End-binding Protein Research Articles

3.: Microtubule dysfunction in whispering dysphonia (DYT4 dystonia)

Whispering dysphonia (DYT4 dystonia) is a dominantly inherited disorder previously described in an Australian family. The clinical phenotype is of spasmodic dysphonia associated with additional focal or generalised dystonia and a characteristic facies and body habitus. Recently, the cause of whispering dysphonia was found to be a mutation in the TUBB4 gene, which encodes for a neuronally expressed tubulin. We aimed to determine whether microtubular function was impaired as a consequence of this TUBB4 mutation. We studied olfactory neurosphere cell lines derived from three affected individuals and three controls. Tubulin end-binding protein (EB3) was fluorescently tagged, allowing us to assess microtubule dynamics using time-lapse imaging in live cells. We found that there was a significant reduction in the density of microtubules and in the speed of microtubule formation. The results suggest that the TUBB4 mutation leads to a reduction in microtubule density and a decrease in the speed of microtubule formation, supporting the notion that microtubule dysfunction can play a role in the pathophysiology of dystonia.

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.

Doublecortin Recognizes the Longitudinal Curvature of the Microtubule End and Lattice

Microtubule ends have distinct biochemical and structural features from those of the lattice. Several proteins that control microtubule behavior can distinguish the end of a microtubule from the lattice. The end-binding protein EB1, for example, recognizes the nucleotide state of microtubule ends, which are enriched in GTP-tubulin. EB1 shares its binding site with Doublecortin (DCX), a protein expressed in developing neurons. We showed recently that DCX binds with highest affinity to microtubule ends. Here we show that DCX recognizes microtubule ends by a novel mechanism based on lattice curvature. Using single-molecule microscopy, we show that DCX "comets" do not elongate at faster microtubule growth rates and DCX does not recognize two out of three GTP analogs. We demonstrate that DCX binds with higher affinity to curved microtubule lattices than to straight ones. We find that curvature recognition is a property of single DCX molecules. Straightening of protofilaments (pfs) at microtubule ends with paclitaxel significantly attenuates end-recognition by DCX, but not EB1. Mutations in DCX found in patients with double cortex syndrome disrupted curvature recognition. We propose a model in which DCX recognizes microtubule ends through specific interactions with their structure. We conclude that microtubule ends have two distinct features that proteins can recognize independently, namely a structural feature related to curvature and nucleotide state.

Abstract 5464: Distinct effects of eribulin on axonal microtubule composition and modifications relative to paclitaxel, vincristine and ixabepilone in preclinical models: correlations with lower levels of eribulin-induced peripheral neuropathy

Abstract Microtubule targeting agents (MTAs) are widely used anti-cancer drugs. They suppress microtubule dynamics, prevent complete spindle formation and induce cell cycle arrest and cell death in rapidly dividing tumor cells. Unfortunately, systemic administration of different MTAs can also lead to significant side effects including debilitating, dose-limiting peripheral neuropathy. Interestingly, different MTAs vary with respect to the frequency and severity of neuropathic symptoms. MTAs are hypothesized to induce neuropathy by disrupting the normal functions of microtubules, including maintenance of elongated axonal structures and mediating axonal transport of cellular components between the cell body and nerve endings. Consistent with this notion, different MTAs vary in their mechanisms of microtubule binding and regulation of microtubule dynamics. To begin testing the hypothesis that differing molecular mechanisms underlie the variable nature of peripheral neuropathy, we examined biochemical changes in microtubule composition, modification and the levels of selected microtubule associated proteins, all of which affect microtubule dynamics and microtubule action. We compared sciatic nerves from mice treated with a Q2Dx3 schedule for 2 weeks at the maximum tolerated doses of paclitaxel, ixabepilone, vincristine or eribulin. Sciatic nerves were fixed, sectioned and immunostained with antibodies directed against α-tubulin, acetylated-tubulin and tyrosinated tubulin as well as the microtubule (+) end binding proteins EB1 and EB3. Images, acquired by confocal microscopy, were analyzed with Imaris software to quantitate drug-induced changes. The most marked effects were observed with eribulin treatment. Sciatic nerves from eribulin treated mice exhibited ∼2.5 fold increase in α-tubulin signal, ∼12 fold increase in acetylated tubulin signal, ∼1.5 fold increase in tyrosinated tubulin signal and ∼2 fold increase in EB1 signal. No significant change was observed for EB3. In contrast, with the exception of a ∼5 fold increase in the acetylated tubulin signal in paclitaxel treated mice, no other statistically significant effects were observed with paclitaxel, ixabepilone or vincristine treatment. The data suggest that the mechanisms of eribulin action alter normal axonal cell biology, leading to increased abundance of tubulin and EB1. Increased levels of acetylated tubulin observed with paclitaxel and eribulin treatment suggests increased microtubule stabilization. Taken together, the data suggest that eribulin affects neuronal microtubules differently than paclitaxel, ixabepilone or vincristine, correlating with its relatively low level of induced peripheral neuropathy. We gratefully thank Eisai Inc. for support of this research. Citation Format: Sarah J. Benbow, Brett M. Cook, Krystyna M. Wozniak, Barbara S. Slusher, Bruce A. Littlefield, Leslie Wilson, Stuart C. Feinstein, Mary A. Jordan. Distinct effects of eribulin on axonal microtubule composition and modifications relative to paclitaxel, vincristine and ixabepilone in preclinical models: correlations with lower levels of eribulin-induced peripheral neuropathy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5464. doi:10.1158/1538-7445.AM2014-5464

Inhibition of microtubule dynamics affects podosome belt formation during osteoclast induction.

Osteoclast is the only cell that can degrade bone tissue in vivo. Recent studies have shown the important role of cytoskeleton dynamics in osteolysis and the formation of podosome belt in osteoclasts. This process is regulated by the dynamic microtubule (MT) network. We treated osteoclast precursor cells Raw264.7 with low concentration of nocodazole (10nM) and antineoplastic drug taxol (10nM) to block MT turnover, and used end binding protein 1 fused to GFP to track the movement of microtubules in induced osteoclasts. We show that low concentrations of nocodazole and taxol interfere with the formation of podosome belt, and reduce TRAP activity of induced osteoclasts. These results suggest that the effect of taxol on MT dynamics may be used clinically to reduce osteoclast activity and potentially prevent development of osteoporosis and other metabolic bone diseases.

An assay to image neuronal microtubule dynamics in mice.

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics(1,2). One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization(1). Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs(1-3). Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker(4), following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones(5). This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types(6-8).

The NAP motif of activity-dependent neuroprotective protein (ADNP) regulates dendritic spines through microtubule end binding proteins.

The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1-3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP=SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities.

IQGAP1 regulates endothelial barrier function via EB1-cortactin cross talk.

Cross talk between the actin cytoskeleton and microtubules (MT) has been implicated in the amplification of agonist-induced Rho signaling, leading to increased vascular endothelial permeability. This study tested the involvement of actin-MT cross talk in the mechanisms of barrier enhancement induced by hepatocyte growth factor (HGF) and evaluated the role of the adaptor protein IQGAP1 in integrating the MT- and actin-dependent pathways of barrier enhancement. IQGAP1 knockdown by small interfering RNA attenuated the HGF-induced increase in endothelial barrier properties and abolished HGF-activated cortical actin dynamics. IQGAP1 reduction abolished HGF-induced peripheral accumulation of Rac cytoskeletal effector cortactin and cortical actin remodeling. In addition, HGF stimulated peripheral MT growth in an IQGAP1-dependent fashion. HGF also induced Rac1-dependent IQGAP1 association with the MT fraction and the formation of a protein complex containing end-binding protein 1 (EB1), IQGAP1, and cortactin. Decreasing endogenous IQGAP1 abolished HGF-induced EB1-cortactin colocalization at the cell periphery. In turn, expression of IQGAP1ΔC (IQGAP1 lacking the C-terminal domain) attenuated the cortactin association with EB1 and suppressed HGF-induced endothelial cell peripheral actin cytoskeleton enhancement. These results demonstrate for the first time the MT-actin cross talk mechanism of HGF-induced endothelial barrier enhancement and suggest that IQGAP1 functions as a hub linking HGF-induced signaling to MT and actin remodeling via EB1-IQGAP1-cortactin interactions.

Actin-microtubule coordination at growing microtubule ends.

To power dynamic processes in cells, the actin and microtubule cytoskeletons organize into complex structures. Although it is known that cytoskeletal coordination is vital for cell function, the mechanisms by which cross-linking proteins coordinate actin and microtubule activities remain poorly understood. In particular, it is unknown how the distinct mechanical properties of different actin architectures modulate the outcome of actin–microtubule interactions. To address this question, we engineered the protein TipAct, which links growing microtubule ends via end-binding proteins to actin filaments. We show that growing microtubules can be captured and guided by stiff actin bundles, leading to global actin–microtubule alignment. Conversely, growing microtubule ends can transport, stretch and bundle individual actin filaments, thereby globally defining actin filament organization. Our results provide a physical basis to understand actin–microtubule cross-talk, and reveal that a simple cross-linker can enable a mechanical feedback between actin and microtubule organization that is relevant to diverse biological contexts.

CLASPs Are Required for Proper Microtubule Localization of End-Binding Proteins

Microtubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus ends. End-binding proteins (EBs) are master regulators of the+TIP complex; however, it is unknown whether EBs are regulated by other+TIPs. Here, we show that cytoplasmic linker-associated proteins (CLASPs) modulate EB localization at MTs. In CLASP-depleted cells, EBs localized along the MT lattice in addition to plus ends. The MT-binding region of CLASP was sufficient for restoring normal EB localization, whereas neither EB-CLASP interactions nor EB tail-binding proteins are involved. Invitro assays revealed that CLASP directly functions to remove EB from MTs. Importantly, this effect occurs specifically during MT polymerization, but not at preformed MTs. Increased GTP-tubulin content within MTs in CLASP-depleted cells suggests that CLASPs facilitate GTP hydrolysis to reduce EB lattice binding. Together, these findings suggest that CLASPs influence the MT lattice itself to regulate EB and determine exclusive plus-end localization of EBs in cells.

Microtubule initiation from the nuclear surface controls cortical microtubule growth polarity and orientation in Arabidopsis thaliana.

The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth.

B-plexins control microtubule dynamics and dendrite morphology of hippocampal neurons

Semaphorins and their receptors plexins are implicated in various processes in the nervous system, but how B-plexins regulate the growth of dendrites remains poorly characterized. We had previously observed that Plexin-B1 and B3 interact with microtubule end-binding proteins (EBs) that are central adapters at growing microtubule tips, and this interaction is involved in neurite growth. Therefore, we hypothesized that plexins regulate microtubule dynamics and through that also dendritogenesis. The role of all three B-plexins was systematically examined in these processes. B-plexins and their ligand Semaphorin-4D influence the dynamics of microtubule tips both EB-dependently and independendently. EB3 as well as Plexin-B1, B2 and B3 turned out to have a significant role in the development of dendritic arbor of rat hippocampal neurons. Our results clearly indicate that semaphorin-plexin-EB pathway is one molecular mechanism how extracellular guidance cues are translated into intracellular mechanics. Taken together, Semaphorin-4D and B-plexins modulate the dynamic behavior of microtubule tips, and are therefore important in neurite growth.

Crosstalk between Axonal Classical Microtubule-Associated Proteins and End Binding Proteins during Axon Extension: Possible Implications in Neurodegeneration

During neuronal development, spherical neuroblasts differentiate into mature neurons through the extension of a long axon and several shorter dendrites. Morphological changes that underlie neuronal differentiation are mostly driven by the microtubular cytoskeleton. Regulation of microtubule dynamics and stability during axon and dendrite extension relies on the action of different families of microtubular proteins, such as classical microtubule-associated proteins (MAPs) and microtubule plus-end tracking proteins (+TIPs). This review article addresses recent research on the crosstalk between the main axonal MAPs, tau and MAP1B, and end binding proteins (EBs), the core +TIPs, during axon outgrowth in developing neuronal cells. Furthermore, we discuss the potential implications of the dysregulation of the interplay between tau and EBs in neurodegenerative disorders such as Alzheimer's disease.

Extending the Schizosaccharomyces pombe molecular genetic toolbox.

Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1 +, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70 + and pku80 + genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1 + to around 75–80% (a 16-fold increase). We describe how a natMX6/rpl42 + cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4 + selection to direct disruptive integration of leu1 + and lys1 + respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.

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

ABSTRACT 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.

End-binding protein 1 stimulates paclitaxel sensitivity in breast cancer by promoting its actions toward microtubule assembly and stability.

Paclitaxel is a microtubule-targeting agent widely used for the treatment of many solid tumors. However, patients show variable sensitivity to this drug, and effective diagnostic tests predicting drug sensitivity remain to be investigated. Herein, we show that the expression of end-binding protein 1 (EB1), a regulator of microtubule dynamics involved in multiple cellular activities, in breast tumor tissues correlates with the pathological response of tumors to paclitaxel-based chemotherapy. In vitro cell proliferation assays reveal that EB1 stimulates paclitaxel sensitivity in breast cancer cell lines. Our data further demonstrate that EB1 increases the activity of paclitaxel to cause mitotic arrest and apoptosis in cancer cells. In addition, microtubule binding affinity analysis and polymerization/depolymerization assays show that EB1 enhances paclitaxel binding to microtubules and stimulates the ability of paclitaxel to promote microtubule assembly and stabilization. These findings thus reveal EB1 as a critical regulator of paclitaxel sensitivity and have important implications in breast cancer chemotherapy.

End binding protein 3 promotes IP3‐gated calcium release and permeability of the endothelial barrier (893.7)

VE‐cadherin, the major endothelial adhesion molecule of AJs, forms hemophilic adhesion and induces re‐organization of actin and microtubule (MT) cytoskeleton. In the current study, we have addressed the role of MT end binding (EB) protein 3 in regulating stability of VE‐cadherin adhesion and lung tissue‐fluid homeostasis. Depletion of EB3 significantly attenuated inositol 1,4,5‐thriphosphate (IP3)‐gated release of calcium from intracellular stores and cell shape change in response to serine protease thrombin. Using acceptor‐photobleaching FRET and pulldown experiments we demonstrated that EB3 interacted with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) both in cells and in cell‐free system. Immunofluorescent staining of endogenous IP3R3 and live‐cell imaging of exogenously‐expressed IP3R3‐GFP in EB3 depleted cells showed that the interaction between EB3 and IP3R3 at the MT tip is required for IP3R3 clustering. Rescue with EB3 mutant lacking the last four amino acids did not restored IP3R3 clustering suggesting that presence of acidic tail is critical for interaction with IP3R3. Pre‐treatment of cells with membrane permeant variant of IP3R3‐derivative peptide (IPRP), containing the EBs consensus motif, significantly inhibited the interaction between EB3 and IP3R3 as well as attenuated thrombin‐induced release of calcium from intracellular stores. Furthermore, infusion of IPRP in lung microvasculature significantly inhibited increase in lung vascular liquid permeability (Kf,c) in response to activation of Protease‐activated receptors‐1 with an agonist peptide. These results identify a novel function of EB3 in regulating calcium signaling and tissue‐fluid homeostasis in lung.

Stable Microtubule Subsets Facilitate Early HIV-1 Infection

The density of the host cytoplasm presents pathogens such as HIV-1 with a major impediment to reaching the nucleus. As such, they must exploit host cytoskeletal networks to facilitate their movement.1 Within host cells, microtubule (MT) filaments act as “highways” for the intracellular transport of cargoes mediated by MT-associated motor proteins.2 While it has been established that the MT motor dynein mediates inward movement of HIV-1 to the nucleus,3 studies using MT depolymerizing agents such as nocodazole have produced seemingly conflicting suggestions that MTs are not important for HIV-1 infection. Recent findings relating to the role of specialized stable MT subsets now offer a potential explanation. MTs form polarized filaments; their minus ends are anchored in a perinuclear MT organizing center (MTOC) while their plus ends grow outward to explore or “sense” the intracellular environment and form connections to the cell periphery. As such, in many cell types MTs are highly dynamic, undergoing rapid phases of growth and collapse through the addition and loss of tubulin subunits primarily at the plus end. MT stability is controlled by highly specialized plus-end-binding proteins, called+TIPs. Central to this is the end-binding protein EB1, which recognizes the MT plus end and recruits other+TIPs that control MT dynamics. In response to specific signals, subsets of MTs become stabilized and form long-lived filaments that acquire distinguishing posttranslational tubulin modifications such as detyrosination and acetylation. Although these modifications do not seem to impart stability per se, they are thought to enable selective recognition of stable MT subsets by specific MT motor proteins. As such, these filaments can act as specialized tracks for vesicle trafficking to facilitate processes such as cell polarization. A notable feature of these long-lived MT subsets is their relative insensitivity to MT depolymerizing agents, but their potential contribution to HIV-1 infection has until now gone largely unstudied. We recently found that HIV-1 infection rapidly induces MT stabilization in a range of human cell types,4 including natural target cells such as T cells, microglia, and primary macrophages (Fig. 1). This stabilization appears to occur very soon after viral entry and involves viral targeting of EB1 and the EB1-binding+TIP, Kif4. MT stabilization continues to increase as infection progresses, potentially as more virus enters the cell, triggered by viral proteins released during uncoating or by the activation of signal pathways that regulate MT stability, something we are currently studying. Viral particles also exhibited significant localization to stable MTs and when stable MT formation was blocked using either RNA interference (RNAi) or dominant negative approaches infection was potently suppressed, with viral particles failing to reach the nucleus efficiently. These findings suggest that HIV-1 induces the formation of specialized stable MT subsets early after entry in order to facilitate translocation of viral particles to the nucleus. The importance of these stable MT subsets to early HIV-1 infection offers an explanation for why dynein is important for infection yet MT depolymerizing agents have little effect, and identifies new factors to explore as potential therapeutic targets to treat HIV-1. FIG. 1. HIV-1 induces stable microtubule (MT) formation in primary human macrophages. Primary human macrophages were mock infected (inset image lower left) or infected with pseudotyped HIV-1 and then fixed and stained for tyrosinated (red) and acetylated (green) ...

End-binding protein 1 (EB1) up-regulation is an early event in colorectal carcinogenesis

End-binding protein (EB1) is a microtubule protein that binds to the tumor suppressor adenomatous polyposis coli (APC). While EB1 is implicated as a potential oncogene, its role in cancer progression is unknown. Therefore, we analyzed EB1/APC expression at the earliest stages of colorectal carcinogenesis and in the uninvolved mucosa (“field effect”) of human and animal tissue. We also performed siRNA-knockdown in colon cancer cell lines. EB1 is up-regulated in early and field carcinogenesis in the colon, and the cellular/nano-architectural effect of EB1 knockdown depended on the genetic context. Thus, dysregulation of EB1 is an important early event in colon carcinogenesis.