Microtubule-binding Protein Research Articles

Molecular and Mechanical Causes of Microtubule Catastrophe and Aging

Tubulin polymers, microtubules, can switch abruptly from the assembly to shortening. These infrequent transitions, termed “catastrophes”, affect numerous cellular processes but the underlying mechanisms are elusive. We approached this complex stochastic system using advanced coarse-grained molecular dynamics modeling of tubulin-tubulin interactions. Unlike in previous simplified models of dynamic microtubules, the catastrophes in this model arise owing to fluctuations in the composition and conformation of a growing microtubule tip, most notably in the number of protofilament curls. In our model, dynamic evolution of the stochastic microtubule tip configurations over a long timescale, known as the system’s “aging”, gives rise to the nonexponential distribution of microtubule lifetimes, consistent with experiment. We show that aging takes place in the absence of visible changes in the microtubule wall or tip, as this complex molecular-mechanical system evolves slowly and asymptotically toward the steady-state level of the catastrophe-promoting configurations. This new, to our knowledge, theoretical basis will assist detailed mechanistic investigations of the mechanisms of action of different microtubule-binding proteins and drugs, thereby enabling accurate control over the microtubule dynamics to treat various pathologies.

Kinetochore–microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint

The spindle assembly checkpoint (SAC) is a genome surveillance mechanism that protects against aneuploidization. Despite profound progress on understanding mechanisms of its activation, it remains unknown what aspect of chromosome–spindle interactions is monitored by the SAC: kinetochore–microtubule attachment or the force generated by dynamic microtubules that signals stable biorientation of chromosomes? To answer this, we uncoupled these two processes by expressing a non-phosphorylatable version of the main microtubule-binding protein at kinetochores (HEC1-9A), causing stabilization of incorrect kinetochore–microtubule attachments despite persistent activity of the error-correction machinery. The SAC is fully functional in HEC1-9A-expressing cells, yet cells in which chromosomes cannot biorient but are stably attached to microtubules satisfy the SAC and exit mitosis. SAC satisfaction requires neither intra-kinetochore stretching nor dynamic microtubules. Our findings support the hypothesis that in human cells the end-on interactions of microtubules with kinetochores are sufficient to satisfy the SAC without the need for microtubule-based pulling forces.

Defects in Synaptic Plasticity, Reduced NMDA-Receptor Transport, and Instability of Postsynaptic Density Proteins in Mice Lacking Microtubule-Associated Protein 1A.

This work is the first report showing the significance of non-motor microtubule-associated protein in maintaining synaptic plasticity thorough a novel mechanism: anchoring of NMDA receptors to cytoskeleton supports transport of NMDA receptors and stabilizes postsynaptic density scaffolds binding to NMDA receptors. Newly generated mutant mice lacking MAP1A exhibited learning disabilities and reduced synaptic plasticity attributable to disruptions of the anchoring machinery.

The Tau-Induced Reduction of mRNA Levels of Kv Channels in Human Neuroblastoma SK-N-SH Cells.

Previous findings indicated that microtubule-binding protein tau and voltage-gated K(+) (Kv) channels exhibit a regulatory role in cell proliferation. However, the possible interaction of tau with Kv channels remained obscure. In this report, transfection of tau plasmids into human neuroblastoma SK-N-SH cells caused a significant reduction in the messenger RNA (mRNA) levels of several Kv channels, including Kv2.1, Kv3.1, Kv5.1, Kv9.2, and KCNH4. Correspondingly, the Kv currents recorded using patch-clamp techniques were substantially declined in the tau-transfected SK-N-SH cells. Moreover, tau induction and treatment with the Kv channel blocker TEA (tetraethylammonium) were able to improve proliferation rates of SK-N-SH cells by 43.1 and 66.2%, respectively. These data suggested that the tau-mediated alteration of Kv channels could be involved in its action on neural proliferation.

Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy

Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

Tauopathies as clinicopathological entities

Tauopathies are a class of neurodegenerative disorders characterized by neuronal and/or glial inclusions composed of the microtubule-binding protein, tau. Several lines of evidence suggest tau aggregation is central to the neurodegenerative process in tauopathies. First, recent animal and cell model studies find abnormally-modified tau alone may be transmitted between adjacent neurons and spread to anatomically connected brain regions to recapitulate human disease. Further, staging efforts in human autopsy cases suggest a sequential distribution of tau aggregation in the central nervous system that could reflect this observed cell-to-cell transmission of pathogenic tau species in animal models. Finally, pathogenic mutations in the MAPT gene encoding tau protein cause hereditary forms of tauopathy.Clinically, tauopathies can present with a range of phenotypes that include both movement- and cognitive/behavioral-disorders (i.e. frontotemporal dementia spectrum disorders) or non-specific amnestic symptoms in advanced age. A major limitation is that current clinical diagnostic criteria for these disorders do not reliably differentiate underlying tauopathy from other neurodegenerative diseases, such as TDP-43 proteinopathies. Thus, current research efforts are focused on improving the ante mortem diagnosis of tauopathies, including pre-clinical stages of disease, as many therapeutic strategies for emerging disease-modifying therapies focus on preventing abnormal folding and spread of tau pathology.

A novel ER-microtubule-binding protein, ERLIN2, stabilizes Cyclin B1 and regulates cell cycle progression.

The gene encoding endoplasmic reticulum (ER) lipid raft-associated protein 2 (ERLIN2) is amplified in human breast cancers. ERLIN2 gene mutations were also found to be associated with human childhood progressive motor neuron diseases. Yet, an understanding of the physiological function and mechanism for ERLIN2 remains elusive. In this study, we reveal that ERLIN2 is a spatially and temporally regulated ER–microtubule-binding protein that has an important role in cell cycle progression by interacting with and stabilizing the mitosis-promoting factors. Whereas ERLIN2 is highly expressed in aggressive human breast cancers, during normal development ERLIN2 is expressed at the postnatal stage and becomes undetectable in adulthood. ERLIN2 interacts with the microtubule component α-tubulin, and this interaction is maximal during the cell cycle G2/M phase where ERLIN2 simultaneously interacts with the mitosis-promoting complex Cyclin B1/Cdk1. ERLIN2 facilitates K63-linked ubiquitination and stabilization of Cyclin B1 protein in G2/M phase. Downregulation of ERLIN2 results in cell cycle arrest, represses breast cancer proliferation and malignancy and increases sensitivity of breast cancer cells to anticancer drugs. In summary, our study revealed a novel ER–microtubule-binding protein, ERLIN2, which interacts with and stabilizes mitosis-promoting factors to regulate cell cycle progression associated with human breast cancer malignancy.

MDM1 is a microtubule-binding protein that negatively regulates centriole duplication.

Mouse double-minute 1 (Mdm1) was originally identified as a gene amplified in transformed mouse cells and more recently as being highly up-regulated during differentiation of multiciliated epithelial cells, a specialized cell type having hundreds of centrioles and motile cilia. Here we show that the MDM1 protein localizes to centrioles of dividing cells and differentiating multiciliated cells. 3D-SIM microscopy showed that MDM1 is closely associated with the centriole barrel, likely residing in the centriole lumen. Overexpression of MDM1 suppressed centriole duplication, whereas depletion of MDM1 resulted in an increase in granular material that likely represents early intermediates in centriole formation. We show that MDM1 binds microtubules in vivo and in vitro. We identified a repeat motif in MDM1 that is required for efficient microtubule binding and found that these repeats are also present in CCSAP, another microtubule-binding protein. We propose that MDM1 is a negative regulator of centriole duplication and that its function is mediated through microtubule binding.

Microtubule-Binding Proteins as Promising Biomarkers of Paclitaxel Sensitivity in Cancer Chemotherapy.

Microtubules, tirelessly animated and highly dynamic structures, are vital for most cellular processes and their intricacies are still being revealed even after a century since their discovery. The importance of microtubules as chemotherapeutic targets cannot be overstated, and their clinical role is unlikely to abate in the near future. Indeed, improved understanding of microtubule biology could herald a new epoch of anticancer drug design by permitting fine-tuning of microtubule-targeting agents, the clinical utility of which is presently often limited by primary or acquired resistance. Paclitaxel, one such agent belonging to the taxane family, has proven a resoundingly successful treatment for many cancer patients; however, for too many others with paclitaxel-refractory tumors, the drug has offered nothing but side effects. Accumulating evidence suggests that microtubule-binding proteins (MBPs) can regulate paclitaxel sensitivity in a wide range of cancer types. Improved understanding of how these proteins can be assayed to predict treatment responses or manipulated pharmacologically to improve clinical outcomes could transform modern chemotherapy and is urgently awaited.

Epigallocatechin-3-gallate enhances clearance of phosphorylated tau in primary neurons

Objectives: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by intracellular accumulations of phosphorylated forms of the microtubule binding protein tau. This study aimed to explore a novel mechanism for enhancing the clearance of these pathological tau species using the green tea flavonoid epigallocatechin-3-gallate (EGCG). EGCG is a potent antioxidant and an activator of the Nrf2 transcriptional pathway. Nrf2 activators including EGCG have shown promise in mitigating amyloid pathology in vitro and in vivo. This study assessed whether EGCG could also alter tau clearance.Methods: Rat primary cortical neuron cultures were treated on day in vitro 8 with EGCG and analyzed for changes in gene and protein expression using luciferase assay, q-PCR, and western blotting.Results: EGCG treatment led to a significant decrease in the protein levels of three AD-relevant phospho-tau epitopes. Unexpectedly, EGCG does not appear to be facilitating this effect through the Nrf2 pathway or by increasing autophagy in general. However, EGCG did significantly increase mRNA expression of the key autophagy adaptor proteins NDP52 and p62.Discussion: In this study, we show that EGCG enhances the clearance of AD-relevant phosphorylated tau species in primary neurons. Interestingly, this result appears to be independent of both Nrf2 activation and enhanced autophagy – two previously reported mechanisms of phytochemical-induced tau clearance. EGCG did significantly increase expression of two autophagy adaptor proteins. Taken together, these results demonstrate that EGCG has the ability to increase the clearance of phosphorylated tau species in a highly specific manner, likely through increasing adaptor protein expression.

α- and β-Tubulin Lattice of the Axonemal Microtubule Doublet and Binding Proteins Revealed by Single Particle Cryo-Electron Microscopy and Tomography

Microtubule doublet (MTD) is the main skeleton of cilia/flagella. Many proteins, such as dyneins and radial spokes, bind to MTD, and generate or regulate force. While the structure of the reconstituted microtubule has been solved at atomic resolution, nature of the axonemal MTD is still unclear. There are a few hypotheses of the lattice arrangement of its α- and β-tubulins, but it has not been described how dyneins and radial spokes bind to MTD. In this study, we analyzed the three-dimensional structure of Tetrahymena MTD at ∼19 Å resolution by single particle cryo-electron microscopy. To identify α- and β-tubulins, we combined image analysis of MTD with specific kinesin decoration. This work reveals that α- and β-tubulins form a B-lattice arrangement in the entire MTD with a seam at the outer junction. We revealed the unique way in which inner arm dyneins, radial spokes, and proteins inside MTD bind and bridge protofilaments.

Identification of novel microtubule-binding proteins by taxol-mediated microtubule stabilization and mass spectrometry analysis.

Microtubule-binding proteins (MBPs) are structurally and functionally diverse regulators of microtubule-mediated cellular processes. Alteration of MBPs has been implicated in the pathogenesis of human diseases, including cancer. MBPs can stabilize or destabilize microtubules or move along microtubules to transport various cargoes. In addition, MBPs can control microtubule dynamics through direct interaction with microtubules or coordination with other proteins. To better understand microtubule structure and function, it is necessary to identify additional MBPs. In this study, we isolated microtubules and MBPs from mammalian cells by a taxol-based method and then profiled a panel of MBPs by mass spectrometry. We discovered a number of previously uncharacterized MBPs, including several membrane-associated proteins and proteins involved in post-translational modifications, in addition to several structural components. These results support the notion that microtubules have a wide range of functions and may undergo more exquisite regulation than previously recognized.

The oligodendroglia cytoskeleton in health and disease.

Oligodendrocytes, the myelin forming cells of the CNS, enwrap neuronal axons and form multilamellar myelin sheets. They are derived from oligodendrocyte precursor cells which migrate from the subventricular zone into the different regions of the brain. Differentiation from the early progenitor to the mature multiprocessed oligodendrocyte is characterized by different morphological stages. To support cell morphology and establish and maintain the myelin membrane, an intact, spatially organized cytoskeleton with dynamic properties is essential. In particular microtubules and their associated proteins play an important role. A variety of microtubule binding proteins, including tau, are present in oligodendrocytes. Oligodendrocytes in culture express all six isoforms of tau which are developmentally regulated. Tau proteins are present in immature and mature oligodendrocytes and specifically prominent in the branching points of the cellular processes. Downregulation of tau impairs cell differentiation and the process of early myelination. In neurodegenerative diseases collectively termed tauopathies, fibrillary tau accumulations occur not only in neurons but also in glia. Tau positive coiled bodies originating in oligodendrocytes are characteristic for the brains of patients with frontotemporal dementias, such as corticobasal degeneration and progressive supranuclear palsy. These aggregates are further characterized by the presence of heat shock proteins and ubiquitin, indicating that stress situations are causally related to the pathogenesis. In this respect, proteasomal dysfunctions have been discussed to be involved in neurodegenerative disorders and the aging process. Data on the consequences of proteolytic stress in oligodendrocytes and the protein aggregation process will be presented. Our data demonstrate that an intact cytoskeleton is essential for cellular defense mechanisms.

EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells.

(-)-epigallocatechin-3-gallate (EGCG) is a well-known cancer chemopreventive agent. The potential mechanisms include regulation of multiple molecules. Carcinogenesis in lung cancer is related to the imbalance of tumor suppressor and oncogene. JWA is a structurally novel microtubule-binding protein and is a potential tumor suppressor. DNA topoisomerase IIα is a nuclear enzyme that governs DNA topology and is usually highly expressed in many types of cancer. It serves as a target of anticancer drugs. In the current study, the regulation of JWA and topoisomerase IIα by EGCG, and thereafter the mutual interaction between them was investigated. The results revealed that EGCG up-regulated JWA while decreased topoisomerase IIα expression in both human non-small cell lung cancer (NSCLC) cells and an NSCLC xenograft mice model. There was a negative correlation between JWA and topoisomerase IIα in NSCLC as well as in human NSCLC tissue specimens. Topoisomerase IIα overexpression reduced JWA at the translational level. Meanwhile, JWA-induced topoisomerase IIα degradation was regulated both in the transcriptional and post-translational level. Interestingly, JWA and topoisomerase IIα regulated each other in the cells arrested in G2/M. Furthermore, JWA and topoisomerase IIα synergistically affected NCI-H460 cells invasion. These results may serve a novel mechanism for cancer prevention.

Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line

Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5YTNAPhigh neuroblastoma cells compared to SH-SY5YTNAPlow cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5YTNAPhigh cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5YTNAPhigh cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5YTNAPhigh cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies.

HkRP3 is a microtubule-binding protein regulating lytic granule clustering and NK cell killing.

NK cells provide host defense by killing viral-infected and cancerous cells through the secretion of preformed lytic granules. Polarization of the lytic granules toward the target cell is dependent on an intact microtubule (MT) network as well as MT motors. We have recently shown that DOCK8, a gene mutated in a primary immunodeficiency syndrome, is involved in NK cell killing in part through its effects on MT organizing center (MTOC) polarization. In this study, we identified Hook-related protein 3 (HkRP3) as a novel DOCK8- and MT-binding protein. We further show that HkRP3 is present in lytic granule fractions and interacts with the dynein motor complex and MTs. Significantly, depletion of HkPR3 impaired NK cell cytotoxicity, which could be attributed to a defect in not only MTOC polarity, but also impaired clustering of lytic granules around the MTOC. Our results demonstrate an important role for HkRP3 in regulating the clustering of lytic granules and MTOC repositioning during the development of NK cell-mediated killing.

Determining a Functional Mechanism for a Dysfunctional Protein

Tau is a microtubule binding protein that forms pathological aggregates in the brain in Alzheimer's disease and other tauopathies. Disease etiology is thought to arise from loss of native interactions between tau and microtubules, as well as from gain of toxicity tied to tau aggregation, although neither mechanism is well-understood. We have investigated the link between function and disease using disease-associated and designed disease-motivated mutants of tau. We find that while the mutants bind microtubules with comparable affinities, they demonstrate an increased affinity for tubulin dimers. Moreover, the mutant forms of tau are impaired in their ability to promote microtubule assembly. Using single molecule FRET, we measure conformational changes in tau upon binding to tubulin that provide a structural framework for the observed altered affinity and function. We propose a model that describes tau binding to tubulin dimers and a mechanism by which disease-relevant alterations to tau impact its function. Together, these results draw attention to the relevance of the interaction between tau and free tubulin as playing an important role in the molecular mechanisms of tau pathology.

Functional Interactions of Disease‐Linked Disordered Proteins: Alpha‐Synuclein, Tau and Complexin

The proteins alpha‐synuclein and tau are linked to Parkinson's and Alzheimer's disease, respectively, yet little is known about their normal physiological functions, while complexin, a protein more loosely linked to neurological and psychiatric disorders, has several opposing functions whose mechanisms remain poorly understood. All three proteins are completely or highly disordered when free in solution. Structural characterization of different states of alpha‐synuclein has lead us to propose a model in which the protein can bridge between the synaptic vesicle and plasma membranes. This model also has implications for membrane‐induced synuclein aggregation. These topics will be discussed in the context of one of the most recent Parkinson's disease associated synuclein mutations. Tau is normally a microtubule binding protein, but can also bind to membrane surfaces, an interaction that may be either physiologically or pathologically relevant. Recent work in the lab has confirmed that tau‐membrane interactions are mediated by short amphipathic helices, which may also play a role in mediating membrane‐ or microtubule‐induced aggregation. Complexin contains a central helix motif that binds to assembled SNARE complexes, and this interaction is key to two opposing functions, enhancing synchronous exocytosis and inhibiting spontaneous vesicle fusion. Binding of the disordered C‐terminal domain of complexin to membranes is critical for its inhibitory function. We have shown that such binding is highly dependent on membrane curvature, and that a local structural transition to a helical conformation, again necessary for function, only occurs upon binding to highly curved membranes such as synaptic vesicles. Thus complexin's function is regulated by a membrane curvature dependent structural transition.

Tau assembly: the dominant role of PHF6 (VQIVYK) in microtubule binding region repeat R3.

Self-aggregation of the microtubule-binding protein Tau reduces its functionality and is tightly associated with Tau-related diseases, termed tauopathies. Tau aggregation is also strongly associated with two nucleating six-residue segments, namely PHF6 (VQIVYK) and PHF6* (VQIINK). In this paper, using experiments and computational modeling, we study the self-assembly of individual and binary mixtures of Tau fragments containing PHF6* (R2/wt; (273)GKVQIINKKLDL(284)) and PHF6 (R3/wt; (306)VQIVYKPVDLSK(317)) and a mutant R2/ΔK280 associated with a neurodegenerative tauopathy. The initial stage of aggregation is probed by ion-mobility mass spectrometry, the kinetics of aggregation monitored with Thioflavin T assays, and the morphology of aggregates visualized by transmission electron microscopy. Insights into the structure of early aggregates and the factors stabilizing the aggregates are obtained from replica exchange molecular dynamics simulations. Our data suggest that R3/wt has a much stronger aggregation propensity than either R2/wt or R2/ΔK280. Heterodimers containing R3/wt are less stable than R3/wt homodimers but much more stable than homodimers of R2/wt and R2/ΔK280, suggesting a possible role of PHF6*-PHF6 interactions in initiating the aggregation of full-length Tau. Lastly, R2/ΔK280 binds more strongly to R3/wt than R2/wt, suggesting a possible mechanism for a pathological loss of normal Tau function.

Engagement, Reception and Breakup: Three Steps of Cell Motility regulated by HPIP signaling

Tumor metastasis is the prime cause for increased morbidity and mortality for majority of the cancers. Invasive or infiltrative ductal carcinoma (IDC) is the predominant form of breast cancer, accounting approximately 80% of all breast cancers. The molecular portraits and the mechanism by which they promote IDC remain largely unknown. Hematopoietic PBX interacting protein (HPIP/PBXIP1), a microtubule binding protein, regulates cancer cell migration and invasion. However, functional mechanism underlying HPIP-mediated cell migration in cancer remains uncertain. Here, we describe our recent studies in which we identified the new mechanisms by which HPIP regulates cell migration in breast cancer cells. Our recent studies confirmed that HPIP expression is elevated in breast infiltrative ductal carcinoma and positively correlated with poor patient survival. We reported that HPIP directly interacts and activates FAK to promote the cell migration. Mechanistic studies further revealed that HPIP induces focal adhesion disassembly by enhancing calpain2 activity through MAPK pathway that led to talin proteolysis, focal adhesion turnover and cell migration. Interestingly, the activated calpain2 in turn cleaves HPIP suggesting the tight regulation of HPIP-FAK-calpain2 signaling cascade during cell migration.