Drosophila EB1 is important for proper assembly,dynamics, and positioning of the mitotic spindle

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.     

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.     

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. In this paper, we showed in vitro that XMAP215^msps had a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerated growth and also increased catastrophe frequency. When all three factors were combined, the growth rate was synergistically enhanced, and rescue events were observed most frequently, but frequent catastrophes restrained the lengthening of the MTs. We propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215^msps polymerase and the EB1–Sentin duo.     

EB1 Recognizes the Nucleotide State of Tubulin in the Microtubule Lattice

Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1.     

Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis

Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility.     

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.     

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.     

Dissecting the Nanoscale Distributions and Functions of Microtubule-End-Binding Proteins EB1 and ch-TOG in Interphase HeLa Cells

Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other’s localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.     

Making microtubules and mitotic spindles in cells without functional centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.     

The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, we conducted an in vivo structure-function analysis of the Drosophila homologue Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNA interference rescue experiments revealed that the NH(2)-terminal four TOG domains of Msps function as paired units and were sufficient to promote microtubule dynamics and EB1 comet formation. We also identified TOG5 and novel inter-TOG linker motifs that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT binding that underlies the ability of Msps to promote MT dynamic instability.     

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.     

Regulation of Focal Adhesion Dynamics and Cell Motility by the EB2 and Hax1 Protein Complex

Cell migration is a fundamental cellular process requiring integrated activities of the cytoskeleton, membrane, and cell/extracellular matrix adhesions. Many cytoskeletal activities rely on microtubule filaments. It has been speculated that microtubules can serve as tracks to deliver proteins essential for focal adhesion turnover. Three microtubule end-binding proteins (EB1, EB2, and EB3) in mammalian cells can track the plus ends of growing microtubules. EB1 and EB3 together can regulate microtubule dynamics by promoting microtubule growth and suppressing catastrophe, whereas, in contrast, EB2 does not play a direct role in microtubule dynamic instability, and little is known about the cellular function of EB2. By quantitative proteomics, we identified mammalian HCLS1-associated protein X-1 (HAX1) as an EB2-specific interacting protein. Knockdown of HAX1 and EB2 in skin epidermal cells stabilizes focal adhesions and impairs epidermal migration in vitro and in vivo. Our results further demonstrate that cell motility and focal adhesion turnover require interaction between Hax1 and EB2. Together, our findings provide new insights for this critical cellular process, suggesting that EB2 association with Hax1 plays a significant role in focal adhesion turnover and epidermal migration.     

Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end

EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. Here, we have defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1's COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. We show that MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, we solved the crystal structure of the EB1 COOH-terminal domain. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity.     

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, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized 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. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.     

Microtubule plus-end dynamics in Xenopus egg extract spindles

Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus egg extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at approximately 11 microm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization ( approximately 30 microm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by alpha-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.     

"Search-and-capture" of microtubules through plus-end-binding proteins (+TIPs)

The generation of a polarized microtubule organization is critically important for proper cellular functions, such as cell division, differentiation and migration. Microtubules themselves are highly dynamic structures, and this dynamic property is temporally and spatially regulated within cells, especially at their plus ends. To explain how microtubules set up and make contacts with cellular structures, a "search-and-capture" mechanism has been proposed, in which the microtubule plus ends dynamically search for and capture specific sites, such as mitotic kinetochores and cell cortex. To date, several classes of proteins have been shown to be associated with microtubule plus ends in a wide range of organisms from fungi to humans and to play critical roles in the "search-and-capture" mechanism. In this review, we overview our current understanding of the "plus-end-binding proteins" (+TIPs), including APC (adenomatous polyposis coli) tumor suppressor protein, cytoplasmic linker proteins (CLIPs), CLIP-associating proteins (CLASPs), cytoplasmic dynein/dynactin, and EB1, an APC-interacting protein.     

Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1

Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtubules, where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1, and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we report crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. We propose that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end.     

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. In this study, we focus 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 reduced microtubule length; this was complemented by readdition of EB1. By time-lapse microscopy, EB1 increased 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 correlated with the abrupt onset of polymerization. Fluorescent EB1 localized 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 predicted 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.     

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. Here, we report 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. Our in vivo data provide evidence of a cell cycle–specific microtubule-binding protein that promotes microtubule dynamicity.     

EBs recognize a nucleotide-dependent structural cap at growing microtubule ends

Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions?of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.