Tubulin Mutations Research Articles

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.

Tubulin mutations and ophthalmic manifestations: a pediatric case series

Tubulinopathies are a wide and overlapping range of brain malformations caused by six known gene mutations of different tubulin isotypes. Brain abnormalities include a range of lissencephalies and polymicrogyria-like cortical dysplasias with additional structural cerebral anomalies. Ocular findings involve associated cortical visual impairment, Congenital fibrosis of extraocular muscles (CFEOM) and associated ptosis, strabismus, and less commonly optic nerve hypoplasia. We present an observational case series of children diagnosed with a tubulinopathy, with emphasis on ophthalmic manifestations.

Control of Growth Cone Polarity, Microtubule Accumulation, and Protrusion by UNC-6/Netrin and Its Receptors in Caenorhabditis elegans.

UNC-6/Netrin has a conserved role in dorsal-ventral axon guidance, but the cellular events in the growth cone regulated by UNC-6/Netrin signaling during outgrowth are incompletely understood. Previous studies showed that, in growth cones migrating away from UNC-6/Netrin, the receptor UNC-5 regulates growth cone polarity, as observed by polarized F-actin, and limits the extent of growth cone protrusion. It is unclear how UNC-5 inhibits protrusion, and how UNC-40 acts in concert with UNC-5 to regulate polarity and protrusion. New results reported here indicate that UNC-5 normally restricts microtubule (MT) + end accumulation in the growth cone. Tubulin mutant analysis and colchicine treatment suggest that stable MTs are necessary for robust growth cone protrusion. Thus, UNC-5 might inhibit protrusion in part by restricting growth cone MT accumulation. Previous studies showed that the UNC-73/Trio Rac GEF and UNC-33/CRMP act downstream of UNC-5 in protrusion. Here, we show that UNC-33/CRMP regulates both growth cone dorsal asymmetric F-actin accumulation and MT accumulation, whereas UNC-73/Trio Rac GEF activity only affects F-actin accumulation. This suggests an MT-independent mechanism used by UNC-5 to inhibit protrusion, possibly by regulating lamellipodial and filopodial actin. Furthermore, we show that UNC-6/Netrin and the receptor UNC-40/DCC are required for excess protrusion in unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of, or in parallel to, F-actin asymmetry and MT + end entry. F-actin accumulation might represent a polarity mark in the growth cone where protrusion will occur, and not protrusive lamellipodial and filopodial actin per se Our data suggest a model in which UNC-6/Netrin first polarizes the growth cone via UNC-5, and then regulates protrusion based upon this polarity (the polarity/protrusion model). UNC-6/Netrin inhibits protrusion ventrally via UNC-5, and stimulates protrusion dorsally via UNC-40, resulting in dorsally-directed migration. The polarity/protrusion model represents a novel conceptual paradigm in which to understand axon guidance and growth cone migration away from UNC-6/Netrin.

Regulation of microtubule dynamic instability by the carboxy-terminal tail of β-tubulin.

Dynamic instability is an intrinsic property of microtubules; however, we do not understand what domains of αβ-tubulins regulate this activity or how these regulate microtubule networks in cells. Here, we define a role for the negatively charged carboxy-terminal tail (CTT) domain of β-tubulin in regulating dynamic instability. By combining in vitro studies with purified mammalian tubulin and in vivo studies with tubulin mutants in budding yeast, we demonstrate that β-tubulin CTT inhibits microtubule stability and regulates the structure and stability of microtubule plus ends. Tubulin that lacks β-tubulin CTT polymerizes faster and depolymerizes slower in vitro and forms microtubules that are more prone to catastrophe. The ends of these microtubules exhibit a more blunted morphology and rapidly switch to disassembly after tubulin depletion. In addition, we show that β-tubulin CTT is required for magnesium cations to promote depolymerization. We propose that β-tubulin CTT regulates the assembly of stable microtubule ends and provides a tunable mechanism to coordinate dynamic instability with ionic strength in the cell.

The α-Tubulin gene TUBA1A in Brain Development: A Key Ingredient in the Neuronal Isotype Blend.

Microtubules are dynamic cytoskeletal polymers that mediate numerous, essential functions such as axon and dendrite growth and neuron migration throughout brain development. In recent years, sequencing has revealed dominant mutations that disrupt the tubulin protein building blocks of microtubules. These tubulin mutations lead to a spectrum of devastating brain malformations, complex neurological and physical phenotypes, and even fatality. The most common tubulin gene mutated is the α-tubulin gene TUBA1A, which is the most prevalent α-tubulin gene expressed in post-mitotic neurons. The normal role of TUBA1A during neuronal maturation, and how mutations alter its function to produce the phenotypes observed in patients, remains unclear. This review synthesizes current knowledge of TUBA1A function and expression during brain development, and the brain malformations caused by mutations in TUBA1A.

Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans.

Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the Caenorhabditis elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.

Abstract 4169: A novel, short isoform of the +Tip microtubule binding protein CLIP170 confers taxane resistance in gastric cancer

Abstract The microtubule (MT) cytoskeleton is one of the most validated therapeutic targets in clinical oncology, as evidenced by the wide clinical activity of taxanes and other MT inhibitors. However, patients with intrinsic drug resistance do not benefit from taxane chemotherapy, such as patients with diffuse gastric cancer (GC). Currently, the molecular mechanisms underlying clinical taxane resistance remain poorly elucidated. Using a panel of GC cell lines with intrinsic sensitivity or resistance to taxanes, we showed lack of drug-target engagement in the resistant lines, despite unimpaired intracellular accumulation of the drug and in the absence of tubulin mutations or altered tubulin isotype expression. Herein, we show for the first time that the resistant cells expressed a novel, short isoform of the MT plus-end binding protein, CLIP170 (CLIP1). CLIP1 binds to the plus-ends of microtubules (+TIPs) and regulates dynein-mediated MT-based trafficking and the tubulin tyrosination cycle. We showed by both mass-spec proteomics and 5’-RACE that the short CLIP1 isoform (CLIP1S) lacks the first 150 amino acids, thus, missing the first Cap-Gly (Cytoskeleton-Associated Protein-Glycine) domain, which is required for proper +TIP localization. Indeed, confocal microscopy experiments showed that CLIP1S was mislocalized to the microtubule lattice in contrast to the canonical comet-like pattern of CLIP1 seen in taxane sensitive cell lines. CLIP1S expression was specifically correlated with taxane resistance (docetaxel, cabazitaxel) as no correlation was observed with other DNA-damaging agents. Since, CLIP1S expression has never been reported before, to establish causation, we stably knocked down CLIP1 and CLIP1S in taxane-sensitive and resistant cells, respectively. CLIP1SKD entirely reversed taxane-resistance (~300 fold) while CLIP1KD had no effect in the taxane-sensitive cell lines. These data suggest a gain-of-function of CLIP1S that leads to taxane resistance. To study the binding kinetics of taxanes to MTs in the presence or absence of CLIP1S we performed live cell imaging of native cytoskeletons using fluorescently-labeled paclitaxel (Flutax). Our data revealed significantly faster dissociation rates of Flutax from MTs in the resistant cells, indicating transient interaction with MTs. Taxane binding to MTs is a two-step process. First taxanes bind to the MT-pore low affinity surface site, which then facilitates access to the high affinity luminal site. Using a small molecule that binds only to MT-pore site, we showed that we showed that taxol binding to the pore is significantly slower in resistant cells expressing CLIP1S, which together with mislocalization of this variant along the MT lattice suggests that it obstructs access to the MT-pore thus restricting entry of taxane into the lumen of microtubules. This finding will have profound implications for taxane resistance as well as microtubule biology broadly. Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Isabel Barasoain, Jose Fernando Diaz, Manish A. Shah, Paraskevi Giannakakou. A novel, short isoform of the +Tip microtubule binding protein CLIP170 confers taxane resistance in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2017-4169

Towards an understanding of the isotype-specific functions of tubulin in neurons: Technical advances in tubulin expression and purification

Microtubules are cytoskeletal filaments critical for determining the complex morphology of neurons, as well as the basic architecture and organization of mitosis in all eukaryotic cells. Microtubules in humans are composed of 8 α- and 9 β-tubulin isotypes, each of which is encoded by different members of a multi-gene family. The expression pattern of tubulin isotypes, in addition to isotype-specific post-translational modifications, is thought to be critical for the morphogenesis of axons and dendrites. Recent studies revealed that several neurodevelopmental disorders are caused by mutations of specific tubulin isotypes, suggesting that each tubulin isotype has distinct functions. Therefore, in vitro and in vivo functional analyses of tubulin isotypes are important to understand the pathogenesis of developmental disorders. Likewise, analysis of developmental disorders may clarify the function of different tubulin isotypes. In this respect, both the preparation of specific tubulin isotypes and of specific mutant tubulin proteins is critical to understanding the function of tubulin. In the last 20 years, various methods have been developed to study functional differences between tubulin isotypes and the functional defects caused by tubulin mutations. These technical achievements have been discussed in this review. The function of tubulin/microtubules in neuronal morphogenesis as revealed through these techniques has also been described.

Abstract 2932: Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer

Abstract Successful clinical application of taxanes (microtubule-stabilizers) is limited due to intrinsic or acquired drug resistance. Thus, it is critical to unveil the molecular mechanisms of taxane resistance to significantly improve clinical outcomes. Retrospective analysis of the TAX-325 gastric cancer (GC) trial revealed that the addition of docetaxel (DTX) to cisplatin/fluorouracil increased progression-free plus overall survival, in intestinal (INT) but not in diffuse (DIF) GC subtypes. Our preclinical data confirmed that DIF GC cell lines are intrinsically resistant to taxanes. The incidence of DTX resistance in DIF GC cell lines was 2.5 times higher than INT GC cell lines. Drug efflux, tubulin posttranslational modification and differential β-tubulin isotype expression were ruled out as potential mechanisms of intrinsic taxane resistance. Thus, a new molecular mechanism must underlie the intrinsic taxane resistance in DIF GC. To quantify the kinetics of taxol binding to cellular MTs, we treated both DIF and INT groups of GC lines with the fluorescein-conjugated paclitaxel analog, Flutax-2. The Flutax-2 staining intensity of cellular MTs was assessed by live-cell confocal microscopy at different time points. Following a 3h incubation, there was less than 20% decrease in Flutax-2 intensity in the sensitive cell lines, compared to 59∼89% decrease in the resistant cell lines. These data suggested different binding kinetics between sensitive and resistant cells. We added 1μM Flutax-2 to sensitive/resistant cells’ native cytoskeletons for 0∼60 seconds to determine the association rate (kon) of Flutax-2 binding to MTs. The kon of Flutax-2 in the sensitive cell lines was significantly higher in sensitive (5.8×104M−1s−1) versus resistant cells (0.3∼2.6×104M−1s−1). Next, we measured the dissociation rates (koff) by competing the pre-bound Flutax-2 with DTX (0∼600 seconds). Although 20∼40% faster koff was observed in resistant cells, it appeared that the association rate of Flutax-2 was the dominant factor of differential taxane binding to MTs in GC cells. The binding mode of taxanes to MTs involves 1) binding to MT pores and 2) internalization to the high-affinity binding site at the MT lumen. To differentiate between the two, we used hexaflutax, which binds exclusively to the MT pores. We observed hexaflutax decorating radial MT arrays in sensitive but not in the resistant cells. As tubulin mutations around the high-affinity taxane binding site or tubulin posttranslational modifications/differential expression of β-tubulin isotype is not the case in DIF GC cell lines, our data suggest that modifications of the MT pore conformation or occlusion of the pore site is responsible for the intrinsic taxane resistance in DIF GC. Determining the origin of the defect at the pores will help design better MT-stabilizing drugs to overcome chemo-resistance, the major obstacle hindering overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2932.

Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells

The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated bytubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cellsand describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains[4], bind tubulin efficiently, as does the Golgi-associatedprotein GCC185, which interacts with the CLASPs [5]. The N-terminal TOGL domain of CLASP1 contributes to tubulin binding and allows CLASP1 to function as an autonomous MT-growth-promoting factor. Interestingly, mutant tubulins bind less well to a number of TAPs, including CLASPs and GCC185, and incorporate less efficiently into cellular MTs. Moreover, expression of these mutants in cells impairs several MT-growth-related processes involving TAPs. Thus, stable tubulin-TAP interactions regulate MT nucleation and growth in cells. Combined, our results provide a resource for investigating tubulin interactions and functions and widen the spectrum of tubulin-related disease mechanisms.

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation.

Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.

De novo TUBB2B mutation causes fetal akinesia deformation sequence with microlissencephaly: An unusual presentation of tubulinopathy

Tubulinopathies are increasingly emerging major causes underlying complex cerebral malformations, particularly in case of microlissencephaly often associated with hypoplastic or absent corticospinal tracts. Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement.We report on an early foetal case with FADS and microlissencephaly due to TUBB2B mutation. Neuropathological examination disclosed virtually absent cortical lamination, foci of neuronal overmigration into the leptomeningeal spaces, corpus callosum agenesis, cerebellar and brainstem hypoplasia and extremely severe hypoplasia of the spinal cord with no anterior and posterior horns and almost no motoneurons.At the cellular level, the p.Cys239Phe TUBB2B mutant leads to tubulin heterodimerization impairment, decreased ability to incorporate into the cytoskeleton, microtubule dynamics alteration, with an accelerated rate of depolymerization.To our knowledge, this is the first case of microlissencephaly to be reported presenting with a so severe and early form of FADS, highlighting the importance of tubulin mutation screening in the context of FADS with microlissencephaly.

Disorders of Microtubule Function in Neurons: Imaging Correlates.

A number of recent studies have described malformations of cortical development with mutations of components of microtubules and microtubule-associated proteins. Despite examinations of a large number of MRIs, good phenotype-genotype correlations have been elusive. Additionally, most of these studies focused exclusively on cerebral cortical findings. The purpose of this study was to characterize imaging findings associated with disorders of microtubule function. MRIs from 18 patients with confirmed tubulin mutations (8 TUBA1A, 5 TUBB2B, and 5 TUBB3) and 15 patients with known mutations of the genes encoding microtubule-associated proteins (5 LIS1, 4 DCX, and 6 DYNC1H1) were carefully visually analyzed and compared. Specific note was made of the cortical gyral pattern, basal ganglia, and white matter to assess internal capsular size, cortical thickness, ventricular and cisternal size, and the size and contours of the brain stem, cerebellar hemispheres and vermis, and the corpus callosum of patients with tubulin and microtubule-associated protein gene mutations. Results were determined by unanimous consensus of the authors. All patients had abnormal findings on MR imaging. A large number of patients with tubulin gene mutations were found to have multiple cortical and subcortical abnormalities, including microcephaly, ventriculomegaly, abnormal gyral and sulcal patterns (termed "dysgyria"), a small or absent corpus callosum, and a small pons. All patients with microtubule-associated protein mutations also had abnormal cerebral cortices (predominantly pachygyria and agyria), but fewer subcortical abnormalities were noted. Comparison of MRIs from patients with known mutations of tubulin genes and microtubule-associated proteins allows the establishment of some early correlations of phenotype with genotype and may assist in identification and diagnosis of these rare disorders.

Abstract 3600: Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation

Abstract The taxanes (i.e., paclitaxel, docetaxel (DTX) and cabazitaxel) are microtubule (MT)-stabilizing drugs widely used to treat solid tumor malignancies. Their success is limited by the presence of intrinsic or acquired drug resistance. Understanding the molecular mechanisms of taxane resistance is a key to significantly improve clinical outcomes of taxane-based chemotherapy. In gastric cancer (GC), retrospective analysis of the TAX-325 trial revealed that the addition of docetaxel (DTX) to standard cisplatin/fluorouracil increased progression-free and overall survival, primarily only in intestinal subtype (INT) GC, suggesting that diffuse (DIF) GC may be intrinsically taxane resistant. In fact, our previous data supported this hypothesis; DIF GC cell lines had higher incidence (63%) of DTX resistance than INT GC cell lines (25%). Flow cytometric analyses showed negligible P-glycoprotein expression on the cell surface of all cell types, and accumulation of C14-labeled DTX in cells was observed in both sensitive and resistant GC cell lines. These results ruled out drug efflux as a possible mechanism that confers taxane resistance in DIF GC cells. Next, we used fluorescein-conjugated paclitaxel (Flutax) as a probe to investigate the affinity of taxanes to MTs. Live cell imaging showed strong binding of Flutax to MTs in a sensitive cell line at least for 60 minutes. In contrast, Flutax failed to stay bound to MTs in resistant GC cell lines,. In addition, discontinuous decoration of MTs with Flutax was seen in resistant but not in sensitive cell lines. Our observation suggests that MTs of resistant GC cell lines have lower affinity for taxanes, and there might be subtle structural changes of MTs. We are currently conducting the competition of Flutax/DTX to quantify the affinity of DTX in both types of GC cell lines. Because tubulin mutations have been reported in many taxane-resistant cell lines, we sequenced the different tubulin isotypes in GC cell lines using both Sanger and next generation sequencing. No mutations were identified. In addition, we did not see any significant changes in the expression of βIII-tubulin or tubulin post-translational modifications between the sensitive and resistant cell lines. Pathway analyses on the RNA-Seq datasets derived from the panels of sensitive and resistant cell lines before and after taxane treatment is ongoing to identify the key molecular events underlying taxane resistance.Taken together, our data indicate that new molecular pathway(s) or change of overall MT structure and/or function may contribute to the impaired taxane binding to MTs in resistant GC cells. Our study will lead us to novel mechanistic insights in intrinsic drug resistance, and will ultimately allow us to target key molecule(s) to overcome chemo-resistance, the key determinant to improve overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Prashant V. Thakkar, Ada Gjyrezi, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2015-3600

Structural basis for drug resistance conferred by β-tubulin mutations: a molecular modeling study on native and mutated tubulin complexes with epothilone B

Using molecular modeling, we have investigated the structure and dynamic properties of epothilone B–tubulin complexes with wild-type and mutated tubulin, aimed at identifying the molecular factors involved in the emergence of drug resistance induced by four protein mutations at Phe270Val, Thr274Ile, Arg282Gln, and Gln292Glu. Our results revealed that tubulin mutations render significant changes in the protein conformation in regions involved either in the binding of the ligand or in interdimer contacts that are relevant to the assembly of stable microtubules. In addition, point mutations induce drastic changes in the binding pose of the ligand and in the interaction networks responsible for the epothilone–tubulin association. Large ligand displacements inside the binding pocket and an overall decrease in the strength of drug-receptor polar contacts suggest a looser binding of the ligand in tubulin mutants. These results explain the loss of activity for epothilone B against cancer cells that contain tubulin mutants and provide valuable information to enhance the understanding of the atomic source of epothilones’ activity, which can be helpful to conduct further research on the rational design of more potent therapeutic tubulin-binding agents.

Recognizable cerebellar dysplasia associated with mutations in multiple tubulin genes.

Mutations in alpha- and beta-tubulins are increasingly recognized as a major cause of malformations of cortical development (MCD), typically lissencephaly, pachygyria and polymicrogyria; however, sequencing tubulin genes in large cohorts of MCD patients has detected tubulin mutations in only 1-13%. We identified patients with a highly characteristic cerebellar dysplasia but without lissencephaly, pachygyria and polymicrogyria typically associated with tubulin mutations. Remarkably, in seven of nine patients (78%), targeted sequencing revealed mutations in three different tubulin genes (TUBA1A, TUBB2Band TUBB3), occurring de novo or inherited from a mosaic parent. Careful re-review of the cortical phenotype on brain imaging revealed only an irregular pattern of gyri and sulci, for which we propose the term tubulinopathy-related dysgyria. Basal ganglia (100%) and brainstem dysplasia (80%) were common features. On the basis of in silico structural predictions, the mutations affect amino acids in diverse regions of the alpha-/beta-tubulin heterodimer, including the nucleotide binding pocket. Cell-based assays of tubulin dynamics reveal various effects of the mutations on incorporation into microtubules: TUBB3 p.Glu288Lys and p.Pro357Leu do not incorporate into microtubules at all, whereas TUBB2B p.Gly13Ala shows reduced incorporation and TUBA1A p.Arg214His incorporates fully, but at a slower rate than wild-type. The broad range of effects on microtubule incorporation is at odds with the highly stereotypical clinical phenotype, supporting differential roles for the three tubulin genes involved. Identifying this highly characteristic phenotype is important due to the low recurrence risk compared with the other (recessive) cerebellar dysplasias and the apparent lack of non-neurological medical issues.

Characterization of tub4(P287L) , a β-tubulin mutant, revealed new aspects of microtubule regulation in shade.

When sun plants, such as Arabidopsis thaliana, are under canopy shade, elongation of stems/petioles will be induced as one of the most prominent responses. Plant hormones mediate the elongation growth. However, how environmental and hormonal signals are translated into cell expansion activity that leads to the elongation growth remains elusive. Through forward genetic study, we identified shade avoidance2 (sav2) mutant, which contains a P287L mutation in β‐TUBULIN 4. Cortical microtubules (cMTs) play a key role in anisotropic cell growth. Hypocotyls of sav2 are wild type‐like in white light, but are short and highly swollen in shade and dark. We showed that shade not only induces cMT rearrangement, but also affects cMT stability and dynamics of plus ends. Even though auxin and brassinosteroids are required for shade‐induced hypocotyl elongation, they had little effect on shade‐induced rearrangement of cMTs. Blocking auxin transport suppressed dark phenotypes of sav2, while overexpressing EB1b‐GFP, a microtubule plus‐end binding protein, rescued sav2 in both shade and dark, suggesting that tub4P287L represents a unique type of tubulin mutation that does not affect cMT function in supporting cell elongation, but may affect the ability of cMTs to respond properly to growth promoting stimuli.

βI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug–tubulin interactions and microtubule stability

Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to β-tubulin at a site that is different from the classical taxoid site. Multiple βI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M βI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian βI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug–tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on β-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel directions for rational structure-based design of new and more potent agents for cancer treatment that target the LAU/PLA site.

Macrocyclic lactones and their relationship to the SNPs related to benzimidazole resistance

Haemonchus contortus is an abomasal nematode of ruminants that is widely present across the world. Its ability to cause death of infected animals and rapidly develop anthelmintic resistance makes it a dangerous pathogen. Ivermectin (IVM) and moxidectin (MOX) are macrocyclic lactones (MLs). They have been successfully used to treat parasitic nematodes over the last three decades. A genetic association between IVM selection and single nucleotide polymorphisms (SNPs) on the β-tubulin isotype 1 gene was reported in H. contortus. These SNPs result in replacing phenylalanine (F, TTC) with tyrosine (Y, TAC) at position 167 or 200 on the β-tubulin protein. Recently we reported a direct interaction of IVM with α- and β-tubulin. It had been hypothesized that the SNPs (F167Y and F200Y) may change tubulin dynamics and directly affect IVM binding. The goal of the current study was to observe the effects of SNPs (F167Y and F200Y) on tubulin polymerization and IVM binding. It was also of interest to evaluate the differences between IVM and MOX on tubulin polymerization. We conclude that the SNPs cause no difference in the polymerization of wild and mutant tubulins. Furthermore, neither of the SNPs reduced IVM binding. Varying results were obtained in the degree of polymerization of parasitic and mammalian tubulin for IVM and MOX, i.e., the extent of polymerization was greater for IVM compared with MOX, for H. contortus tubulin, and vice versa for mammalian tubulin. Molecular modeling showed that IVM and MOX docked into the taxane binding pocket of both mammalian and parasitic wild type and mutant tubulins. However the binding was stronger for mammalian tubulin as compared to parasitic tubulin.

A Mechanical Switch from Diffusion to Directional Motion Activates ATPase in Dynein Motor

Dynein is a motor protein that moves along microtubule tracks via the energy from ATP hydrolysis. Unlike other processive cytoskeletal motors, the dynein step size is highly variable with a significant level of diffusion. To investigate the molecular basis of the stochastic nature of dynein stepping, we here characterized the structure, physical properties, and effects of site-directed mutations of the dynein-microtubule interface.We found that mutation of either the R403 or E416 residue of α-tubulin to alanine changed the directional movement of the microtubules on a dynein-coated surface to undirected thermal diffusion, resulting in a loss of dynein ATPase activity. Biochemical and cryo-electron microscopy analyses of the microtubule binding domain (MTBD)-microtubule complex revealed that these tubulin residues switch dynein from diffusional to stationary binding by forming salt bridges with the residue in H1 and H6 of the MTBD. The formation of two salt bridges then triggers a registry change in the stalk coiled coil required for ATPase activation, and thus leads to directional movement. In this mechanism, the previously undescribed interaction between α-R403 and E3390 in H1 of the MTBD plays a key role, and is likely to explain the fact that the equivalent tubulin mutation in mammals (R402) can cause lissencephaly (Keays et al., Cell 128, 45-).Compared to kinesin-microtubule interactions, where the weak-to-strong state transition is mediated by several contact sites involving a few tubulin residues (Uchimura et al., EMBO. J., 29, 1167-), for dynein, the mechanical switch from diffusional to stationary binding is controlled by only two salt bridges. Because of this pinpoint regulation, the stepping motion of dynein might be only loosely coupled with the reaction of ATP hydrolysis, resulting in the variable step size.