RESUMO
Microtubules form a major cytoskeleton and exhibit dynamic instability through the repetitive polymerization/depolymerization of tubulin dimers. Although microtubule stability should be precisely controlled to maintain various cellular functions, it has been difficult to assess its status in vivo. Here, we propose a tubulin fractionation method reflecting the stability of microtubules in mouse tissues. Analyses of tubulin fractionated by two-step of ultracentrifugation demonstrated three distinct pools of tubulin, that appeared to be stable microtubule, labile microtubule, and free tubulin. Using this method, we were able to show the specific binding of different microtubule-associated proteins onto each pool of microtubules. Also, there were clear differences in the population of stable microtubule among tissues depending on the proliferative capacity of the constituent cells. These findings indicate that this method is useful for broad analysis of microtubule stability in physiological and pathological conditions.
Assuntos
Microtúbulos/metabolismo , Animais , Encéfalo/metabolismo , Química Encefálica , Fracionamento Celular , Feminino , Masculino , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Tubulina (Proteína)/análise , Tubulina (Proteína)/isolamento & purificação , UltracentrifugaçãoRESUMO
Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and ß-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The ß-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the ß-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.
Assuntos
Babesia/genética , Babesiose/epidemiologia , Cervos/parasitologia , Theileria/genética , Theileriose/epidemiologia , Tubulina (Proteína)/isolamento & purificação , Animais , Animais Selvagens , Babesia/classificação , Babesia/isolamento & purificação , Babesiose/parasitologia , Japão/epidemiologia , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Theileria/classificação , Theileria/isolamento & purificação , Theileriose/parasitologia , Tubulina (Proteína)/classificação , Tubulina (Proteína)/genéticaRESUMO
Cytoplasmic extracts from unfertilized Xenopus eggs have made important contributions to our understanding of microtubule dynamics, spindle assembly, and scaling. Until recently, these in vitro studies relied on the use of heterologous tubulin. This protocol allows for the purification of physiologically relevant Xenopus tubulins in milligram yield, which are a complex mixture of isoforms with various post-translational modifications. The protocol is applicable to any cell or tissue of interest. For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020).
Assuntos
Extratos Celulares/química , Cromatografia de Afinidade/métodos , Óvulo/citologia , Coloração e Rotulagem , Tubulina (Proteína)/isolamento & purificação , Xenopus/metabolismo , Animais , Feminino , Domínios Proteicos , Tubulina (Proteína)/químicaRESUMO
One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The "tubulin code" is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs. To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps. We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.
Assuntos
Polimerização , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Animais , Encéfalo/metabolismo , Adesão Celular , Densitometria , Células HEK293 , Células HeLa , Humanos , Camundongos , Microtúbulos/metabolismo , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
In the plant cytoskeleton research, mammalian brain tubulin has been widely used to study plant microtubule-interacting proteins in vitro since purification of tubulins from plant sources is generally considered to be challenging and time-consuming. A convenient method for affinity purification of tubulins was devised, which utilized the TOG domains of yeast Stu2 tubulin-binding protein as an affinity ligand (Widlund et al., 2012). We showed that this so-called TOG tubulin affinity chromatography worked efficiently with plant materials, especially actively-dividing cultured cells (Hotta et al., 2016). Plant tubulins purified with the TOG method is highly assembly-competent and thus can be used in various in vitro experiments. Here, we summarize purification strategies of native or tagged plant tubulins as well as an in vitro pull-down assay to monitor their polymerization activity.
Assuntos
Arabidopsis/metabolismo , Cromatografia de Afinidade/métodos , Proteínas de Plantas/isolamento & purificação , Tubulina (Proteína)/isolamento & purificação , Sequência de Aminoácidos , Arabidopsis/genética , Histidina/metabolismo , Oligopeptídeos/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/metabolismo , Tubulina (Proteína)/químicaRESUMO
Studies reported that Δ9-tetrahydrocannabinol (Δ9-THC) is an essential drug as an anti-cancer, neuroprotective, anti-inflammatory, and immune-modulatory agent. However, the mechanism by which Δ9-THC causes these events remains to be elucidated. We attempted to investigate the in vivo studies of Δ9-THC on brain microtubule dynamicity, and acetylcholinesterase (AChE) activity. The microtubule polymerization, secondary and tertiary structures of α/ß-tubulins, as well as the AChE activity, were evaluated in the experimental groups. The significantly lowest optical density and initial rate of polymerization was observed in THC 3 mg/kg, THC 9 mg/kg, and THC 18 mg/kg treated groups. The content of secondary and tertiary structures of α/ß-tubulins was significantly affected in treated groups. The AChE activity was significantly lower in treated groups in a dose-dependent manner. These data highlight the microtubule dynamicity as a molecular target for Δ9-THC, which affects memory dysfunction. However, Δ9-THC can be inhibited the AChE activity and provide an improved therapeutics for neurodegenerative diseases.
Assuntos
Dronabinol/farmacologia , Microtúbulos/efeitos dos fármacos , Acetilcolinesterase/efeitos dos fármacos , Animais , Inibidores da Colinesterase/farmacologia , Dicroísmo Circular , Relação Dose-Resposta a Droga , Polimerização , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Tubulina (Proteína)/química , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
α/ß-tubulin heterodimers, which can harbor diverse isotypes and post-translational modifications, polymerize into microtubules that are fundamental to many cellular processes. Due to long-standing challenges in generating recombinant tubulin, however, it has been difficult to examine the properties of specific tubulin isotypes. Here, we provide a protocol for purifying milligrams of affinity tag-free, isotypically pure recombinant tubulin. Our method can be applicable to any tubulin of interest, opening the door to dissecting how tubulin diversity regulates microtubule function. For complete details on the use and execution of this protocol, please see Ti et al. (2018).
Assuntos
Clonagem Molecular/métodos , Tubulina (Proteína)/isolamento & purificação , Animais , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Células Sf9 , Tubulina (Proteína)/genéticaRESUMO
Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends.
Assuntos
Microtúbulos/metabolismo , Modelos Biológicos , Tubulina (Proteína)/metabolismo , Animais , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Simulação de Dinâmica Molecular , Polimerização/efeitos dos fármacos , Suínos , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/ultraestrutura , Moduladores de Tubulina/farmacologiaRESUMO
Cilia and flagella play essential roles in environmental sensing, cell locomotion, and development. These organelles possess a central microtubule-based structure known as the axoneme, which serves as a scaffold and is crucial for the function of cilia. Despite their key roles, the biochemical and biophysical properties of the ciliary proteins are poorly understood. To address this issue, we have developed a novel method to purify functional tubulins from different parts of the axoneme, namely the central pair and B-tubule. We use the biflagellate green alga Chlamydomonas reinhardtii, a model organism for studying cilia due to the conserved structure of this organelle, availability of genetic tools and a large collection of mutant strains. Our method yields highly purified functional axonemal tubulins in sufficient quantities to be used for in vitro biochemical and biophysical studies, such as microtubule dynamic assays. It takes 7 to 8 days to grow enough cells; the isolation of the flagella and the purification of the axonemal tubulins require an additional two full days.© 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth and harvest of large volume of cell culture Support Protocol: Assembly of homemade concentration apparatus Basic Protocol 2: Isolation of flagella Basic Protocol 3: Tubulin extraction and purification.
Assuntos
Chlamydomonas reinhardtii/química , Cílios/química , Proteínas de Plantas/isolamento & purificação , Tubulina (Proteína)/isolamento & purificação , Proteínas de Plantas/química , Tubulina (Proteína)/químicaRESUMO
The microtubule cytoskeleton plays a crucial role in a myriad of cellular events, including mitosis, cell differentiation, migration, and the maintenance of cell shape. Microtubules are assembled from α- and ß-tubulin heterodimers, whose biosynthesis is a complex process requiring the balanced production of α- and ß-tubulin subunits. This chapter focuses on a method for the combined expression of tagged α- and ß-tubulin dimers, their purification, and the isolation of co-purifying tubulin-associated proteins (TAPs) in mammalian cells. This approach is currently used in our laboratory to study tubulin function and to identify and characterize TAPs.
Assuntos
Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Recombinantes , Tubulina (Proteína)/genética , Tubulina (Proteína)/isolamento & purificação , Animais , Linhagem Celular , Cromatografia de Afinidade , Expressão Gênica , Humanos , Proteínas Associadas aos Microtúbulos/química , Microtúbulos/metabolismo , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Transfecção , Tubulina (Proteína)/químicaRESUMO
Tau is an intrinsically disordered, microtubule-associated protein that has a role in regulating microtubule dynamics. Despite intensive research, the molecular mechanisms of Tau-mediated microtubule polymerization are poorly understood. Here we used single-molecule fluorescence to investigate the role of Tau's N-terminal domain (NTD) and proline-rich region (PRR) in regulating interactions of Tau with soluble tubulin. We assayed both full-length Tau isoforms and truncated variants for their ability to bind soluble tubulin and stimulate microtubule polymerization. We found that Tau's PRR is an independent tubulin-binding domain that has tubulin polymerization capacity. In contrast to the relatively weak interactions with tubulin mediated by sites distributed throughout Tau's microtubule-binding region (MTBR), resulting in heterogeneous Tau: tubulin complexes, the PRR bound tubulin tightly and stoichiometrically. Moreover, we demonstrate that interactions between the PRR and MTBR are reduced by the NTD through a conserved conformational ensemble. On the basis of these results, we propose that Tau's PRR can serve as a core tubulin-binding domain, whereas the MTBR enhances polymerization capacity by increasing the local tubulin concentration. Moreover, the NTD appears to negatively regulate tubulin-binding interactions of both of these domains. The findings of our study draw attention to a central role of the PRR in Tau function and provide mechanistic insight into Tau-mediated polymerization of tubulin.
Assuntos
Prolina/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Animais , Sítios de Ligação , Bovinos , Transferência Ressonante de Energia de Fluorescência , Polimerização , Tubulina (Proteína)/isolamento & purificação , Proteínas tau/genética , Proteínas tau/isolamento & purificaçãoRESUMO
In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6-7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins.
Assuntos
Processamento de Proteína Pós-Traducional/genética , Tubulina (Proteína)/química , Tubulina (Proteína)/isolamento & purificação , Animais , Reatores Biológicos , Química Encefálica , Linhagem Celular , Células HeLa , Humanos , Camundongos , Polimerização , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , UltracentrifugaçãoRESUMO
Cilia and flagella play essential roles in cell motility, sensing and development. These organelles have tightly controlled lengths, and the axoneme, which forms the core structure, has exceptionally high stability. This is despite being composed of microtubules that are often characterized as highly dynamic. To understand how ciliary tubulin contribute to stability, we develop a procedure to differentially extract tubulins from different components of axonemes purified from Chlamydomonas reinhardtii, and characterize their properties. We find that the microtubules support length stability by two distinct mechanisms: low dynamicity, and unusual stability of the protofilaments. The high stability of the protofilaments manifests itself in the formation of curved tip structures, up to a few microns long. These structures likely reflect intrinsic curvature of GTP or GDP·Pi tubulin and provide structural insights into the GTP-cap. Together, our study provides insights into growth, stability and the role of post-translational modifications of axonemal microtubules.
Assuntos
Axonema/metabolismo , Movimento Celular , Cílios/fisiologia , Proteínas de Plantas/metabolismo , Tubulina (Proteína)/metabolismo , Chlamydomonas reinhardtii , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Plantas/isolamento & purificação , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
Theaflavin (TF) and epigallocatechin-3-gallate (EGCG) both have been reported previously as microtubule depolymerizing agents that also have anticancer effects on various cancer cell lines and in animal models. Here, we have applied TF and EGCG in combination on HeLa cells to investigate if they can potentiate each other to improve their anticancer effect in lower doses and the underlying mechanism. We found that TF and EGCG acted synergistically, in lower doses, to inhibit the growth of HeLa cells. We found the combination of 50 µg/mL TF and 20 µg/mL EGCG to be the most effective combination with a combination index of 0.28. The same combination caused larger accumulation of cells in the G 2 /M phase of the cell cycle, potent mitochondrial membrane potential loss, and synergistic augmentation of apoptosis. We have shown that synergistic activity might be due to stronger microtubule depolymerization by simultaneous binding of TF and EGCG at different sites on tubulin: TF binds at vinblastine binding site on tubulin, and EGCG binds near colchicines binding site on tubulin. A detailed mechanistic analysis revealed that stronger microtubule depolymerization caused effective downregulation of PI3K/Akt signaling and potently induced mitochondrial apoptotic signals, which ultimately resulted in the apoptotic death of HeLa cells in a synergistic manner.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Biflavonoides/farmacologia , Catequina/análogos & derivados , Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Polimerização/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antioxidantes/metabolismo , Biflavonoides/metabolismo , Sítios de Ligação , Catequina/metabolismo , Catequina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Cabras , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/metabolismoRESUMO
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 ß-tubulin isoforms were cloned by reverse transcription PCR from protoscolex RNA. Then, these two tubulin isoforms (α9 and ß4) 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 ß4-tubulin encode corresponding proteins of 451 and 443 amino acids. The average yields of α9- and ß4-tubulin were 2.0-3.0 mg/L and 3.5-5.0 mg/L of culture, respectively. Moreover, recombinant α9- and ß4-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.
Assuntos
Echinococcus granulosus/genética , Polimerização , Tubulina (Proteína)/genética , Animais , Clonagem Molecular , Equinococose/parasitologia , Echinococcus granulosus/química , Expressão Gênica , Microtúbulos , Isoformas de RNA/genética , Isoformas de RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Tubulina (Proteína)/química , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/metabolismoRESUMO
Microtubules are non-covalent cylindrical polymers formed by alpha- and beta-tubulin heterodimer units, crucial for cell division, intracellular transport, motility and differentiation. This makes them very attractive pharmacological targets exploited to develop different drugs such as anthelmintics, antifungals, and antineoplastics. In this work, in order to establish an in vitro target-based screen to integrate to the search for new anthelmintics, we explored the extraction of native assembly-competent tubulin from two helminth parasites: Mesocestoides vogae tetrathyridia (syn. corti, Cestoda: Cyclophyllidea), a useful cestode biological model, and Haemonchus contortus, a sheep gastrointestinal nematode of interest in livestock production. For this purpose, a novel tubulin affinity chromatography procedure was employed, based on the binding capacity of TOG (Tumor Overexpressed Gene) domain from MAPs (microtubule-associated proteins). The TOG domain of the protein Stu2 from Saccharomyces cerevisiae fused to GST (glutathione S- transferase) were produced in E. coli, and the immobilized recombinant proteins allowed for native tubulin extraction from parasites. The binding capacity of TOG1 affinity column (3.6%) was estimated using commercial porcine brain tubulin. A total amount of up to 126 µg of M. vogae tubulin was purified, whereas H. contortus tubulin co-eluted with glutamate dehydrogenase enzyme. The identity of tubulins was confirmed by western blotting and mass spectrometry. The abundance of tubulin estimated in M. vogae was 10% soluble extract, which probably could explain differences observed between tubulin purification results of both helminth parasites.
Assuntos
Cromatografia de Afinidade/métodos , Haemonchus/química , Mesocestoides/química , Proteínas Associadas aos Microtúbulos/metabolismo , Tubulina (Proteína)/isolamento & purificação , Sequência de Aminoácidos , Animais , Infecções por Cestoides/parasitologia , Escherichia coli/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hemoncose/parasitologia , Hemoncose/veterinária , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ovinos , Doenças dos Ovinos/parasitologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMO
Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely understood. To further our understanding of the protein composition of human airway cilia, we performed a semiquantitative analysis of ciliary axonemes using label-free LC/MSE, which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence of 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the nontubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, â¼70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia-specific expression for eight uncharacterized proteins, and fluorescence microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.
Assuntos
Axonema/genética , Cílios/genética , Proteínas/genética , Proteoma/genética , Dineínas/genética , Dineínas/isolamento & purificação , Regulação da Expressão Gênica , Humanos , Proteínas/isolamento & purificação , Proteômica , Tubulina (Proteína)/genética , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
Microtubules are essential cellular structures in plant cells. They are polymerized from tubulin dimers and are regulated by microtubule-associated proteins (MAPs). Here, we describe a protocol for purifying tubulin dimers and MAPs from plant cells. The protocol involves preparing vacuole-free mini-protoplasts, a high quality cytoplasmic extract, cycles of microtubule polymerization and depolymerization to increase tubulin and MAP concentration, separation of tubulin and MAPs by column chromatography. We also present tubulin purification methods for biochemical assays.
Assuntos
Arabidopsis/química , Fracionamento Celular/métodos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Nicotiana/química , Proteínas de Plantas/isolamento & purificação , Protoplastos/química , Técnicas de Cultura de Células , Fracionamento Celular/instrumentação , Centrifugação com Gradiente de Concentração/instrumentação , Centrifugação com Gradiente de Concentração/métodos , Cromatografia por Troca Iônica , Meios de Cultura/química , Eletroforese em Gel de Poliacrilamida , Proteínas Associadas aos Microtúbulos/química , Microtúbulos , Células Vegetais/química , Proteínas de Plantas/química , Povidona/química , Multimerização Proteica , Dióxido de Silício/química , Tubulina (Proteína)/química , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation playing important functions in various biological processes. By using MALDI-ICR-mass-spectrometry and Western-blot analysis, we have shown the presence of shortened isoform of a-tubulin in complex with the affinity-purified proteasomes from stable cell lines K562 and HEK293.
Assuntos
Complexo de Endopeptidases do Proteassoma , Tubulina (Proteína) , Células HEK293 , Humanos , Células K562 , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tubulina (Proteína)/química , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/metabolismoRESUMO
For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog egg tubulin incorporates into meiotic spindles assembled in egg extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus egg extract experiments. Frog egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro.