RESUMO
Cilia are microtubule-based hair-like structures that project from the surfaces of eukaryotic cells. Cilium formation relies on intraflagellar transport (IFT) to move ciliary proteins such as tubulin from the site of synthesis in the cell body to the site of function in the cilium. A large protein complex (the IFT complex) is believed to mediate interactions between cargoes and the molecular motors that walk along axonemal microtubules between the ciliary base and tip. A recent study using purified IFT complexes has identified a tubulin-binding module in the two core IFT proteins IFT74 and IFT81 that likely serves to bind and transport tubulin within cilia. Here, we calculate the amount of tubulin required to support the observed cilium assembly kinetics and explore the possibility of multiple tubulin binding sites within the IFT complex.
Assuntos
Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Tubulina (Proteína)/químicaRESUMO
Mutations in Hydin cause hydrocephalus in mice, and HYDIN is a strong candidate for causing hydrocephalus in humans. The gene is conserved in ciliated species, including Chlamydomonas reinhardtii. An antibody raised against C. reinhardtii hydin was specific for an approximately 540-kD flagellar protein that is missing from axonemes of strains that lack the central pair (CP). The antibody specifically decorated the C2 microtubule of the CP apparatus. An 80% knock down of hydin resulted in short flagella lacking the C2b projection of the C2 microtubule; the flagella were arrested at the switch points between the effective and recovery strokes. Biochemical analyses revealed that hydin interacts with the CP proteins CPC1 and kinesin-like protein 1 (KLP1). In conclusion, C. reinhardtii hydin is a CP protein required for flagellar motility and probably involved in the CP-radial spoke control pathway that regulates dynein arm activity. Hydrocephalus caused by mutations in hydin likely involves the malfunctioning of cilia because of a defect in the CP.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Flagelos/metabolismo , Locomoção/fisiologia , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Chlamydomonas reinhardtii/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/fisiologia , Flagelos/ultraestrutura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrocefalia/genética , Hidrocefalia/metabolismo , Hidrocefalia/fisiopatologia , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mutação/genética , Proteínas de Protozoários/genéticaRESUMO
Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagellar proteins. The genes encoding nine of these not previously characterized plus the previously described PACRG protein were cloned, inserted into a vector adding a triple-HA tag to the C-terminus of the gene product, and transformed into C. reinhardtii. Expression was confirmed by western blotting. Indirect immunofluorescence located all 10 fusion proteins in the flagellum; PACRG was localized to a subset of outer doublet microtubules. For some proteins, additional signal was observed in the cell body. Among the latter was FAP232-HA, which showed a spotted distribution along the flagella and an accumulation at the basal bodies. This pattern is characteristic for intraflagellar transport (IFT) proteins. FAP232-HA co-localized with the IFT protein IFT46 and co-sedimented with IFT particles in sucrose gradients. Furthermore, it co-immunoprecipitated with IFT complex B protein IFT46, but not with IFT complex A protein IFT139. We conclude that FAP232 is a novel component of IFT complex B and rename it IFT25. Homologues of IFT25 are encoded in the genomes of a subset of organisms that assemble cilia or flagella; C. reinhardtii IFT25 is 37% identical to the corresponding human protein. Genes encoding IFT25 homologues are absent from the genomes of organisms that lack cilia and flagella and, interestingly, also from those of Drosophila melanogaster and Caenorhabditis elegans, suggesting that IFT25 has a specialized role in IFT that is not required for the assembly of cilia or flagella in the worm and fly. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Flagelos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Chlamydomonas reinhardtii/genética , Técnica Indireta de Fluorescência para Anticorpo , Imunoprecipitação , Modelos Genéticos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de AminoácidosRESUMO
During ciliogenesis, centrioles convert to membrane-docked basal bodies, which initiate the formation of cilia/flagella and template the nine doublet microtubules of the flagellar axoneme. The discovery that many human diseases and developmental disorders result from defects in flagella has fueled a strong interest in the analysis of flagellar assembly. Here, we will review the structure, function, and development of basal bodies in the unicellular green alga Chlamydomonas reinhardtii, a widely used model for the analysis of basal bodies and flagella. Intraflagellar transport (IFT), a flagella-specific protein shuttle critical for ciliogenesis, was first described in C. reinhardtii. A focus of this review will be on the role of the basal bodies in organizing the IFT machinery.
RESUMO
Bardet-Biedl syndrome (BBS) is a rare inherited disease caused by defects in the BBSome, an octameric complex of BBS proteins. The BBSome is conserved in most organisms with cilia, which are microtubule (MT)-based cell organelles that protrude from the cell surface and function in motility and sensing. Cilia assembly, maintenance, and function require intraflagellar transport (IFT), a bidirectional motility of multi-megadalton IFT trains propelled by molecular motors along the ciliary MTs. IFT has been shown to transport structural proteins, including tubulin, into growing cilia. The BBSome is an adapter for the transport of ciliary membrane proteins and cycles through cilia via IFT. While both the loss and the abnormal accumulation of ciliary membrane proteins have been observed in bbs mutants, recent data converge on a model where the BBSome mainly functions as a cargo adapter for the removal of certain transmembrane and peripheral membrane proteins from cilia. Here, we review recent data on the ultrastructure of the BBSome and how the BBSome recognizes its cargoes and mediates their removal from cilia.
Assuntos
Síndrome de Bardet-Biedl/fisiopatologia , Membrana Celular/metabolismo , Cílios/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Animais , Transporte Biológico , HumanosRESUMO
The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Centrossomo/metabolismo , Proteínas do Citoesqueleto , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteínas de Ancoragem à Quinase A , Antígenos/metabolismo , Fracionamento Celular , Clonagem Molecular , Ácidos Graxos Insaturados/farmacologia , Imunofluorescência , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Microscopia Imunoeletrônica , Modelos Moleculares , Transporte Proteico , Combinação Trimetoprima e Sulfametoxazol/metabolismo , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Most motile cilia and flagella have nine outer doublet and two central pair (CP) microtubules. Outer doublet microtubules are continuous with the triplet microtubules of the basal body, are templated by the basal body microtubules, and grow by addition of new subunits to their distal ("plus") ends. In contrast, CP microtubules are not continuous with basal body microtubules, raising the question of how these microtubules are assembled and how their polarity is established. METHODS: CP assembly in Chlamydomonas reinhardtii was analyzed by electron microscopy and wide-field and super-resolution immunofluorescence microscopy. To analyze CP assembly independently from flagellar assembly, the CP-deficient katanin mutants pf15 or pf19 were mated to wild-type cells. HA-tagged tubulin and the CP-specific protein hydin were used as markers to analyze de novo CP assembly inside the formerly mutant flagella. RESULTS: In regenerating flagella, the CP and its projections assemble near the transition zone soon after the onset of outer doublet elongation. During de novo CP assembly in full-length flagella, the nascent CP was first apparent in a subdistal region of the flagellum. The developing CP replaces a fibrous core that fills the axonemal lumen of CP-deficient flagella. The fibrous core contains proteins normally associated with the C1 CP microtubule and proteins involved in intraflagellar transport (IFT). In flagella of the radial spoke-deficient mutant pf14, two pairs of CPs are frequently present with identical correct polarities. CONCLUSIONS: The temporal separation of flagellar and CP assembly in dikaryons formed by mating CP-deficient gametes to wild-type gametes revealed that the formation of the CP does not require proximity to the basal body or transition zone, or to the flagellar tip. The observations on pf14 provide further support that the CP self-assembles without a template and eliminate the possibility that CP polarity is established by interaction with axonemal radial spokes. Polarity of the developing CP may be determined by the proximal-to-distal gradient of precursor molecules. IFT proteins accumulate in flagella of CP mutants; the abnormal distribution of IFT proteins may explain why these flagella are often shorter than normal.
RESUMO
Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule-membrane links there. Ultrastructural analysis revealed defects in these microtubule-membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule-membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.
Assuntos
Estruturas Celulares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Animais , Transporte Biológico , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/fisiologia , Cílios/metabolismo , Flagelos/metabolismo , Flagelos/fisiologia , Flagelos/ultraestrutura , Deleção de Genes , Membranas/metabolismo , Microscopia Imunoeletrônica , Proteínas Associadas aos Microtúbulos/genética , MutaçãoAssuntos
Apicomplexa , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/química , Proteínas de Protozoários/análise , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Apicomplexa/química , Apicomplexa/classificação , Sequência de Bases , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Clonagem Molecular , Primers do DNA/genética , Proteínas Associadas aos Microtúbulos/classificação , Dados de Sequência Molecular , Filogenia , Proteínas de Protozoários/classificação , Toxoplasma/química , Toxoplasma/genética , Toxoplasma/ultraestruturaRESUMO
The development and health of mammals requires proper ciliary motility. Ciliated epithelia are found in the airways, the uterus and Fallopian tubes, the efferent ducts of the testes, and the ventricular system of the brain. A technique is described for the motion analysis of ependymal cilia in the murine brain. Vibratome sections of the brain are imaged by differential interference contrast microscopy and recorded by high-speed digital imaging. Side views of individual cilia are traced to establish their bending pattern. Tracking of individual cilia recorded in top view allows determination of bend planarity and beat direction. Ciliary beat frequency is determined from line scans of image sequences. The capacity of the epithelium to move fluid and objects is revealed by analyzing the velocity of polystyrene beads added to brain sections. The technique is useful for detailed assessment of how various conditions or mutations affect the fidelity of ciliary motility at the ependyma. The methods are also applicable to other ciliated epithelia, for example, in airways.
Assuntos
Encéfalo/citologia , Cílios , Epêndima/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Animais , Cílios/metabolismo , Cílios/ultraestrutura , Processamento de Imagem Assistida por Computador/instrumentação , Camundongos , MovimentoRESUMO
The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualize the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies.
Assuntos
Chlamydomonas reinhardtii/citologia , Flagelos/ultraestrutura , Microscopia de Fluorescência/métodos , Animais , Axonema/ultraestrutura , Transporte Biológico/fisiologia , Chlamydomonas reinhardtii/metabolismo , Flagelos/metabolismo , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência/instrumentação , Proteínas Motores Moleculares/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.
Assuntos
Proteínas de Algas/fisiologia , Chlamydomonas reinhardtii/metabolismo , Flagelos/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Axonema/genética , Axonema/fisiologia , Chlamydomonas reinhardtii/genética , Transporte Proteico/fisiologia , Transdução de SinaisRESUMO
Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility, and mice with Hydin defects develop lethal hydrocephalus. To determine if defects in Hydin cause hydrocephalus through a mechanism involving cilia, we compared the morphology, ultrastructure, and activity of cilia in wild-type and hydin mutant mice strains. The length and density of cilia in the brains of mutant animals is normal. The ciliary axoneme is normal with respect to the 9 + 2 microtubules, dynein arms, and radial spokes but one of the two central microtubules lacks a specific projection. The hydin mutant cilia are unable to bend normally, ciliary beat frequency is reduced, and the cilia tend to stall. As a result, these cilia are incapable of generating fluid flow. Similar defects are observed for cilia in trachea. We conclude that hydrocephalus in hydin mutants is caused by a central pair defect impairing ciliary motility and fluid transport in the brain.
Assuntos
Movimento Celular/genética , Ventrículos Cerebrais/metabolismo , Cílios/metabolismo , Epêndima/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Ventrículos Cerebrais/fisiopatologia , Ventrículos Cerebrais/ultraestrutura , Líquido Cefalorraquidiano/metabolismo , Cílios/ultraestrutura , Epêndima/anormalidades , Epêndima/ultraestrutura , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Predisposição Genética para Doença/genética , Hidrocefalia/genética , Hidrocefalia/metabolismo , Hidrocefalia/fisiopatologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica de Transmissão , Mutação/genética , Mucosa Respiratória/metabolismo , Mucosa Respiratória/fisiopatologia , Mucosa Respiratória/ultraestrutura , Traqueia/metabolismo , Traqueia/fisiopatologia , Traqueia/ultraestruturaRESUMO
An inverted repeat corresponding to parts of the centrin gene of Chlamydomonas reinhardtii was placed downstream of the NIT1 promoter, which is induced by ammonium starvation. After induction, transformants developed centrin deficiency as assayed by immunofluorescence, Western blotting, and Northern blotting. The effect was reversible, demonstrating that the NIT1 promoter allowed controlled RNA interference in Chlamydomonas reinhardtii.
Assuntos
Aminoidrolases/genética , Chlamydomonas reinhardtii/genética , Inativação Gênica , Regiões Promotoras Genéticas , Combinação Trimetoprima e Sulfametoxazol/metabolismo , Aminoidrolases/metabolismo , Animais , Chlamydomonas reinhardtii/metabolismo , Interferência de RNARESUMO
Green fluorescent protein (GFP) was used to analyse three proteins in the flagellar basal apparatus of C. reinhardtii: (1) Striated fiber assemblin (SFA), the major component of the striated microtubule-associated fibers; (2) Centrin, present in the nucleus basal body connectors (NBBCs) and the distal connecting fiber (dCF) between the two basal bodies; and (3) DIP13, the Chlamydomonas homologue of human autoantigen NA14. The fusions co-localized with the wild-type proteins when expressed moderately. Overexpression of centrin-GFP and DIP13-GFP resulted in the formation of large aggregates and disturbed the distribution of the respective wild-type proteins. The amount of wild-type DIP13 was significantly reduced in cells overexpressing DIP13-GFP. Moreover, the cells frequently failed to assemble full-length flagella and flagellar regeneration was delayed, indicating a role of DIP13 during flagellar assembly. In contrast, overexpression of GFP-SFA, which retained more wild-type properties than SFA-GFP, increased the size of the striated fibers without altering the cross-shaped pattern. Abnormal patterns were observed in centrin-deficient cells, suggesting that centrin is required for proper localization of SFA. Photobleaching of GFP-SFA fibers indicated that GFP-SFA in the fibers is turned over slowly. Conditionally expressed centrin-GFP was incorporated into NBBCs in regions close to the basal bodies, but underrepresented in the dCF, indicative of a different dynamic of these two centrin fibers. Bending of the NBBCs was observed in vivo during flagellar motion, indicating that the filaments are flexible. In conclusion, in Chlamydomonas GFP-tagging is a useful tool for yielding new insights into the function and properties of the analyzed proteins.
Assuntos
Proteínas de Algas/biossíntese , Proteínas de Ligação ao Cálcio/biossíntese , Chlamydomonas reinhardtii/ultraestrutura , Proteínas Cromossômicas não Histona/biossíntese , Flagelos/fisiologia , Proteínas de Fluorescência Verde , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Chlamydomonas reinhardtii/metabolismo , Flagelos/ultraestrutura , Regeneração/fisiologia , SolubilidadeRESUMO
Centrin, a 20 kDa calcium-binding protein, is a constituent of contractile basal body-associated fibers in protists and of various centrosomal structures. A construct inducing centrin RNAi was used to study the effect of centrin deficiency in Chlamydomonas. Transformants contained variable amounts of residual centrin (down to 5% of wild-type) and lacked centrin fibers. They displayed a variable flagellar number phenotype with mostly nonflagellate cells, suggesting that centrin is required for basal body assembly. Furthermore, basal bodies often failed to dock to the plasma membrane and to assemble flagella, and displayed defects in the flagellar root system indicating that centrin deficiency interferes with basal body development. Multiple basal bodies caused the formation of additional microtubular asters, whereas the microtubular cytoskeleton was disordered in most cells without basal bodies. The number of multinucleated cells was increased, indicating that aberrant numbers of basal bodies interfered with the cytokinesis of Chlamydomonas. In contrast to wild-type cells, basal bodies in centrin-RNAi cells were separated from the spindle poles, suggesting a role of centrin in tethering basal bodies to the spindle. To test whether an association with the spindle poles is required for correct basal body segregation, we disrupted centrin fibers in wild-type cells by over-expressing a nonfunctional centrin-GFP. In these cells, basal bodies were disconnected from the spindle but segregation errors were not observed. We propose that basal body segregation in Chlamydomonas depends on an extranuclear array of microtubules independent of the mitotic spindle.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Diferenciação Celular/fisiologia , Centríolos/metabolismo , Chlamydomonas/metabolismo , Proteínas Cromossômicas não Histona/deficiência , Flagelos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Divisão Celular/fisiologia , Centríolos/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Chlamydomonas/citologia , Proteínas Cromossômicas não Histona/genética , Flagelos/ultraestrutura , Microscopia Eletrônica , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Interferência de RNA/fisiologia , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
Striated fiber assemblin (SF-assemblin or SFA) is the major component of the striated microtubule-associated fibers (SMAFs) in the flagellar basal apparatus of green flagellates. We generated nuclear transformants of Chlamydomonas expressing green fluorescent protein (GFP) fused to the C-terminus of SFA. SFA-GFP assembled into striated fibers that exceeded those of wild-type cells in size by several fold. At elevated temperatures (>or=32 degrees C) SFA-GFP was mostly soluble and heat shock depolymerized the SMAFs. C-terminal deletions of 18 or only six residues disturbed the ability of SFA-GFP to polymerize, indicating an important role of the C-terminal domain for fiber formation. The exchange of the penultimate Ser275 with alanine made SFA-GFP highly insoluble, causing aberrant fiber formation and conferring heat stability to the fibers. By contrast, a replacement with glutamic acid increased the solubilty of the molecule, indicating that phosphorylation on Ser275 might control solubility of SFA. In vivo observation of GFP fluorescence showed that SFA-GFP fibers were disassembled during mitosis. In cells overexpressing full-length or truncated SFA-GFP, the amount of wild-type protein was reduced. Elevated temperatures dissolved SFA-GFP fibers and induced the synthesis of SFA, suggesting that cells control both the amount of soluble and polymeric SFA. By expressing constructs consisting of cDNA and genomic DNA for parts of SFA in antiparallel configuration, the amount of SFA was severely reduced. In these strains we observed defects in flagellar assembly, indicating an important role for noncontractile striated roots in the flagella apparatus.
Assuntos
Chlamydomonas reinhardtii/ultraestrutura , Proteínas Luminescentes/genética , Proteínas Associadas aos Microtúbulos/análise , Proteínas de Protozoários/análise , Sequência de Aminoácidos , Animais , Ciclo Celular , Células Cultivadas , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , DNA Antissenso/metabolismo , DNA Antissenso/farmacologia , Proteínas de Fluorescência Verde , Temperatura Alta , Indicadores e Reagentes/análise , Proteínas Luminescentes/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/ultraestrutura , Modelos Genéticos , Dados de Sequência Molecular , Proteínas de Protozoários/genética , RNA/metabolismo , Proteínas Recombinantes de Fusão/análise , Análise de Sequência de DNA , Fatores de TempoRESUMO
The posttranslational modifications of alpha-tubulin of Toxoplasma gondii were characterized by antibodies and biochemical analysis of the carboxy-terminal peptide. Alpha-Tubulin is acetylated and glutamylated. Side chains with up to three glutamate residues are linked to Glu445 of T. gondii alpha-tubulin. The data suggest that the site of glutamylation on alpha-tubulin is conserved over a broad range of species.