Tubulin Isoforms Research Articles

Tubulin mutations in neurodevelopmental disorders as a tool to decipher microtubule function.

Malformations of cortical development (MCDs) are a group of severe brain malformations associated with intellectual disability and refractory childhood epilepsy. Human missense heterozygous mutations in the 9 α-tubulin and 10 β-tubulin isoforms forming the heterodimers that assemble into microtubules (MTs) were found to cause MCDs. However, how a single mutated residue in a given tubulin isoform can perturb the entire microtubule population in a neuronal cell remains a crucial question. Here, we examined 85 MCD-associated tubulin mutations occurring in TUBA1A, TUBB2, and TUBB3 and their location in a three-dimensional (3D) microtubule cylinder. Mutations hitting residues exposed on the outer microtubule surface are likely to alter microtubule association with partners, while alteration of intradimer contacts may impair dimer stability and straightness. Other types of mutations are predicted to alter interdimer and lateral contacts, which are responsible for microtubule cohesion, rigidity, and dynamics. MCD-associated tubulin mutations surprisingly fall into all categories, thus providing unexpected insights into how a single mutation may impair microtubule function and elicit dominant effects in neurons.

Analysis of polymorphisms in the colchicine binding site of tubulin in colchicine-resistant familial Mediterranean fever patients.

Familial Mediterranean fever is a hereditary autoinflammatory syndrome. The typical treatment for the disease is colchicine. However, a subset of patients are not responsive to colchicine. In this study, polymorphisms in the colchicine-binding site of the TUBB1 gene, which encodes a tubulin isoform specific to leukocytes, were investigated in patients with colchicine-resistant disease. FMF patients who were followed in the Department of Pediatric Rheumatology at Hacettepe University were included in this study. Colchicine resistance was defined as ongoing disease activity (≥ 1 attack/month over 3months or persistently elevated CRP) while taking the maximum tolerated dose of colchicine. A total of 62 Turkish FMF patients (42 colchicine-responsive and 20 colchicine-resistant) and a control group of healthy children were included in the study. DNA was extracted for analysis of TUBB1, and the colchicine binding site was sequenced. We did not observe A248T (rs148237574) or M257V (rs759579888), two variations that were previously associated with colchicine resistance in an in silico analysis. We did detect T274M (rs35565630), R306H (rs772479017), and R307H (rs6070697) variants in the FMF patients, but there was no statistically significant difference between the colchicine-responsive and colchicine-resistant groups. This is the first study to evaluate TUBB1 gene polymorphisms in the colchicine binding site in patients with FMF. Our data do not support the hypothesis that these polymorphisms are a possible cause of colchicine resistance in FMF patients.

Diversity of α-tubulin transcripts in Lolium rigidum.

Tubulin, the target site of dinitroaniline herbicides, is encoded by small gene families in plants. To better characterize the mechanisms of target-site resistance to dinitroaniline herbicides in the globally important weedy species Lolium rigidum, attempts were made to amplify and sequence α-tubulin transcripts. Four α-tubulin isoforms (TUA1, TUA2, TUA3 and TUA4) were identified in L. rigidum. Variations in the number and sequence of transcripts encoding these α-tubulin proteins were found in individuals from the two L. rigidum populations examined. Within and among populations, differences in the 5'- and 3'-untranslated regions of cDNA in TUA3 and TUA4 were identified. Furthermore, a novel double mutation, Arg-390-Cys+Asp-442-Glu, in the TUA3 transcript was identified and has the potential to confer dinitroaniline resistance. This research reveals the complexity of the α-tubulin gene family in individuals/populations of the cross-pollinated weedy species L. rigidum, and highlights the need for better understanding of the molecular architecture of tubulin gene families for detecting resistance point mutations. Although TUA4 is a commonly expressed α-tubulin isoform containing most frequently reported resistance mutations, other mutant tubulin isoforms may also have a role in conferring dinitroaniline resistance.

Intrinsic and Extrinsic Factors Affecting Microtubule Dynamics in Normal and Cancer Cells.

Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule–MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the consequences of such a phenomenon, including drug resistance development.

Abstract 1924: Genetic analysis of responses to eribulin versus vinorelbine and paclitaxel in 100 cancer cell lines from the cancer cell line Encyclopedia (CCLE)

Abstract Background: Eribulin mesylate, a synthetic analog of the marine sponge natural product halichondrin B, is a microtubule dynamics inhibitor that acts via both cytotoxic antimitotic mechanisms as well as non-cytotoxic effects on tumor vasculature, tumor phenotype and the tumor microenvironment. Eribulin is approved in the US for treatment of certain patients with advanced breast cancer or liposarcoma. Here we examined in vitro antiproliferative responses to eribulin versus vinorelbine and paclitaxel in 100 human cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE), followed by analysis of CCLE baseline gene expression data with the goal of assessing overlapping and non-overlapping pathways and genes associated with drug response and resistance. Methods: In vitro proliferation assays were conducted on 100 CCLE cell lines. Cancer types represented by ≥5 cell lines include breast, lung, blood, colorectum, ovary, pancreas, skin and kidney. Comparative systems-level analyses of genes, networks and pathways associated with drug response and/or resistance were performed to identify gene and pathway-level determinants of drug response that were either unique to eribulin or shared with vinorelbine and/or paclitaxel. Results: We evaluated genes and molecular pathways driving sensitivity to eribulin versus vinorelbine and paclitaxel in a panel of 100 CCLE cell lines and identified key molecular determinants of response and resistance unique to eribulin. Pathway expression analysis revealed key pathways significantly associated with response to eribulin, including those associated with oxidative stress-induced senescence, histone deacetylation, DNA methylation and Notch signaling. The oxidative stress-induced senescence pathway was significantly enriched for genes whose pretreatment/basal mRNA expression levels were positively associated with response to eribulin but not vinorelbine or paclitaxel. Expression levels of various tubulin isoforms were also identified as positively correlated with eribulin response. Higher and lower pretreatment expression of E-cadherin and N-cadherin, respectively, were also found in cell lines sensitive to eribulin treatment. Conclusion: Pretreatment gene expression analysis demonstrated key gene sets and molecular pathways that may drive response and resistance to eribulin and may differentiate this drug from vinorelbine and paclitaxel. Detailed analysis will be presented at the meeting. Citation Format: Pallavi Sachdev, Roy Ronen, Janusz Dutkowski, Bruce A. Littlefield. Genetic analysis of responses to eribulin versus vinorelbine and paclitaxel in 100 cancer cell lines from the cancer cell line Encyclopedia (CCLE) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1924.

A year in the salt marsh: Seasonal changes in gill protein expression in the temperate intertidal mussel Geukensia demissa

Organisms living in temperate and polar regions experience extensive seasonal changes in the physical and biotic environment, including temperature, insolation, and food availability, among other factors. Sessile intertidal organisms respond to such seasonal fluctuations largely through physiological and biochemical means, because their behavioral responses are severely limited. In this study, we used a proteomic approach to examine changes in seasonal protein expression of gill from the intertidal mussel Geukensia demissa, a keystone species of the western Atlantic salt marsh, over the course of one year. Gill tissue of mussels collected in summer had the greatest number of proteins significantly increased in abundance (37 of 592 spots detected on two-dimensional polyacrylamide gels), although autumn mussels revealed a comparable proportion of up-regulated proteins (31 spots). In contrast, the number of proteins changing in abundance in winter and spring mussels were substantially smaller (15 and 9, respectively). Identification of these proteins revealed both expected and unanticipated changes to the proteome. Maintenance of gill cilia dominates in the summer when filter-feeding is most active, as evidenced by cytoskeletal proteins such as tektin-4 and tubulin isoforms; a signal of protection from heat stress is also present in summer (e.g., heat shock cognate 70). In autumn oxidative stress protection (peroxiredoxin-5 and manganese-containing superoxide dismutase) and aerobic ATP synthetic capacity (ATP synthase subunits a and delta) appear to increase. In winter a signal of cold-induced oxidative stress is apparent (Mn-SOD and NADP-dependent isocitrate dehydrogenase), perhaps in association with heavy metal toxicity and exposure to pathogens. Gill tissue from spring shows relatively little environmental acclimatization, other than a possible increase in protein synthesis capacity.

The Tubulin Code in Microtubule Dynamics and Information Encoding.

Microtubules are non-covalent mesoscale polymers central to the eukaryotic cytoskeleton. Microtubule structure, dynamics, and mechanics are modulated by a cell's choice of tubulin isoforms and post-translational modifications, a "tubulin code," which is thought to support the diverse morphology and dynamics of microtubule arrays across various cell types, cell cycle, and developmental stages. We give a brief historical overview of research into tubulin diversity and highlight recent progress toward uncovering the mechanistic underpinnings of the tubulin code. As a large number of essential pathways converge upon the microtubule cytoskeleton, understanding how cells utilize tubulin diversity is crucial to understanding cellular physiology and disease.

Differences in Intrinsic Tubulin Dynamic Properties Contribute to Spindle Length Control in Xenopus Species.

The function of cellular organelles relates not only to their molecular composition but also to their size. However, how the size of dynamic mesoscale structures is established and maintained remains poorly understood [1-3]. Mitotic spindle length, for example, varies several-fold among cell types and among different organisms [4]. Although most studies on spindle size control focus on changes in proteins that regulate microtubule dynamics [5-8], the contribution of the spindle's main building block, the αβ-tubulin heterodimer, has yet to be studied. Apart from microtubule-associated proteins and motors, two factors have been shown to contribute to the heterogeneity of microtubule dynamics: tubulin isoform composition [9, 10] and post-translational modifications [11]. In the past, studying the contribution of tubulin and microtubules to spindle assembly has been limited by the fact that physiologically relevant tubulins were not available. Here, we show that tubulins purified from two closely related frogs, Xenopus laevis and Xenopus tropicalis, have surprisingly different microtubule dynamics invitro. X.laevis microtubules combine very fast growth and infrequent catastrophes. In contrast, X.tropicalis microtubules grow slower and catastrophe more frequently. We show that spindle length and microtubule mass can be controlled by titrating the ratios of the tubulins from the two frog species. Furthermore, we combine our invitro reconstitution assay and egg extract experiments with computational modeling to show that differences in intrinsic properties of different tubulins contribute to the control of microtubule mass and therefore set steady-state spindle length.

Crosstalk between Mitochondria and Cytoskeleton in Cardiac Cells.

Elucidation of the mitochondrial regulatory mechanisms for the understanding of muscle bioenergetics and the role of mitochondria is a fundamental problem in cellular physiology and pathophysiology. The cytoskeleton (microtubules, intermediate filaments, microfilaments) plays a central role in the maintenance of mitochondrial shape, location, and motility. In addition, numerous interactions between cytoskeletal proteins and mitochondria can actively participate in the regulation of mitochondrial respiration and oxidative phosphorylation. In cardiac and skeletal muscles, mitochondrial positions are tightly fixed, providing their regular arrangement and numerous interactions with other cellular structures such as sarcoplasmic reticulum and cytoskeleton. This can involve association of cytoskeletal proteins with voltage-dependent anion channel (VDAC), thereby, governing the permeability of the outer mitochondrial membrane (OMM) to metabolites, and regulating cell energy metabolism. Cardiomyocytes and myocardial fibers demonstrate regular arrangement of tubulin beta-II isoform entirely co-localized with mitochondria, in contrast to other isoforms of tubulin. This observation suggests the participation of tubulin beta-II in the regulation of OMM permeability through interaction with VDAC. The OMM permeability is also regulated by the specific isoform of cytolinker protein plectin. This review summarizes and discusses previous studies on the role of cytoskeletal proteins in the regulation of energy metabolism and mitochondrial function, adenosine triphosphate (ATP) production, and energy transfer.

Abstract 3619: Nanoparticle formulation of VERU 111 for pancreatic cancer treatment

Abstract Background: Pancreatic cancer (PanCa) is one of the leading causes of cancer-related mortality in the United States due to very limited therapeutic options. Thus, developing novel therapeutic strategies will help for the management of this disease. We recently identified VERU-111, a novel synthetic molecule which showed potent anti-cancer effect against PanCa via targeting clinically important βIII and βIV tubulin isoforms. In this study, we synthesized and characterized its novel nanoformulation (MNP-VERU) and evaluated its therapeutic effects in vitro and xenograft mouse model. Methods: MNPs were prepared by chemical precipitation method and loaded with VERU-111 using diffusion method. This formulation was characterized for particle size, chemical composition, and drug loading efficiency, using various physico-chemical methods (TEM, FT-IR, DSC, TGA, and HPLC). The internalization of MNP-VERU was achieved after 6 hours incubation with MNP-VERU in PanCa cells. To determine therapeutic efficacy of MNP-VERU, we performed various in vitro (MTS, wound healing, boyden chamber real-time xCELLigence, and apoptosis assays) and in vivo (mouse tumor xenograft) studies using PanCa. Effect of MNP-VERU on various key oncogenic signaling pathways, and miRNAs was evaluated by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses respectively. Result: Our novel MNP-VERU formulation provided average size of 110 nm in dynamic light scattering (DLS) and exhibited -8.23 to -11.65 mV zeta potential with an outstanding loading efficiency (94%). Cellular uptake and internalization studies demonstrate that MNP-VERU escape lysosomal degradation, providing efficient endosomal release to cytosol. MNP-VERU showed remarkable anti-cancer potential in various PanCa cells (Panc-1, AsPC-1, HPAF-II, BxPC-3, MiaPaca) and more effectively repressed βIII and βIV tubulin isoforms via restoring the expression of miR-200c. MNP-VERU more effectively suppressed AsPC-1 cells derived xenograft tumors in athymic nude mice. Conclusions: Taken together, our results suggest that MNP-VERU has more anti-cancer potential than free VERU-111 against PanCa. MNP-VERU may reduce the toxicity and improve the bioavailability of free VERU-111 and could be used for the management of PanCa. Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Neeraj Chauhan, Prashanth K. Nagesh, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. Nanoparticle formulation of VERU 111 for pancreatic cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3619.

Altered Cytoskeletal Composition and Delayed Neurite Elongation in tau45–230-Expressing Hippocampal Neurons

Calpain-mediated tau cleavage into the neurotoxic tau45–230 fragment plays an important role in Alzheimer's disease (AD). This tau fragment accumulates mainly in the cytoplasm of degenerating neurons. However, subcellular localization studies indicated that a pool of tau45–230 associates with the cytoskeleton in hippocampal neurons. In the present study, we assessed whether such localization could underlie tau45–230 neurotoxic effects. Quantitative Western blot analysis showed decreased levels of full-length tau bound to microtubules in tau45–230-expressing hippocampal neurons when compared to controls. In addition, the presence of this tau fragment induced a transient increase in tyrosinated tubulin, a marker of unstable microtubules, followed by a significant decrease in the levels of this tubulin isoform. The data obtained also showed a significant reduction in actin filaments in tau45–230-expressing neurons. These changes in microtubules and actin filaments correlated with delayed neurite elongation and axonal differentiation in the presence of this tau fragment. Together, these results suggest that tau45–230 could exert its toxic effects, at least in part, by modifying the composition of the neuronal cytoskeleton and impairing neurite elongation in neurons undergoing degeneration.

Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradation

Clinical microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents. Drugs from both classes achieve microtubule inhibition by binding different sites on tubulin and inhibiting or promoting polymerization with no concomitant effects on the protein levels of tubulin heterodimers. Here, we have identified a series of small molecules with diverse structures potentially representing a third class of novel tubulin inhibitors that promote degradation by covalent binding to Cys-239 of β-tubulin. The small molecules highlighted in this study include T0070907 (a peroxisome proliferator-activated receptor γ inhibitor), T007-1 (a T0070907 derivative), T138067, N,N'-ethylene-bis(iodoacetamide) (EBI), and allyl isothiocyanate (AITC). Label-free quantitative proteomic analysis revealed that T007-1 promotes tubulin degradation with high selectivity. Mass spectrometry findings showed covalent binding of both T0070907 and T007-01 to Cys-239 of β-tubulin. Furthermore, T007-1 exerted a degradative effect on tubulin isoforms possessing Cys-239 (β2, β4, and β5(β)) but not those containing Ser-239 (β3, β6) or mutant β-tubulin with a C239S substitution. Three small molecules (T138067, EBI, and AITC) also reported to bind covalently to Cys-239 of β-tubulin similarly induced tubulin degradation. Our results strongly suggest that covalent modification of Cys-239 of β-tubulin by small molecules could serve as a novel strategy to promote tubulin heterodimer degradation. We propose that these small molecules represent a third novel class of tubulin inhibitor agents that exert their effects through degradation activity.

Therapeutic efficacy of a novel \u03b2III/\u03b2IV-tubulin inhibitor (VERU-111) in pancreatic cancer

BackgroundThe management of pancreatic cancer (PanCa) is exceptionally difficult due to poor response to available therapeutic modalities. Tubulins play a major role in cell dynamics, thus are important molecular targets for cancer therapy. Among various tubulins, βIII and βIV-tubulin isoforms have been primarily implicated in PanCa progression, metastasis and chemo-resistance. However, specific inhibitors of these isoforms that have potent anti-cancer activity with low toxicity are not readily available.MethodsWe determined anti-cancer molecular mechanisms and therapeutic efficacy of a novel small molecule inhibitor (VERU-111) using in vitro (MTS, wound healing, Boyden chamber and real-time xCELLigence assays) and in vivo (xenograft studies) models of PanCa. The effects of VERU-111 treatment on the expression of β-tubulin isoforms, apoptosis, cancer markers and microRNAs were determined by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses.ResultsWe have identified a novel small molecule inhibitor (VERU-111), which preferentially represses clinically important, βIII and βIV tubulin isoforms via restoring the expression of miR-200c. As a result, VERU-111 efficiently inhibited tumorigenic and metastatic characteristics of PanCa cells. VERU-111 arrested the cell cycle in the G2/M phase and induced apoptosis in PanCa cell lines via modulation of cell cycle regulatory (Cdc2, Cdc25c, and Cyclin B1) and apoptosis - associated (Bax, Bad, Bcl-2, and Bcl-xl) proteins. VERU-111 treatment also inhibited tumor growth (P < 0.01) in a PanCa xenograft mouse model.ConclusionsThis study has identified an inhibitor of βIII/βIV tubulins, which appears to have excellent potential as monotherapy or in combination with conventional therapeutic regimens for PanCa treatment.

Post-Translational Polymodification of β1 Tubulin Regulates Motor Protein Localisation in Platelet Production and Function

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which β1 tubulin, the platelet and megakaryocyte lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived megakaryocytes, and healthy human donor platelets. We find distinct patterns of polymodification in megakaryocytes and platelets, mediated by the cell specific expression of effecting (Tubulin Tyrosine Ligase Like - TTLL) and reversing (Cytosolic Carboxypeptidase - CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

Proteomic profile associated with cell death induced by androgens in Taenia crassiceps cysticerci: proposed interactome

Androgens have been shown to exert a cysticidal effect upon Taenia crassiceps, an experimental model of cysticercosis. To further inquire into this matter, the Taenia crassiceps model was used to evaluate the expression of several proteins after testosterone (T4) and dihydrotestosterone (DHT) in vitro treatment. Under 2-D proteomic maps, parasite extracts were resolved into approximately 130 proteins distributed in a molecular weight range of 10-250 kDa and isoelectrical point range of 3-10. The resultant proteomic pattern was analysed, and significant changes were observed in response to T4 and DHT. Based on our experience with electrophoretic patterns and proteomic maps of cytoskeletal proteins, alteration in the expression of isoforms of actin, tubulin and paramyosin and of other proteins was assessed. Considering that androgens may exert their biological activity in taeniids through the non-specific progesterone receptor membrane component (PGRMC), we harnessed bioinformatics to propose the identity of androgen-regulated proteins and establish their hypothetical physiological role in the parasites. These analyses yield a possible explanation of how androgens exert their cysticidal effects through changes in the expression of proteins involved in cytoskeletal rearrangement, dynamic vesicular traffic and transduction of intracellular signals.

Genetics and Expression Profile of the Tubulin Gene Superfamily in Breast Cancer Subtypes and Its Relation to Taxane Resistance.

Taxanes are a class of chemotherapeutic agents that inhibit cell division by disrupting the mitotic spindle through the stabilization of microtubules. Most breast cancer (BC) tumors show resistance against taxanes partially due to alterations in tubulin genes. In this project we investigated tubulin isoforms in BC to explore any correlation between tubulin alterations and taxane resistance. Genetic alteration and expression profiling of 28 tubulin isoforms in 6714 BC tumor samples from 4205 BC cases were analyzed. Protein-protein, drug-protein and alterations neighbor genes in tubulin pathways were examined in the tumor samples. To study correlation between promoter activity and expression of the tubulin isoforms in BC, we analyzed the ChIP-seq enrichment of active promoter histone mark H3K4me3 and mRNA expression profile of MCF-7, ZR-75-30, SKBR-3 and MDA-MB-231 cell lines. Potential correlation between tubulin alterations and taxane resistance, were investigated by studying the expression profile of taxane-sensitive and resistant BC tumors also the MDA-MB-231 cells acquired resistance to paclitaxel. All genomic data were obtained from public databases. Results showed that TUBD1 and TUBB3 were the most frequently amplified and deleted tubulin genes in the BC tumors respectively. The interaction analysis showed physical interactions of α-, β- and γ-tubulin isoforms with each other. The most of FDA-approved tubulin inhibitor drugs including taxanes target only β-tubulins. The analysis also revealed sex tubulin-interacting neighbor proteins including ENCCT3, NEK2, PFDN2, PTP4A3, SDCCAG8 and TBCE which were altered in at least 20% of the tumors. Three of them are tubulin-specific chaperons responsible for tubulin protein folding. Expression of tubulin genes in BC cell lines were correlated with H3K4me3 enrichment on their promoter chromatin. Analyzing expression profile of BC tumors and tumor-adjacent normal breast tissues showed upregulation of TUBA1A, TUBA1C, TUBB and TUBB3 and downregulation of TUBB2A, TUBB2B, TUBB6, TUBB7P pseudogene, and TUBGCP2 in the tumor tissues compared to the normal breast tissues. Analyzing taxane-sensitive versus taxane-resistant tumors revealed that expression of TUBB3 and TUBB6 was significantly downregulated in the taxane-resistant tumors. Our results suggest that downregulation of tumor βIII- and βV-tubulins is correlated with taxane resistance in BC. Based on our results, we conclude that aberrant protein folding of tubulins due to mutation and/or dysfunction of tubulin-specific chaperons may be potential mechanisms of taxane resistance. Thus, we propose studying the molecular pathology of tubulin mutations and folding in BC and their impacts on taxane resistance.

Recombinant α- and β-tubulin from Echinococcus granulosus: expression, purification and polymerization.

Echinococcosis, which causes a high disease burden and is of great public health significance, is caused by the larval stage of Echinococcus species. It has been suggested that tubulin is the target of benzimidazoles, the only drugs for the treatment of echinococcosis. This study evaluated the characteristics of tubulins from Echinococcus granulosus. The full-length cDNAs of E. granulosus α- and β-tubulin isoforms were cloned by reverse transcription PCR from protoscolex RNA. Then, these two tubulin isoforms (α9 and β4) were recombinantly expressed as insoluble inclusion bodies in Escherichia coli. Nickel affinity chromatography was used to purify and refold the contents of these inclusion bodies as active proteins. The polymerization of tubulins was monitored by UV spectrophotometry (A350) and confirmed by confocal microscopy and transmission electron microscopy (TEM). Nucleotide sequence analysis revealed that E. granulosus 1356 bp α9-tubulin and 1332 bp β4-tubulin encode corresponding proteins of 451 and 443 amino acids. The average yields of α9- and β4-tubulin were 2.0–3.0 mg/L and 3.5–5.0 mg/L of culture, respectively. Moreover, recombinant α9- and β4-tubulin were capable of polymerizing into microtubule-like structures under appropriate conditions in vitro. These recombinant tubulins could be helpful for screening anti-Echinococcus compounds targeting the tubulins of E. granulosus.

Dissecting the role of the tubulin code in mitosis.

Mitosis is an essential process that takes place in all eukaryotes and involves the equal division of genetic material from a parental cell into two identical daughter cells. During mitosis, chromosome movement and segregation are orchestrated by a specialized structure known as the mitotic spindle, composed of a bipolar array of microtubules. The fundamental structure of microtubules comprises of α/β-tubulin heterodimers that associate head-to-tail and laterally to form hollow filaments. In vivo, microtubules are modified by abundant and evolutionarily conserved tubulin posttranslational modifications (PTMs), giving these filaments the potential for a wide chemical diversity. In recent years, the concept of a "tubulin code" has emerged as an extralayer of regulation governing microtubule function. A range of tubulin isoforms, each with a diverse set of PTMs, provides a readable code for microtubule motors and other microtubule-associated proteins. This chapter focuses on the complexity of tubulin PTMs with an emphasis on detyrosination and summarizes the methods currently used in our laboratory to experimentally manipulate these modifications and study their impact in mitosis.

The Depression/Schizophrenia Continuum: Does Cytoskeletal Tensegrity Play a Role?

There is mounting evidence that schizophrenia and depression are polar extremes of the same processes. The neuronal microtubule cytoskeleton is regulated through the actions of microtubule-associated proteins, the centrosome, tubulin posttranslational modifications and differential expression of tubulin isoforms. It is beginning to be thought that they may be important in the development of consciousness itself. It is proposed here that cytoskeletal tensegrity architecture plays a pivotal role in the etiology of psychiatric conditions. If the subcellular architecture is too tense, schizophrenia, if to loose, depression. This review focuses on the role of the microtubule cytoskeleton in schizophrenia, bipolar disorder and depression. Schizophrenia appears to be a disorder of microtubule architecture, depression one of intracellular transport along microtubules and bipolar disorder shares aspects of both schizophrenia and depression. Both current and potential treatments for these disorders target the microtubule cytoskeleton.

A microtubule bestiary: structural diversity in tubulin polymers.

Microtubules are long, slender polymers of αβ-tubulin found in all eukaryotic cells. Tubulins associate longitudinally to form protofilaments, and adjacent protofilaments associate laterally to form the microtubule. In the textbook view, microtubules are 1) composed of 13 protofilaments, 2) arranged in a radial array by the centrosome, and 3) built into the 9+2 axoneme. Although these canonical structures predominate in eukaryotes, microtubules with divergent protofilament numbers and higher-order microtubule assemblies have been discovered throughout the last century. Here we survey these noncanonical structures, from the 4-protofilament microtubules of Prosthecobacter to the 40-protofilament accessory microtubules of mantidfly sperm. We review the variety of protofilament numbers observed in different species, in different cells within the same species, and in different stages within the same cell. We describe the determinants of protofilament number, namely nucleation factors, tubulin isoforms, and posttranslational modifications. Finally, we speculate on the functional significance of these diverse polymers. Equipped with novel tubulin-purification tools, the field is now prepared to tackle the long-standing question of the evolutionary basis of microtubule structure.