End-binding Protein Research Articles

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.

Regulation of EB1/3 proteins by classical MAPs in neurons

Microtubules (MTs) are key cytoskeletal elements in developing and mature neurons. MT reorganization underlies the morphological changes that occur during neuronal development. Furthermore, MTs contribute to the maintenance of neuronal architecture, enable intracellular transport and act as scaffolds for signaling molecules. Thus, a fine-tuned regulation of MT dynamics and stability is crucial for the correct differentiation and functioning of neurons. Different types of proteins contribute to the regulation of the MT state, such as plus-end tracking proteins (+TIPs), which interact with the plus-ends of growing microtubules, and classical microtubule-associated proteins (MAPs), which bind along the microtubule lattice. Recent evidence indicates that MAPs interplay with End Binding Proteins (EBs), the core +TIPs, in neuronal cells. This might contribute to the orchestrated regulation of MT dynamics in neurons. In this mini-review article, we address recent research on the neuronal crosstalk between EBs and classical MAPs and speculate on its possible functional relevance.

Tracking the biogenesis and inheritance of subpellicular microtubule in Trypanosoma brucei with inducible YFP-α-tubulin.

The microtubule cytoskeleton forms the most prominent structural system in Trypanosoma brucei, undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance during T. brucei cell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP-α-tubulin expression. During new flagellum/flagellum attachment zone (FAZ) biogenesis and cell growth, YFP-α-tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division.

GAS2-like proteins mediate communication between microtubules and actin through interaction with end-binding proteins

ABSTRACTCrosstalk between the microtubule (MT) and actin cytoskeletons is fundamental to many cellular processes including cell polarisation and cell motility. Previous work has shown that members of the growth-arrest-specific 2 (GAS2) family mediate the crosstalk between filamentous actin (F-actin) and MTs, but the molecular basis of this process remained unclear. By using fluorescence microscopy, we demonstrate that three members of this family, GAS2-like 1, GAS2-like 2 and GAS2-like 3 (G2L1, G2L2 and G2L3, also known as GAS2L1, GAS2L2 and GAS2L3, respectively) are differentially involved in mediating the crosstalk between F-actin and MTs. Although all localise to actin and MTs, only the exogenous expression of G2L1 and G2L2 influenced MT stability, dynamics and guidance along actin stress fibres. Biochemical analysis and live-cell imaging revealed that their functions are largely due to the association of these proteins with MT plus-end-binding proteins that bind to SxIP or SxLP motifs located at G2L C-termini. Our findings lead to a model in which end-binding (EB) proteins play a key role in mediating actin–MT crosstalk.

Adhesive interactions of N-cadherin limit the recruitment of microtubules to cell–cell contacts through organization of actomyosin

Adhesive interactions of cadherins induce crosstalk between adhesion complexes and the actin cytoskeleton, allowing strengthening of adhesions and cytoskeletal organization. The underlying mechanisms are not completely understood, and microtubules (MTs) might be involved, as for integrin-mediated cell-extracellular-matrix adhesions. Therefore, we investigated the relationship between N-cadherin and MTs by analyzing the influence of N-cadherin engagement on MT distribution and dynamics. MTs progressed less, with a lower elongation rate, towards cadherin adhesions than towards focal adhesions. Increased actin treadmilling and the presence of an actomyosin contractile belt, suggested that actin relays inhibitory signals from cadherin adhesions to MTs. The reduced rate of MT elongation, associated with reduced recruitment of end-binding (EB) proteins to plus ends, was alleviated by expression of truncated N-cadherin, but was only moderately affected when actomyosin was disrupted. By contrast, destabilizing actomyosin fibers allowed MTs to enter the adhesion area, suggesting that tangential actin bundles impede MT growth independently of MT dynamics. Blocking MT penetration into the adhesion area strengthened cadherin adhesions. Taken together, these results establish a crosstalk between N-cadherin, F-actin and MTs. The opposing effects of cadherin and integrin engagement on actin organization and MT distribution might induce bias of the MT network during cell polarization.

CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions

Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. In this paper, we report that the MT plus end-binding protein CLASP2 localizes to adherens junctions (AJs) via direct interaction with p120-catenin (p120) in primary basal mouse keratinocytes. Reductions in the levels of p120 or CLASP2 decreased the localization of the other protein to cell-cell contacts and altered AJ dynamics and stability. These features were accompanied by decreased MT density and altered MT dynamics at intercellular junction sites. Interestingly, CLASP2 was enriched at the cortex of basal progenitor keratinocytes, in close localization to p120. Our findings suggest the existence of a new mechanism of MT targeting to AJs with potential functional implications in the maintenance of proper cell-cell adhesion in epidermal stem cells.

A Morphometric Screen Identifies Specific Roles for Microtubule-Regulating Genes in Neuronal Development of P19 Stem Cells

The first morphological change after neuronal differentiation is the microtubule-dependent initiation of thin cell protrusions called neurites. Here we performed a siRNA-based morphometric screen in P19 stem cells to evaluate the role of 408 microtubule-regulating genes during this early neuromorphogenesis step. This screen uncovered several novel regulatory factors, including specific complex subunits of the microtubule motor dynein involved in neurite initiation and a novel role for the microtubule end-binding protein EB2 in attenuation of neurite outgrowth. Epistasis analysis suggests that competition between EB1 and EB2 regulates neurite length, which links its expression to neurite outgrowth. We propose a model that explains how microtubule regulators can mediate cellular morphogenesis during the early steps of neuronal development by controlling microtubule stabilization and organizing dynein-generated forces.

HIV-1 Induces the Formation of Stable Microtubules to Enhance Early Infection

Stable microtubule (MT) subsets form distinct networks from dynamic MTs and acquire distinguishing posttranslational modifications, notably detyrosination and acetylation. Acting as specialized tracks for vesicle and macromolecular transport, their formation is regulated by the end-binding protein EB1, which recruits proteins that stabilize MTs. We show that HIV-1 induces the formation of acetylated and detyrosinated stable MTs early in infection. Although the MT depolymerizing agent nocodazole affected dynamic MTs, HIV-1 particles localized to nocodazole-resistant stable MTs, and infection was minimally affected. EB1 depletion or expression of an EB1 carboxy-terminal fragment that acts as a dominant-negative inhibitor of MT stabilization prevented HIV-1-induced stable MT formation and suppressed early viral infection. Furthermore, we show that the HIV-1 matrix protein targets the EB1-binding protein Kif4 to induce MT stabilization. Our findings illustrate how specialized MT-binding proteins mediate MT stabilization by HIV-1 and the importance of stable MT subsets in viral infection.

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells.DOI: http://dx.doi.org/10.7554/eLife.01222.001

Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, <em>Schizosaccharomyces pombe</em> Pot1 and <em>Saccharomyces cerevisiae </em>Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain

Cdc13, the telomere end-binding protein from Saccharomyces cerevisiae, is a multidomain protein that specifically binds telomeric single-stranded DNA (ssDNA) with exquisitely high affinity to coordinate telomere maintenance. Recent structural and genetic data have led to the proposal that Cdc13 is the paralog of RPA70 within a telomere-specific RPA complex. Our understanding of Cdc13 structure and biochemistry has been largely restricted to studies of individual domains, precluding analysis of how each domain influences the activity of the others. To better facilitate a comparison to RPA70, we evaluated the ssDNA binding of full-length S. cerevisiae Cdc13 to its minimal substrate, Tel11. We found that, unlike RPA70 and the other known telomere end-binding proteins, the core Cdc13 ssDNA-binding activity is wholly contained within a single tight-binding oligosaccharide/oligonucleotide/oligopeptide binding (OB)-fold. Because two OB-folds are implicated in dimerization, we also evaluated the relationship between dimerization and ssDNA-binding activity and found that the two activities are independent. We also find that Cdc13 binding exhibits positive cooperativity that is independent of dimerization. This study reveals that, while Cdc13 and RPA70 share similar domain topologies, the corresponding domains have evolved different and specialized functions.

End Binding Proteins Are Obligatory Dimers

End binding (EB) proteins are responsible for the recruitment of an array of microtubule plus-end tracking proteins (+TIPs) to growing microtubules ends. EBs encompass an N-terminal calponin homology domain that confers microtubule tip tracking activity to the protein. The C-terminal domain of EBs contains a coiled coil that mediates the parallel dimerization of EB monomers. This part of the protein is also responsible for partner binding. While dimerization is not essential for microtubule tip tracking by EBs it is a prerequisite for +TIP partner binding. The concentration of EBs in cells has been estimated to be in the range of hundreds of nanomoles. In contrast, in in vitro single molecule experiments EB concentrations of subnanomoles are employed. From a mechanistic point of view it is important to assess the oligomerization state of EBs at physiologically and experimentally relevant protein concentrations, in particular if the goal of a study is to model the behavior of EB-dependent dynamic +TIP networks. Here we have determined the stability of the EB1 and EB3 dimers using multi-angle light scattering and fluorescence analytical ultracentrifugation. We show that these EBs form stable dimers and do not dissociate even at very low nanomolar concentrations. The dimers remained stable at both room temperature as well as at the physiologically relevant temperature of 37°C. Together, our results reveal that EBs are obligatory dimers, a conclusion that has implications for the mechanistic understanding of these key proteins involved in the orchestration of dynamic protein networks at growing microtubule ends.

Many disease-associated variants of hTERT retain high telomerase enzymatic activity

Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays.

Depletion of end-binding protein 1 (EB1) promotes apoptosis of human non-small-cell lung cancer cells via reactive oxygen species and Bax-mediated mitochondrial dysfunction

Although end-binding protein 1 (EB1) is well known to regulate microtubule dynamics, the role of EB1 in apoptosis of non-small cell lung cancer (NSCLC) is poorly understood. Here, we investigated the molecular mechanism by which EB1 regulates apoptosis in H460, A549, and H1299 cells. Depletion of EB1 in A549 and H1299 cells, which express high levels of EB1, induced cell death in a p53-independent manner through over-production of reactive oxygen species (ROS) and Bax induction. This phenomenon was potentiated in radiation-treated EB1-knockdown cells and was largely blocked by N-acetyl-L-cysteine, a scavenger of ROS. ROS accelerated the activation of nuclear factor-kappa B (NF-κB) to promote transcriptional activity of Bax, an action that was accompanied by cytochrome c translocation and apoptosis-inducing factor (AIF) release. The NF-κB inhibitor, BAY 11-7082, potently inhibited the apoptosis induced by EB1 knockdown and radiation treatment, in association with diminished activity of the mitochondrial death pathway. Conversely, ectopic overexpression of EB1 in H460 cells, which express low levels of EB1, remarkably abrogated radiation-induced apoptosis and NF-κB-mediated mitochondrial dysfunction. Our data provide the first demonstration that down-regulation of EB1 promotes NSCLC cell death by inducing ROS-mediated, NF-κB-dependent Bax signaling cascades, a process in which cytochrome c and AIF play important roles, indicating a potential therapeutic benefit of EB1 in lung cancer.

Microtubule-Associated Histone Deacetylase 6 Supports the Calcium Store Sensor STIM1 in Mediating Malignant Cell Behaviors

Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) storage sensor that promotes cell growth, migration, and angiogenesis in breast and cervical cancers. Here, we report that the microtubule-associated histone deacetylase 6 (HDAC6) differentially regulates activation of STIM1-mediated store-operated Ca(2+) entry (SOCE) between cervical cancer cells and normal cervical epithelial cells. Confocal microscopy of living cells indicated that microtubule integrity was necessary for STIM1 trafficking to the plasma membrane and interaction with Orai1, an essential pore subunit of SOCE. Cancer cells overexpressed both STIM1 and Orai1 compared with normal cervical epithelial cells. HDAC6 upregulation in cancer cells was accompanied by hypoacetylated α-tubulin. Tubastatin-A, a specific HDAC6 inhibitor, inhibited STIM1 translocation to plasma membrane and blocked SOCE activation in cancer cells but not normal epithelial cells. Genetic or pharmacologic inhibition of HDAC6 blocked STIM1 membrane trafficking and downstream Ca(2+) influx, as evidenced by total internal reflection fluorescent images and intracellular Ca(2+) determination. In contrast, HDAC6 inhibition did not affect interactions between STIM1 and the microtubule plus end-binding protein EB1. Analysis of surgical specimens confirmed that most cervical cancer tissues overexpressed STIM1 and Orai1, accompanied by hypoacetylated α-tubulin. Together, our results identify HDAC6 as a candidate target to disrupt STIM1-mediated SOCE as a general strategy to block malignant cell behavior.

R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I-SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.

Coordinated Interactions of Multiple POT1-TPP1 Proteins with Telomere DNA

Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5'-TTAGGG-3' in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.

Aurora B spatially regulates EB3 phosphorylation to coordinate daughter cell adhesion with cytokinesis

During mitosis, human cells round up, decreasing their adhesion to extracellular substrates. This must be quickly reestablished by poorly understood cytoskeleton remodeling mechanisms that prevent detachment from epithelia, while ensuring the successful completion of cytokinesis. Here we show that the microtubule end-binding (EB) proteins EB1 and EB3 play temporally distinct roles throughout cell division. Whereas EB1 was involved in spindle orientation before anaphase, EB3 was required for stabilization of focal adhesions and coordinated daughter cell spreading during mitotic exit. Additionally, EB3 promoted midbody microtubule stability and, consequently, midbody stabilization necessary for efficient cytokinesis. Importantly, daughter cell adhesion and cytokinesis completion were spatially regulated by distinct states of EB3 phosphorylation on serine 176 by Aurora B. This EB3 phosphorylation was enriched at the midbody and shown to control cortical microtubule growth. These findings uncover differential roles of EB proteins and explain the importance of an Aurora B phosphorylation gradient for the spatiotemporal regulation of microtubule function during mitotic exit and cytokinesis.

End-binding proteins sensitize microtubules to the action of microtubule-targeting agents

Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process.

Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.