Peptide aptamers define distinct EB1- and EB3-binding motifs and interfere with microtubule dynamics

EB1 is a conserved protein that plays a central role in regulating microtubule dynamics and organization. It binds directly to microtubule plus ends and recruits other plus end–localizing proteins. Most EB1-binding proteins contain a Ser–any residue–Ile-Pro (SxIP) motif. Here we describe the isolation of peptide aptamers with optimized versions of this motif by screening for interaction with the Drosophila EB1 protein. The use of small peptide aptamers to competitively inhibit protein interaction and function is becoming increasingly recognized as a powerful technique. We show that SxIP aptamers can bind microtubule plus ends in cells and functionally act to displace interacting proteins by competitive binding. Their expression in developing flies can interfere with microtubules, altering their dynamics. We also identify aptamers binding to human EB1 and EB3, which have sequence requirements similar to but distinct from each other and from Drosophila EB1. This suggests that EB1 paralogues within one species may interact with overlapping but distinct sets of proteins in cells.     

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.     

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.     

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.     

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.     

EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells

Highly conserved EB1 family proteins bind to the growing ends of microtubules, recruit multiple cargo proteins, and are critical for making dynamic microtubules in vivo. However, it is unclear how these master regulators of microtubule plus ends promote microtubule dynamics. In this paper, we identify a novel EB1 cargo protein, Sentin. Sentin depletion in Drosophila melanogaster S2 cells, similar to EB1 depletion, resulted in an increase in microtubule pausing and led to the formation of shorter spindles, without displacing EB1 from growing microtubules. We demonstrate that Sentin's association with EB1 was critical for its plus end localization and function. Furthermore, the EB1 phenotype was rescued by expressing an EBN-Sentin fusion protein in which the C-terminal cargo-binding region of EB1 is replaced with Sentin. Knockdown of Sentin attenuated plus end accumulation of Msps (mini spindles), the orthologue of XMAP215 microtubule polymerase. These results indicate that EB1 promotes dynamic microtubule behavior by recruiting the cargo protein Sentin and possibly also a microtubule polymerase to the microtubule tip.     

The Microtubule Plus-End Tracking Proteins SPR1 and EB1b Interact to Maintain Polar Cell Elongation and Directional Organ Growth in Arabidopsis

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.     

Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking

Visualization of specific molecules and their interactions in real time and space is essential to delineate how cellular dynamics and the signaling circuit are orchestrated. Spatial regulation of conformational dynamics and structural plasticity of protein interactions is required to rewire signaling circuitry in response to extracellular cues. We introduce a method for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells, using photoactivatable complementary fluorescent (PACF) proteins. Subsets of complementary fluorescent protein molecules were activated, localized, and then bleached; this was followed by the assembly of superresolution images from aggregate position of sum interactive molecules. Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells. We further delineated the structure–function relationship of EB1 by generating EB1-PACF dimers (EB1^wt:EB1^wt, EB1^wt:EB1^mt, and EB1^mt:EB1^mt) and imaging their precise localizations in culture cells. Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells. Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.     

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.     

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Formins regulate phragmoplast expansion, microtubule turnover rate, actin nucleation, and cell plate membrane remodeling during cytokinesis.     

Positioning and capture of cell surface-associated microtubules in epithelial tendon cells that differentiate in primary embryonic Drosophila cell cultures

Using primary embryonic Drosophila cell cultures, we have investigated the assembly of transcellular microtubule bundles in epidermal tendon cells. Muscles attach to the tendon cells of previously undescribed epidermal balls that form shortly after culture initiation. Basal capture of microtubule ends in cultured tendon cells is confined to discrete sites that occupy a relatively small proportion of the basal cell surface. These capturing sites are associated with hemiadherens junctions that link the ends of muscle cells to tendon cell bases. In vivo, muscle attachment and microtubule capture occur across the entire cell base. The cultured tendon cells reveal that the basal ends of their microtubules can be precisely targeted to small, pre-existing, structurally well-defined cortical capturing sites. However, a search and capture targeting procedure, such as that undertaken by kinetochore microtubules, cannot fully account for the precision of microtubule capture and positioning in tendon cells. We propose that cross-linkage of microtubules is also required to zip them into apicobasally oriented alignment, progressing from captured basal plus ends to apical minus ends. This involves repositioning of apical minus ends before they become anchored to an apical set of hemiadherens junctions. The proposal is consistent with our finding that hemiadherens junctions assemble at tendon cell bases before they do so at cell apices in both cultures and embryos. It is argued that control of microtubule positioning in the challenging spatial situations found in vitro involves the same procedures as those that operate in vivo.     

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). We show that 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 was partially rescued by CSN1 or EB1. These data demonstrate that the CSN-dependent protection of EB1 is important for microtubule function.     

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.     

Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint

Kinetochore attachment to the ends of dynamic microtubules is a conserved feature of mitotic spindle organization that is thought to be critical for proper chromosome segregation. Although kinetochores have been described to transition from lateral to end-on attachments, the phase of lateral attachment has been difficult to study in yeast due to its transient nature. We have previously described a kinetochore mutant, DAM1-765, which exhibits lateral attachments and misregulation of microtubule length. Here we show that the misregulation of microtubule length in DAM1-765 cells occurs despite localization of microtubule associated proteins Bik1, Stu2, Cin8 and Kip3 to microtubules. DAM1-765 kinetochores recruit the spindle checkpoint protein Bub1, however Bub1 localization to DAM1-765 kinetochores is not sufficient to cause a cell cycle arrest. Interestingly, the DAM1-765 mutation rescues the temperature sensitivity of a biorientationdeficient ipl1-321 mutant, and DAM1-765 chromosome loss rates are similar to wild-type cells. the spindle checkpoint in DAM1-765 cells responds properly to unattached kinetochores created by nocodazole treatment and loss of tension caused by a cohesin mutant. progression of DAM1-765 cells through mitosis therefore suggests that satisfaction of the checkpoint depends more highly on biorientation of sister kinetochores than on achievement of a specific interaction between kinetochores and microtubule plus ends.Key words: spindle assembly checkpoint, kinetochore-microtubule attachments, biorientation, DAM1-765     

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.     

Vestigial Is Required during Late-Stage Muscle Differentiation in Drosophila melanogaster Embryos

The somatic muscles of Drosophila develop in a complex pattern that is repeated in each embryonic hemi-segment. During early development, progenitor cells fuse to form a syncytial muscle, which further differentiates via expression of muscle-specific factors that induce specific responses to external signals to regulate late-stage processes such as migration and attachment. Initial communication between somatic muscles and the epidermal tendon cells is critical for both of these processes. However, later establishment of attachments between longitudinal muscles at the segmental borders is largely independent of the muscle–epidermal attachment signals, and relatively little is known about how this event is regulated. Using a combination of null mutations and a truncated version of Sd that binds Vg but not DNA, we show that Vestigial (Vg) is required in ventral longitudinal muscles to induce formation of stable intermuscular attachments. In several muscles, this activity may be independent of Sd. Furthermore, the cell-specific differentiation events induced by Vg in two cells fated to form attachments are coordinated by Drosophila epidermal growth factor signaling. Thus, Vg is a key factor to induce specific changes in ventral longitudinal muscles 1–4 identity and is required for these cells to be competent to form stable intermuscular attachments with each other.     

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.     

Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.     

C. elegans ankyrin repeat protein VAB-19 is a component of epidermal attachment structures and is essential for epidermal morphogenesis

Elongation of the epidermis of the nematode Caenorhabditis elegans involves both actomyosin-mediated changes in lateral epidermal cell shape and body muscle attachment to dorsal and ventral epidermal cells via intermediate-filament/hemidesmosome structures. vab-19 mutants are defective in epidermal elongation and muscle attachment to the epidermis. VAB-19 is a member of a conserved family of ankyrin repeat-containing proteins that includes the human tumor suppressor Kank. In epidermal cells, VAB-19::GFP localizes with components of epidermal attachment structures. In vab-19 mutants, epidermal attachment structures form normally but do not remain localized to muscle-adjacent regions of the epidermis. VAB-19 localization requires function of the transmembrane attachment structure component Myotactin. vab-19 mutants also display aberrant actin organization in the epidermis. Loss of function in the spectrin SMA-1 partly bypasses the requirement for VAB-19 in elongation, suggesting that VAB-19 and SMA-1/spectrin might play antagonistic roles in regulation of the actin cytoskeleton.