EB 1 immunofluorescence reveals an increase in growing astral microtubule length and number during anaphase in NRK-52E cells

Spindle positioning in animal cells is thought to rely upon the interaction of astral microtubules with the cell cortex. Information on the dynamics of astral microtubules during this process is scarce, in part because of the difficulty in visualising these microtubules by light microscopy. EB1 is a protein which specifically localises to growing microtubule distal tips. Immunostaining for EB1 therefore represents a powerful method for visualising the distribution of growing microtubule tips within cells. In this study we used EB1 immunostaining in mitotic NRK-52E cells to quantitatively analyse the length and number of growing astral microtubules during metaphase and anaphase. We observed a dramatic increase in growing astral microtubule length and number during anaphase. Furthermore, drug treatments which specifically destroyed astral microtubules resulted in an increase in misaligned anaphase but not metaphase spindles. We suggest that an anaphase-specific increase in growing astral microtubule length and number facilitates the maintenance of a correctly aligned spindle in mitotic NRK-52E cells.     

CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.     

Tyrosine-dependent capture of CAP-Gly domain-containing proteins in complex mixture by EB1 C-terminal peptidic probes

Microtubule dynamics is regulated by an array of microtubule associated proteins of which the microtubule plus-end tracking proteins (+TIPs) are prominent examples. +TIPs form dynamic interaction networks at growing microtubule ends in an EB1-dependent manner. The interaction between the C-terminal domain of EB1 and the CAP-Gly domains of the +TIP CLIP-170 depends on the last tyrosine residue of EB1. In the present study, we generated peptidic probes corresponding to the C-terminal tail of EB1 to affinity-capture binding partners from cell lysates. Using an MS-based approach, we showed that the last 15 amino-acid residues of EB1, either free or immobilized on beads, bound recombinant CAP-Gly domains of CLIP-170. We further demonstrate that this binding was prevented when the C-terminal tyrosine of EB1 was absent in the peptidic probes. Western blotting in combination with a label-free quantitative proteomic analysis revealed that the peptidic probe harboring the C-terminal tyrosine of EB1 effectively pulled-down proteins with CAP-Gly domains from endothelial cell extracts. Additional proteins known to interact directly or indirectly with EB1 and the microtubule cytoskeleton were also identified. Our peptidic probes represent valuable tools to detect changes induced in EB1-dependent +TIP networks by external cues such as growth factors and small molecules.     

Proteomic-based identification of the APC-binding protein EB1 as a candidate of novel tissue biomarker and therapeutic target for colorectal cancer

Novel candidates of biomarker and therapeutic target in colorectal cancer (CRC) were investigated using a proteomic approach. The proteome of normal colorectal epithelial tissues was compared with that of the tumor ones in 59 CRC patients using two-dimensional difference gel electrophoresis. Of 3458 protein spots, 110 exhibited statistically significant (p<0.01) differences in intensity (more than 2.5-folds) between the normal and tumor tissue groups. Of 67 unique gene products that were identified for 105 of the 110 protein spots, we focused on the higher expression of the adenoma polyposis coli-binding protein EB1 (EB1). EB1 was originally discovered as a binding protein of APC, which is a tumor suppressor gene product, and the expression of EB1 has been associated with poor prognosis in several malignancies but not in CRC. Immunohistochemical analysis of the 132 CRC cases revealed that EB1 was overexpressed in tumor cells in correlation with poor prognosis. Suppression of EB1 by RNAi inhibited CRC cell proliferation and invasion. In this study, the overexpression of EB1 in CRC tissues correlating with prognosis, and its functional contribution to the malignant phenotypes of CRC cells are described. The present findings indicate that EB1 is a potential biomarker and therapeutic target in CRC.     

The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood-testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150(Glued), EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.     

The Microtubule-associated Protein EB1 Links AIM2 Inflammasomes with Autophagy-dependent Secretion

Inflammasomes are multi-protein complexes that regulate chronic inflammation-associated diseases by inducing interleukin-1 β (IL-1β) secretion. Numerous components involved in inflammasome activation have been identified, but the mechanisms of inflammasome-mediated IL-1β secretion have not yet been fully explored. Here, we demonstrate that end-binding protein 1 (EB1), which is required for activation of AIM2 inflammasome complex, links the AIM2 inflammasome to autophagy-dependent secretion. Imaging studies revealed that AIM2 inflammasomes colocalize with microtubule organizing centers and autophagosomes. Biochemical analyses showed that poly(dA-dT)-activated AIM2 inflammasomes induce autophagy and IL-1β secretion in an LC3-dependent fashion. Furthermore, depletion of EB1 decreases autophagic shedding and intracellular trafficking. Finally, we found that the 5′-AMP activated protein kinase may regulate this EB1-mediated autophagy-based inflammasome-induced secretion of IL-1β. These findings reveal a novel EB1-mediated pathway for the secretion of IL-1β.     

TAXOL REDUCES THE RATE OF CYTOKINESIS IN PtK1CELLS

Mitotic PtK₁cells were treated both during mid-anaphase and at furrow initiation with the potent microtubule (MT) stabilizing agent, taxol, to determine the role of MTs in the rate of cytokinetic events. Rates of cytokinesis (μm/min) were measured by changes in furrow diameter. Incubation of PtK₁cells during mid-anaphase with 5 μg/ml taxol slows the rate of cytokinesis by an average of 43%. Instead of furrow initiation to midbody formation taking an average of 10.7 min (1.6 μm/min), furrowing to midbody formation was completed in an average of 19.0 min (0.9 μm/min), which does not include the 7-min period between taxol application in mid-anaphase and furrow initiation. Application of 5 μg/ml taxol to cells at furrow initiation had a reduced effect on decreasing the rate of cytokinesis and midbody formation; furrowing to midbody formation took an average of 14.6 min (1.2 μm/min). These data suggest that delays in the rate of cytokinesis is dependent on the mitotic stage at which taxol is applied. Ultrastructural analysis shows that taxol treatment of anaphase cells prevents midbody formation during early G₁, yet MT number and organization in the furrowed region is not significantly altered from untreated cells. There is little change in the organization and amount of contractile ring microfilaments, yet filaments are also found parallel to midbody MTs. Our results may be explained by the fact that taxol tends to stabilize MTs which probably affects the rate at which they depolymerize in the terminal phases of cytokinesis. Reduction in depolymerization rates of a stable population of MTs could serve to regulate the rate of cytokinesis.     

Multisite phosphorylation disrupts arginine-glutamate salt bridge networks required for binding of cytoplasmic linker-associated protein 2 (CLASP2) to end-binding protein 1 (EB1)

A group of diverse proteins reversibly binds to growing microtubule plus ends through interactions with end-binding proteins (EBs). These +TIPs control microtubule dynamics and microtubule interactions with other intracellular structures. Here, we use cytoplasmic linker-associated protein 2 (CLASP2) binding to EB1 to determine how multisite phosphorylation regulates interactions with EB1. The central, intrinsically disordered region of vertebrate CLASP proteins contains two SXIP EB1 binding motifs that are required for EB1-mediated plus-end-tracking in vitro. In cells, both EB1 binding motifs can be functional, but most of the binding free energy results from nearby electrostatic interactions. By employing molecular dynamics simulations of the EB1 interaction with a minimal CLASP2 plus-end-tracking module, we find that conserved arginine residues in CLASP2 form extensive hydrogen-bond networks with glutamate residues predominantly in the unstructured, acidic C-terminal tail of EB1. Multisite phosphorylation of glycogen synthase kinase 3 (GSK3) sites near the EB1 binding motifs disrupts this electrostatic "molecular Velcro." Molecular dynamics simulations and (31)P NMR spectroscopy indicate that phosphorylated serines participate in intramolecular interactions with and sequester arginine residues required for EB1 binding. Multisite phosphorylation of these GSK3 motifs requires priming phosphorylation by interphase or mitotic cyclin-dependent kinases (CDKs), and we find that CDK- and GSK3-dependent phosphorylation completely disrupts CLASP2 microtubule plus-end-tracking in mitosis.     

Giardia lamblia EB1 is a functional homolog of yeast Bim1p that binds to microtubules

Giardia lamblia, with two nuclei and a distinct polarized morphology, is an interesting organism for investigating how distribution of its microtubule (MT) is controlled during its cell cycle. In this study, we identified the end-binding protein 1 (EB1) of G. lamblia, a well-known microtubule-associated protein that organizes MTs in eukaryotes. Immunofluorescence assays using recombinant EB1 (rEB1)-specific antibodies demonstrated EB1 localization in nuclear membrane as well as in some cytoskeletal structures such as axomenes and median bodies of trophozoites of G. lamblia. Complementation experiments using the BIM1 knock-out mutant of yeast, the yeast homolog of mammalian EB1, showed that giardial EB1 was able to carry out a homologous function in controlling MT dynamics. In addition, rEB1 of G. lamblia co-precipitated with MTs by an in vitro binding assay, thereby demonstrating that G. lamblia EB1 is a MT-associated protein. These results, taken together, suggest that G. lamblia EB1 is a functional homolog of eukaryotic EB1 and is likely to be a determinant for MT distribution.     

Controlled and stochastic retention concentrates dynein at microtubule ends to keep endosomes on track

Bidirectional transport of early endosomes (EEs) involves microtubules (MTs) and associated motors. In fungi, the dynein/dynactin motor complex concentrates in a comet-like accumulation at MT plus-ends to receive kinesin-3-delivered EEs for retrograde transport. Here, we analyse the loading of endosomes onto dynein by combining live imaging of photoactivated endosomes and fluorescent dynein with mathematical modelling. Using nuclear pores as an internal calibration standard, we show that the dynein comet consists of ～55 dynein motors. About half of the motors are slowly turned over (T(1/2): ～98 s) and they are kept at the plus-ends by an active retention mechanism involving an interaction between dynactin and EB1. The other half is more dynamic (T(1/2): ～10 s) and mathematical modelling suggests that they concentrate at MT ends because of stochastic motor behaviour. When the active retention is impaired by inhibitory peptides, dynein numbers in the comet are reduced to half and ～10% of the EEs fall off the MT plus-ends. Thus, a combination of stochastic accumulation and active retention forms the dynein comet to ensure capturing of arriving organelles by retrograde motors.     

EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation

Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.     

Structural and functional differences between porcine brain and budding yeast microtubules

The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.     

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.     

MAP1B‐LC1 prevents autophagosome formation by linking syntaxin 17 to microtubules

In fed cells, syntaxin 17 (Stx17) is associated with microtubules at the endoplasmic reticulum–mitochondria interface and promotes mitochondrial fission by determining the localization and function of the mitochondrial fission factor Drp1. Upon starvation, Stx17 dissociates from microtubules and Drp1, and binds to Atg14L, a subunit of the phosphatidylinositol 3‐kinase complex, to facilitate phosphatidylinositol 3‐phosphate production and thereby autophagosome formation, but the mechanism underlying this phenomenon remains unknown. Here we identify MAP1B‐LC1 (microtubule‐associated protein 1B‐light chain 1) as a critical regulator of Stx17 function. Depletion of MAP1B‐LC1 causes Stx17‐dependent autophagosome accumulation even under nutrient‐rich conditions, whereas its overexpression blocks starvation‐induced autophagosome formation. MAP1B‐LC1 links microtubules and Stx17 in fed cells, and starvation causes the dephosphorylation of MAP1B‐LC1 at Thr217, allowing Stx17 to dissociate from MAP1B‐LC1 and bind to Atg14L. Our results reveal the mechanism by which Stx17 changes its binding partners in response to nutrient status.     

Analysis of centrosome localization of BRCA1 and its activity in suppressing centrosomal aster formation

BRCA1, a product of a familial breast and ovarian cancer susceptibility gene, localizes to centrosomes and physically interacts with γ-tubulin, a key centrosomal protein for microtubule nucleation and anchoring at centrosomes. Here, we performed a rigorous analysis of centrosome localization of BRCA1, and found that BRCA1 is specifically associated with mother centrioles in unduplicated centrosomes, and daughter centrioles acquire BRCA1 prior to initiation of duplication, and thus duplicated centrosomes are both bound by BRCA1. We further found that BRCA1 suppresses centrosomal aster formation. In addition, we identified a new domain of BRCA1 critical for γ-tubulin binding, which confers not only its localization to centrosomes, but also its activity to suppress centrosomal aster formation.     

EB病毒包膜糖蛋白gp85真核表达载体的构建

用基因工程技术克隆EB病毒中抗原性较强的膜蛋白gp85的编码基因BXLF2,构建真核表达载体。以EB病毒B95—8细胞培养上清为模板,PCR扩增出BXLF2基因。PCR产物经SnaBⅠ和NotⅠ双酶切后克隆至毕赤酵母表达载体pPIC9K,用双酶切和DNA测序鉴定重组质粒。重组质粒双酶切的片段大小与预期符合,重组克隆外源基因的测序结果与献报道一致。结果表明,EB病毒gp85的编码基因BXLF2被成功地克隆入真核表达载体pPIC9K,为下一步在毕赤酵母中表达EB病毒gp85蛋白建立了基础。     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.     

Mutant DLX 3 disrupts odontoblast polarization and dentin formation

Tricho-dento-osseous (TDO) syndrome is an autosomal dominant disorder characterized by abnormalities in the thickness and density of bones and teeth. A 4-bp deletion mutation in the Distal-Less 3 (DLX3) gene is etiologic for most cases of TDO. To investigate the in vivo role of mutant DLX3 (MT-DLX3) on dentin development, we generated transgenic (TG) mice expressing MT-DLX3 driven by a mouse 2.3 Col1A1 promoter. Dentin defects were radiographically evident in all teeth and the size of the nonmineralized pulp was enlarged in TG mice, consistent with clinical characteristics in patients with TDO. High-resolution radiography, microcomputed tomography, and SEM revealed a reduced zone of mineralized dentin with anomalies in the number and organization of dentinal tubules in MT-DLX3 TG mice. Histological and immunohistochemical studies demonstrated that the decreased dentin was accompanied by altered odontoblast cytology that included disruption of odontoblast polarization and reduced numbers of odontoblasts. TUNEL assays indicated enhanced odontoblast apoptosis. Expression levels of the apoptotic marker caspase-3 were increased in odontoblasts in TG mice as well as in odontoblastic-like MDPC-23 cells transfected with MT-DLX3 cDNA. Expression of Runx2, Wnt 10A, and TBC1D19 colocalized with DLX3 expression in odontoblasts, and MT-DLX3 significantly reduced expression of all three genes. TBC1D19 functions in cell polarity and decreased TBC1D19 expression may contribute to the observed disruption of odontoblast polarity and apoptosis. These data indicate that MT-DLX3 acts to disrupt odontoblast cytodifferentiation leading to odontoblast apoptosis, and aberrations of dentin tubule formation and dentin matrix production, resulting in decreased dentin and taurodontism.In summary, this TG model demonstrates that MT-DLX3 has differential effects on matrix production and mineralization in dentin and bone and provides a novel tool for the investigation of odontoblast biology.