003).

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

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. This study shows 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. It is concluded mal3p and tip1p form a system allowing microtubules to target cell ends. It is proposed 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 (Busch, 2004a)

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. Tea2p interacts with tip1p, and 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. These results suggest a model in which kinesins regulate microtubule growth by transporting regulatory factors such as tip1p to the growing microtubule tips (Busch, 2004b).

The fission yeast kinetochore component Spc7 associates with the EB1 family member Mal3 and is required for kinetochore-spindle association

A critical aspect of mitosis is the interaction of the kinetochore with spindle microtubules. Fission yeast Mal3 is a member of the EB1 family of microtubule plus-end binding proteins that have been implicated in this process. However, the Mal3 interaction partner at the kinetochore had not been identified. This study shows that the mal3 mutant phenotype can be suppressed by the presence of extra Spc7, an essential kinetochore protein associated with the central centromere region. Mal3 and Spc7 interact physically as both proteins can be coimmunoprecipitated. Overexpression of a Spc7 variant severely compromises kinetochore-microtubule interaction, indicating that the Spc7 protein plays a role in this process. Spc7 function seems to be conserved because, Spc105, a Saccharomyces cerevisiae homolog of Spc7, identified by mass spectrometry as a component of the conserved Ndc80 complex, can rescue mal3 mutant strains (Kerres, 2004; full text of article).

The EB1 homolog Mal3 stimulates the ATPase of the kinesin Tea2 by recruiting it to the microtubule

Tea2 is a kinesin family member from Schizosaccharomyces pombe that is targeted to microtubule tips and cell ends in a process that depends on EB1 homolog Mal3. Constructs of Tea2 containing the motor domain only or the motor domain plus the N-terminal extension are monomeric, whereas a construct including the first predicted coiled coil region is dimeric. These constructs have a low basal rate of ATP, but microtubules stimulate the rate of ATP hydrolysis to a maximum of approximately 15 s(-1). Hydrodynamic analysis of Mal3 indicates that it is dimeric. Mal3 is known to associate with Tea2, and analysis with the above Tea2 constructs indicates that the principal site of interaction of Mal3 with Tea2 is the N-terminal extension, although a weaker interaction is also observed with the motor domain alone. In parallel to the binding studies, Mal3 strongly stimulates the ATPase of constructs containing the N-terminal extension by decreasing the K0.5(MT) for stimulation by microtubules but only weakly stimulates motor domains without the N-terminal extension. Mal3 reduces the K0.5(MT) values without affecting the k(cat) value at saturating microtubule level. Binding of Mal3 to microtubules induces an increase in the binding of Tea2 and a reciprocal stimulation of Mal3 binding by Tea2 is also observed. Tea2 is a plus end directed motor that drives sliding of axonemes when adsorbed to a glass surface. The sliding rate is initially unaffected by Mal3, but axonemes stop moving on continued exposure to Mal3 (Browning, 2005).

Gamma-tubulin is required for proper recruitment and assembly of Kar9-Bim1 complexes in budding yeast

Microtubule plus-end-interacting proteins (+TIPs) promote the dynamic interactions between the plus ends (+ends) of astral microtubules and cortical actin that are required for preanaphase spindle positioning. Paradoxically, +TIPs such as the EB1 orthologue Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Deletion of four C-terminal residues of the budding yeast gamma-tubulin Tub4 (tub4-delta dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependent spindle positioning. Surprisingly, Kar9 localizes to microtubule +ends in tub4-delta dsyl cells, but these microtubules fail to position the spindle when targeted to the bud. Using cofluorescence and coaffinity purification, it has been showm Kar9 complexes in tub4-delta dsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-delta dsyl cells, but it are restored by deletion of Kar9. Moreover, Myo2- and F-actin-dependent dwelling of Kar9 in the bud is observed in tub4-delta dsyl cells, suggesting defective Kar9 complexes tether microtubule +ends to the cortex. Overproduction of Bim1, but not Kar9, restores Kar9-dependent spindle positioning in the tub4-delta dsyl mutant, reduces cortical dwelling, and promotes Bim1-Kar9 interactions. It is proposed that SPBs, via the tail of Tub4, promote the assembly of functional +TIP complexes before their deployment to microtubule +ends (Cuschieri, 2006).

Dictyostelium EB1 is a genuine centrosomal component required for proper spindle formation

EB1 proteins are ubiquitous microtubule-associated proteins involved in microtubule search and capture, regulation of microtubule dynamics, cell polarity, and chromosome stability. A complete cDNA has been cloned of Dictyostelium EB1 (DdEB1), the largest known EB1 homolog (57 kDa). Immunofluorescence analysis and expression of a green fluorescent protein-DdEB1 fusion protein revealed that DdEB1 localizes along microtubules, at microtubule tips, centrosomes, and protruding pseudopods. During mitosis, it was found at the spindle, spindle poles, and kinetochores. DdEB1 is the first EB1-homolog that is also a genuine centrosomal component, because it was localized at isolated centrosomes that are free of microtubules. Furthermore, centrosomal DdEB1 distribution is unaffected by nocodazole treatment. DdEB1 colocalizes with DdCP224, the XMAP215 homolog, at microtubule tips, the centrosome, and kinetochores. Furthermore, both proteins are part of the same cytosolic protein complex, suggesting that they may act together in their functions. DdEB1 deletion mutants expressed as green fluorescent protein or maltose-binding fusion proteins indicate that microtubule binding requires homo-oligomerization, which is mediated by a coiled-coil domain. A DdEB1 null mutant is viable but retarded in prometaphase progression due to a defect in spindle formation. Because spindle elongation is normal, DdEB1 seems to be required for the initiation of the outgrowth of spindle microtubules (Rehberg, 2002; full text of article).

The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing the microtubule-binding properties of the fission yeast EB1 homolog Mal3p have been studied. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. Structural data is provided showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam; the data suggests that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. These findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes (Sandblad, 2006).

Crystal structure of the amino-terminal microtubule-binding domain of EB1

The end-binding protein 1 (EB1) family is a highly conserved group of proteins that localizes to the plus-ends of microtubules. EB1 has been shown to play an important role in regulating microtubule dynamics and chromosome segregation, but its regulation mechanism is poorly understood. This study determined the 1.45-Å resolution crystal structure of the amino-terminal domain of EB1, which is essential for microtubule binding; it forms a calponin homology (CH) domain fold that is found in many proteins involved in the actin cytoskeleton. The functional CH domain for actin binding is a tandem pair, whereas EB1 is the first example of a single CH domain that can associate with the microtubule filament. Although this biochemical study shows that microtubule binding of EB1 is electrostatic in part, mutational analysis suggests that the hydrophobic network, which is partially exposed in the crystal structure, is also important for the association. It is proposed that, like other actin-binding CH domains, EB1 employs the hydrophobic interaction to bind to microtubules (Hayashi, 2003; full text of article).

EB1-microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules

EB1 targets to polymerizing microtubule ends, where it is favorably positioned to regulate microtubule polymerization and confer molecular recognition of the microtubule end. This study focused on two aspects of the EB1-microtubule interaction: regulation of microtubule dynamics by EB1 and the mechanism of EB1 association with microtubules. Immunodepletion of EB1 from cytostatic factor-arrested M-phase Xenopus egg extracts dramatically reduces microtubule length; this is complemented by readdition of EB1. By time-lapse microscopy, EB1 increases the frequency of microtubule rescues and decreased catastrophes, resulting in increased polymerization and decreased depolymerization and pausing. Imaging of EB1 fluorescence revealed a novel structure: filamentous extensions on microtubule plus ends that appeared during microtubule pauses; loss of these extensions correlate with the abrupt onset of polymerization. Fluorescent EB1 localizes to comets at the polymerizing plus ends of microtubules in cytostatic factor extracts and uniformly along the lengths of microtubules in interphase extracts. The temporal decay of EB1 fluorescence from polymerizing microtubule plus ends predicts a dissociation half-life of seconds. Fluorescence recovery after photobleaching also revealed dissociation and rebinding of EB1 to the microtubule wall with a similar half-life. EB1 targeting to microtubules is thus described by a combination of higher affinity binding to polymerizing ends and lower affinity binding along the wall, with continuous dissociation. The latter is likely to be attenuated in interphase. The highly conserved effect of EB1 on microtubule dynamics suggests it belongs to a core set of regulatory factors conserved in higher organisms, and the complex pattern of EB1 targeting to microtubules could be exploited by the cell for coordinating microtubule behaviors (Tirnauer, 2002a; full text of article).

EB1 targets to kinetochores with attached, polymerizing microtubules

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, its targeting was visualized in mitotic PtK1 cells. Fluorescent EB1, which localizes to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associates with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurs in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 does not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targets to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localizes independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. These results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment (Tirnauer, 2002b).

Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome

EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. The C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 forms mutually exclusive complexes with APC and dynactin and a direct interaction between EB1 and p150(Glued). EB1-GFP deletion mutants demonstrate a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibits microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes is also inhibited. None of these effects are seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression does not induce Golgi apparatus fragmentation. It is propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array (Askham, 2002; full text of article).

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. This study clarifies the interaction between EB1 and dynactin and shows 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 differences in microtubule dynamics (Ligon, 2003).

Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex

Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. This study reports the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, these studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. It is therefore proposed that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator (Hayashi, 2005).

APC and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes

Adenomatous polyposis coli (APC) and End-binding protein 1 (EB1) localize to centrosomes independently of cytoplasmic microtubules (MTs) and purify with centrosomes from mammalian cell lines. Localization of EB1 to centrosomes is independent of its MT binding domain and is mediated by its C-terminus. Both APC and EB1 preferentially localize to the mother centriole and EB1 forms a cap at the end of the mother centriole that contains the subdistal appendages as defined by epsilon-tubulin localization. Like endogenous APC and EB1, fluorescent protein fusions of APC and EB1 localize preferentially to the mother centriole. Depletion of EB1 by RNA interference reduces MT minus-end anchoring at centrosomes and delays MT regrowth from centrosomes. In summary, these data indicate that APC and EB1 are functional components of mammalian centrosomes and that EB1 is important for anchoring cytoplasmic MT minus ends to the subdistal appendages of the mother centriole (Louie, 2004).

Structural insights into the EB1-APC interaction

EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. EB1 is a stable dimer with a parallel coiled coil, and dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 microM affinity. The conserved APC Ile2805-Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789-Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC-EB1 is post-translationally regulated in cells. These findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms (Honnappa, 2005; full text of article).

The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein, a domain of APC that is commonly deleted in colorectal neoplasia. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 and Saccharomyces cerevisiae Bim1p. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle, in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis. This study demonstrated that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 is also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex is independent of APC and is preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes (Berrueta, 1999).

APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment

A cancer causing truncation in adenomatous polyposis coli (APC), APC(1-1450), dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. This study examined the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. APC(1-1450) acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC(1-1450) compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells (Green, 2005; full text of article).

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. A nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin were comicroinjected to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and have a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associates briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, EB1-green fluorescent protein and endogenous APC were monitored concomitantly in living cells. Only a small fraction of EB1 colocalizes 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 does not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remains bound to MTs stabilized with low nocodazole, whereas EB1 does not. Thus, the association of endogenous APC with MT ends correlates directly with their increased growth stability, this can occur independently of its association with EB1, and APC and EB1 can associate with MT plus ends by distinct mechanisms (Kita, 2006; full text of article).

Regulated binding of adenomatous polyposis coli protein to actin

Adenomatous polyposis coli (APC) protein is a large tumor suppressor that is truncated in most colorectal cancers. The carboxyl-terminal third of APC protein mediates direct interactions with microtubules and the microtubule plus-end tracking protein EB1. In addition, APC has been localized to actin-rich regions of cells, but the mechanism and functional significance of this localization have remained unclear. This study shows that purified carboxyl-terminal basic domain of human APC protein (APC-basic) binds directly to and bundled actin filaments and associates with actin stress fibers in microinjected cells. Actin filaments and microtubules compete for binding to APC-basic, but APC-basic also can cross-link actin filaments and microtubules at specific concentrations, suggesting a possible role in cytoskeletal cross-talk. APC interactions with actin in vitro are inhibited by its ligand EB1, and co-microinjection of EB1 preventes APC association with stress fibers. Point mutations in EB1 that disrupt APC binding relieves the inhibition in vitro and restores APC localization to stress fibers in vivo, demonstrating that EB1-APC regulation is direct. Because tumor formation and metastasis involve coordinated changes in the actin and microtubule cytoskeletons, this novel function for APC and its regulation by EB1 may have direct implications for understanding the molecular basis of tumor suppression (Moseley, 2007).

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

EBs and CLIPs are evolutionarily conserved proteins that associate with the tips of growing microtubules, and regulate microtubule dynamics and their interactions with intracellular structures. This study investigated the functional relationship of CLIP-170 and CLIP-115 with the three EB family members, EB1, EB2(RP1), and EB3 in mammalian cells. 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 (Komarova, 2005; full text of article).

Microtubule plus-end loading of p150Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells

Microtubule dynamics and function are regulated, at least in part, by a family of proteins that localize to microtubule plus-ends, and include EB1, CLIP-170 and the dynactin component p150(Glued). Plus-end pools of these proteins, notably dynactin, have been invoked in a number of 'search-and-capture' mechanisms, including the attachment of microtubules to kinetochores during mitosis and to endomembranes prior to the initiation of intracellular transport. This study shows that, in mammalian cells, EB1 is required for the plus-end localization of CLIP-170, and that this is in turn required to localize p150(Glued) to plus-ends. Specific depletion of CLIP-170 results in defects in microtubule dynamics, cell polarization in response to scratch wounding and a loss of p150(Glued) from plus ends. By contrast, removal of p150(Glued) from plus-ends by depletion of either EB1 or CLIP-170 caused no defects in the localization of intracellular organelles, the dynamics of ER-to-Golgi transport, the efficiency of transferrin uptake or the motility of early endosomes or lysosomes. In addition to labelling microtubule plus-ends, GFP-p150(Glued) becomes incorporated into the dynactin complex and labels small, highly dynamic, punctate structures that move along microtubules. A subset of these structures colocalizes with ER-Golgi transport intermediates. Together, these data show that the function of CLIP-170 and p150(Glued) in membrane trafficking is not associated with their plus-end localization (Watson, 2006; full text of article).

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. Whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization was investigated. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functions as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevents microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, is 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, inhibits 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 (Wen, 2004).

Melanophilin and myosin Va track the microtubule plus end on EB1

In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. This study shows that Mlp is also a microtubule plus end-tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp-microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end indirectly [corrected] by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast. It is suggested that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end (Wu, 2005; full text of article).

The microtubule plus-end tracking protein EB1 is required for Kv1 voltage-gated K+ channel axonal targeting

Axonal Kv1 channels regulate action potential propagation -- an evolutionarily conserved function important for the control of motor behavior as evidenced from the linkage of human Kv1 channel mutations to myokymia/episodic ataxia type 1 (EA1) and the Shaker mutant phenotype in Drosophila. To search for the machinery that mediates axonal targeting of Kv1 channels composed of both α and α subunits, it was first demonstrate that Kvβ2 is responsible for targeting Kv1 channels to the axon. Next, it was shown that Kvβ2 axonal targeting depends on its ability to associate with the microtubule (MT) plus-end tracking protein (+TIP) EB1. Not only do Kvß2 and EB1 move in unison down the axon, Brefeldin A-sensitive Kv1-containing vesicles can also be found at microtubule ends near the cell membrane. In addition, it was found that Kvβ2 associates with KIF3/kinesin II as well. Indeed, Kv1 channels rely on both KIF3/kinesin II and EB1 for their axonal targeting (Gu, 2006).

Axonal voltage-gated potassium (Kv1, KCNA, Shaker) channels control action potential waveform and regulate the fidelity of action potential propagation. Multiple Kv1 family members including Kv1.1, Kv1.2, and Kv1.4, which share ~70% amino acid identity, give rise to Kv channels in mammalian axons with functions important for normal behavior and neuronal excitability. Indeed, mutations of the human KCNA1 gene cause episodic ataxia type 1, most likely due to abnormally robust action potential invasion of axonal branches of central neurons, as well as hypokalemic myokymia -- a likely consequence of action potential reflection (backfiring) from motor nerve terminals. Myokymic and neuromyotonic discharges of motor axons have also been associated with autoantibodies against Kv1 channels in patients with autoimmune, sometimes paraneoplastic, conditions. Intriguingly, Kv1 channel function is important not only for movement but also for sleep; Shaker flies are short sleepers. To better understand the physiological function and regulation of Kv1 channels, it will be important to learn how these channels target to the axon (Gu, 2006).

In the mammalian brain, Kv1 channels reside in the axons and terminal fields in multiple brain regions and in cultured neurons. Their axonal targeting requires the highly conserved Kv1 T1 tetramerization domain. T1 mutations that disrupt T1-Kvβ interaction abolish the ability of T1 to mediate axonal targeting thus implicating Kvβ, which belongs to the eldo-keto reductase family (Heinemann, 2006). These Kvβ subunits regulate Kv1 channel inactivation and promote channel forward trafficking; however, it is not known how Kvβ might target Kv1 channels to the axon (Gu, 2006).

Microtubule (MT)-interacting proteins are good candidates for mediating axonal targeting, since MT extends down the axon with its plus end pointing distally. A number of +TIPs including EB1 specifically track the rapidly growing MT plus end and concentrate in distal axons and growth cones. Whether axonal targeting involves the association of cargos with +TIPs as well as kinesin, perhaps in a way analogous to protein targeting to the cell end in yeast, is an interesting open question (Gu, 2006).

Initially identified as a binding partner of the tumor suppressor adenomatous polyposis coli (APC), EB1 binds to MT plus end and promotes MT extension. The evolutionarily conserved EB1 contains an N-terminal calponin homology domain for MT binding (Hayashi, 2003) and a C-terminal domain for dimerization and for interaction with EB1-binding proteins including other +TIPs such as APC). EB1 can display plus-end accumulation in the absence of other +TIPs in yeast and in mammals. Moreover, EB1 may enhance kinesin interaction with microtubules (Browning, 2005; Galjart, 2005) and help localizing kinesin to MT plus ends in yeast. Similarly, APC, which is transported along the microtubule via KIF3/kinesin II, may be anchored at the MT plus end by EB1 in mammalian cells (Gu, 2006 and references therein).

How membrane proteins such as Kv1 channels are transported down the axon is an interesting open question. Whereas KIF3/kinesin II is involved in the axonal transport of 90-160 nm vesicles and ~40% of these vesicles contain the KIF3-interacting protein fodrin, it is unknown how most of the cargo vesicles are recognized by KIF3, nor have the molecular contents of these vesicles been identified. There is also no precedent for the involvement of EB1 in axonal targeting of membrane proteins (Gu, 2006).

In this study, it is first shown that axonal targeting of YFP-Kvβ2 is independent of its ability to interact with Kv1 α subunits, and then a novel interaction was identified between Kvβ2 and EB1. Fluorescence resonance energy transfer (FRET) and live-cell imaging studies further revealed that Kvβ2 and EB1 closely associated with each other as they moved together anterogradely along the axon. Also KIF3/kinesin II was identified as the microtubule-based motor that associates with Kvβ2. Finally, reduction of endogenous EB1 or KIF3 blocked axonal targeting of Kv1 channels but not voltage-gated sodium (Nav) channels (Gu, 2006).

This study has shown that Kvβ is the Kv1 channel subunit primarily responsible for axonal targeting, and EB1, the +TIP that binds microtubule via its N-terminal domain and several other +TIPs via its C-terminal domain, as a Kvβ2 binding partner crucial for the axonal transport of Kv1 channels. Finally, this study shows that KIF3/kinesin II also associates with Kvβ2 and is required for Kv1 channel axonal targeting (Gu, 2006).

Axonal targeting of Kv1 channels likely entails physical interaction between Kv1 channel subunits and the axonal targeting machinery. When T1, the highly conserved Kv1 tetramerization domain, is fused with single span membrane proteins such as CD4 and transferrin receptor, its ability to promote axonal targeting is abolished by mutations that prevents its binding to Kvβ2. Together with the finding of Kvβ mutations disrupting Kv1 targeting to axons, these studies raise the possibility that Kvβ subunits are responsible for Kv1 channel axonal targeting (Gu, 2006).

Having found endogenous Kvβ2 in the axons of cultured hippocampal neurons within the first week in vitro, it was next shown that the K235E mutation of Kvβ2 at the T1-Kvβ2 interface eliminates T1 binding but does not affect axonal targeting. Whereas YFP-Kvβ2 expression enhances targeting of Kv1.2HA to the axonal membrane, expression of the YFP-K235E mutant incapable of assembling with Kv1.2 nearly eliminates the surface expression as well as axonal targeting of Kv1.2HA. Taken together, these findings suggest that Kvβ2 is responsible for targeting Kv1 channels to the axon (Gu, 2006).

To look for molecules involved in Kv1 channel axonal targeting, proteins in complex with Kvβ2 in the rat brain were identified. Following up the finding that YFP-Kvβ2 axonal targeting was hampered by paclitaxel, the microtubule stabilizing agent that also causes EB1 dissociation from microtubules, it was found in CoIP studies association of EB1 with Kv1.2 and Kvβ2 in the rat brain. Moreover, bacterially expressed His-EB1 pulled down Kvβ2 and bacterially expressed GST-Kvβ2 pulled down EB1. In addition, Kv1.2 partially colocalized with EB1 in axons of hippocampal neurons. These Kv1.2-containing puncta, likely post-Golgi carrier vesicles given their sensitivity to Brefeldin A, could be found at the MT ends near the cell membrane of neuronal soma, as expected from their partial colocalization with EB1. Given the ability of EB1 to track the plus ends of growing microtubules while they extend distally in the axons, its association with Kv1 channel subunits in central neurons raises the intriguing question concerning the role of EB1 in Kv1 channel axonal targeting (Gu, 2006).

Several lines of evidence were obtained implicating EB1 in axonal targeting of Kv1 channels: (1) there was excellent correlation between the ability of truncation mutants of Kvβ2 to associate with EB1 and their axonal targeting; (2) expression of a fluorescent fusion protein of EB1 C-terminal domain (YFP-EB1C) had dominant-negative effects over Kv1 channel axonal targeting; (3) suppressing EB1 level via siRNA impaired the axonal targeting of endogenous Kv1.1/Kvβ2, but not Nav channels and Tau1. Taken together, these findings strongly suggest that EB1 is crucial for Kv1 channel axonal targeting. In future studies, it would be of interest to find out whether other +TIPs are involved in axonal targeting of Kv1 or other ion channels (Gu, 2006).

How might axonal targeting of Kv1 channels be dependent on EB1? After assembly of Kv1.2 and Kvβ2 in the endoplasmic reticulum, both endogenous channel subunits colocalize in puncta much the same way as Kv1.2 localization to the EB1-positive puncta, presumably due to the association of EB1 with complexes of Kv1.2/Kvβ2 in post-Golgi carrier vesicles. rhis finding thus lends further support to the notion that the punctate distribution of endogenous EB1 could reflect EB1 association with vesicles or large protein complexes. Whereas two-color live-cell imaging showed anterograde comigration of Kvβ2 and EB1 along axons, at the rate of 0.11 ± 0.05 μm/s at room temperature, reasonably comparable to the rate of EB1-GFP movement, it is important to note that the involvement of EB1 in Kv1 channel axonal targeting does not preclude a likely role of MT-based motors in axonal transport of Kv1 channels or the involvement of other +TIPs. Indeed, interactions were found between Kvβ2 and KIF3/kinesin II and a requirement of KIF3 for axonal targeting of endogenous Kv1 channels but not Nav channels (Gu, 2006).

How could Kv1 channel axonal targeting be dependent on both EB1 and KIF3/kinesin II? The findings indicate that the KIF3/kinesin II motor coimmunoprecipitated with Kvβ2, suggesting that KIF3 either interacts directly with Kvβ2 or at least that the two proteins are associated in a complex and that KIF3 is required for targeting of Kv1.2. Given that a vesicle may contain multiple Kv1 channels each of which containing four Kvβ2 subunits, the same vesicle may be associated with both EB1 and KIF3, which moves at about twice the rate of EB1 tracking at the plus end of microtubules. Given the presence of bundles of microtubules with their growing plus ends pointing distally but distributed all along the axon, interaction between Kvβ2 and EB1 as well as KIF3 may enhance the ability of Kv1-containing vesicles to stay associated with microtubules -- with ends of microtubule presumably via EB1 and with microtubules via the KIF3 motor. The ability of Kvβ2 subunits of the Kv1 channel to associate with the dimeric EB1 and the trimeric KIF3, which contains two motor proteins KIF3A and KIF3B, may further enhance the interaction between these proteins and microtubules, analogous to the ability of the yeast EB1 and kinesin -- both dimeric proteins that associate with each other -- to mutually stabilize their interactions with microtubules, and the ability of EB1 and EB3 to mediate CLIP protein accumulation at the MT tips. In addition, as the KIF3-driven vesicle containing Kv1 channels reaches the MT plus end, its association with EB1, which promotes MT extension, may either allow the vesicle to hold onto the microtubule plus end before shifting to another microtubule in the bundle or (if the MT plus end is within reach of the axonal membrane), to facilitate vesicle delivery to the axonal membrane (Gu, 2006).

Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions

Gap junctions are intercellular channels that connect the cytoplasms of adjacent cells. For gap junctions to properly control organ formation and electrical synchronization in the heart and the brain, connexin-based hemichannels must be correctly targeted to cell-cell borders. While it is generally accepted that gap junctions form via lateral diffusion of hemichannels following microtubule-mediated delivery to the plasma membrane, evidence is provided for direct targeting of hemichannels to cell-cell junctions through the following: (1) a pathway that is dependent on microtubules, (2) the adherens-junction proteins N-cadherin and β-catenin, (3) the microtubule plus-end-tracking protein (+TIP) EB1, and (4) its interacting protein p150(Glued). Based on live cell microscopy that includes fluorescence recovery after photobleaching (FRAP), total internal reflection fluorescence (TIRF), deconvolution, and siRNA knockdown, it is proposed that preferential tethering of microtubule plus ends at the adherens junction promotes delivery of connexin hemichannels directly to the cell-cell border. These findings support an unanticipated mechanism for protein delivery to points of cell-cell contact (Shaw, 2007).

FRAP studies revealed that nocodazole and Taxol both cause disruption of the fast repopulation of Cx43 plaques. While nocodazole depolymerizes microtubules, Taxol stabilizes them but disrupts their interactions with EB1 (Nakata, 2003), which normally associates with rapidly growing microtubule plus ends. EB1-capped microtubules interact more frequently and for a longer period of time at plasma membrane-containing plaque than at regions of plasma membrane that do not contain plaque, EB1 coimmunoprecipitates with Cx43-YFP expressed in HeLa cells, and EB1 knockdown decreases plaque formation at the cell-cell border, thereby implicating EB1 as a major player in gap junction formation (Shaw, 2007).

How might the plus end-binding protein EB1 facilitate connexin delivery? The C terminus of EB1 binds the p150(Glued) subunit of the dynein/dynactin complex. Moreover, dynein/dynactin localizes with AJs at cell-cell contact points through direct binding with β-catenin. In this manner, dynein/dynactin may serve as an anchor for microtubules at the AJ. Knockdown of p150(Glued) or β-catenin disrupts the formation of gap junction plaques, and p150(Glued) knockdown impairs capture of EB1 at cortical regions of cell-cell contact. While it is possible that targeted connexin delivery involves other cytoskeletal elements, +TIPs, and cortical proteins, this study identifies some key components of functional importance: the microtubule plus end-binding protein EB1, the EB1-binding protein p150(Glued) as part of the dynein/dynactin complex that can tether microtubules to the AJ, and β-catenin as the cytoplasmic enforcer of homophilic cadherin-cadherin interaction. Most likely, actin also acts as an important initial sensor of cell-cell interaction and guides the localization of AJs with assistance from Rho-GTPases. It will be interesting to determine how other proteins associated with the cytoskeleton or cell-cell junctions may contribute to targeted delivery of gap junction proteins (Shaw, 2007).

EB1 is required for primary cilia assembly in fibroblasts

EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and different cellular organelles. EB1 also localizes to centrosomes and is required for centrosomal MT anchoring and organization of the MT network. EB1 localizes to the flagellar tip and proximal region of the basal body in Chlamydomonas, but the function of EB1 in the cilium/flagellum is unknown. EB1 was depleted from NIH3T3 fibroblasts by using siRNA, and it was found that EB1 depletion causes a approximately 50% reduction in the efficiency of primary cilia assembly in serum-starved cells. Expression of dominant-negative EB1 also inhibits cilia formation, and expression of mutant dominant-negative EB1 constructs suggests that binding of EB1 to p150(Glued) is important for cilia assembly. Finally, expression of a C-terminal fragment of the centrosomal protein CAP350, which removes EB1 from the centrosome but not MT plus ends, also inhibits ciliogenesis. It is concluded that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts (Schroder, 2007).

Ubiquitin-dependent proteolysis of the microtubule end-binding protein 1, EB1, is controlled by the COP9 signalosome: possible consequences for microtubule filament stability

The COP9 signalosome (CSN) is a regulatory particle of the ubiquitin (Ub) proteasome system (UPS) consisting of eight subunits (CSN1-CSN8). The CSN stabilizes the microtubule end-binding protein 1 (EB1) towards degradation by the UPS. EB1, the master regulator of microtubule plus ends, controls microtubule growth and dynamics. Therefore, regulation of EB1 stability by the CSN has consequences for microtubule function. EB1 binds the CSN via subunit CSN5. The C terminus of EB1 is sufficient for interaction with the CSN. Dimerization of EB1 is a prerequisite for complex association and subsequent CSN-mediated phosphorylation, as revealed by studies with the EB1I224A mutant, which is unable to dimerize. In cells, EB1 and CSN co-localize to the centrosome, as demonstrated by confocal fluorescence microscopy. EB1 is ubiquitinated and its proteolysis can be inhibited by MG132, demonstrating that it is a substrate of the UPS. Its degradation is accelerated by inhibition of CSN-associated kinases. HeLa cells permanently expressing siRNAs against CSN1 (siCSN1) or CSN3 (siCSN3) exhibit reduced levels of the CSN complex accompanied by lower steady-state concentrations of EB1. In siCSN1 cells, EB1 is less phosphorylated as compared with control cells, demonstrating that the protein is most likely protected towards the UPS by CSN-mediated phosphorylation. The CSN-dependent EB1 stabilization is not due to the CSN-associated deubiquitinating enzyme USP15. Treatment with nocodazole revealed a significantly increased sensitivity of siCSN1 and siCSN3 cells towards the microtubule depolymerizing drug accompanied by a collapse of microtubule filaments. A nocodazole-induced cell-cycle arrest is partially rescued by CSN1 or EB1. These data demonstrate that the CSN-dependent protection of EB1 is important for microtubule function (Peth, 2007).

Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner

The orientation of mitotic spindles is tightly regulated in polarized cells, but it has been unclear whether there is a mechanism regulating spindle orientation in nonpolarized cells. This study shows that integrin-dependent cell adhesion to the substrate orients the mitotic spindle of nonpolarized cultured cells parallel to the substrate plane. The spindle is properly oriented in cells plated on fibronectin or collagen, but misoriented in cells on poly-L-lysine or treated with the RGD peptide or anti-beta1-integrin antibody, indicating requirement of integrin-mediated cell adhesion for this mechanism. Remarkably, this mechanism is independent of gravitation or cell-cell adhesion, but requires actin cytoskeleton and astral microtubules. Furthermore, myosin X and the microtubule plus-end-tracking protein EB1 are shown to play a role in this mechanism through remodeling of actin cytoskeleton and stabilization of astral microtubules, respectively. These results thus uncover the existence of a mechanism that orients the spindle parallel to the cell-substrate adhesion plane, and identify crucial factors involved in this novel mechanism (Toyoshima, 2007).

EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B

The Aurora B kinase coordinates kinetochore-microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. This study found that EB1, a microtubule plus end-tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). These data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase (Banerjee, 2014).

Eb1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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