RESUMO
Secondary wall deposition in xylem vessels is activated by Vascular-Related NAC Domain proteins (VNDs) that belong to a group of secondary wall NAC (SWN) transcription factors. By contrast, Xylem NAC Domain1 (XND1) negatively regulates secondary wall deposition in xylem vessels when overexpressed. The mechanism by which XND1 exerts its functions remains elusive. We employed the promoter of the fiber-specific Secondary Wall-Associated NAC Domain1 (SND1) gene to ectopically express XND1 in fiber cells to investigate its mechanism of action on secondary wall deposition. Ectopic expression of XND1 in fiber cells severely diminished their secondary wall deposition and drastically reduced the expression of SWN-regulated downstream transcription factors and secondary wall biosynthetic genes but not that of the SWN genes themselves. Transactivation analyses revealed that XND1 specifically inhibited SWN-activated expression of these downstream genes but not their MYB46-activated expression. Both the NAC domain and the C-terminus of XND1 were required for its inhibitory function and its NAC domain interacted with the DNA-binding domains of SWNs. XND1 was shown to be localized in the cytoplasm and the nucleus and its co-expression with VND6 resulted in the cytoplasmic sequestration of VND6. Furthermore, the C-terminus of XND1 was indispensable for the XND1-mediated cytoplasmic retention of VND6 and its fusion to VND6 was able to direct VND6 to the cytoplasm and render it unable to activate the gene expression. Since the XND1 gene is specifically expressed in xylem cells, these results indicate that XND1 acts through inhibiting VND functions to negatively regulate secondary wall deposition in xylem vessels.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Xilema/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Parede Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Fatores de Transcrição/metabolismo , Xilema/fisiologiaRESUMO
BACKGROUND: The actin cytoskeleton is involved in an array of integral structural and developmental processes throughout the cell. One of actin's best-studied binding partners is the small ubiquitously expressed protein, profilin. Arabidopsis thaliana is known to encode a family of five profilin sequence variants: three vegetative (also constitutive) profilins that are predominantly expressed in all vegetative tissues and ovules, and two reproductive profilins that are specifically expressed in pollen. This paper analyzes the roles of the three vegetative profilin members, PRF1, PRF2, and PRF3, in plant cell and organ development. RESULTS: Using a collection of knockout or severe knockdown T-DNA single mutants, we found that defects in each of the three variants gave rise to specific developmental deficiencies. Plants lacking PRF1 or PRF2 had defects in rosette leaf morphology and inflorescence stature, while those lacking PRF3 led to plants with slightly elongated petioles. To further examine these effects, double mutants and double and triple gene-silenced RNAi epialleles were created. These plants displayed significantly compounded developmental defects, as well as distinct lateral root growth morphological phenotypes. CONCLUSION: These results suggest that having at least one vegetative profilin gene is essential to viability. Evidence is presented that combinations of independent function, quantitative genetic effects, and functional redundancy have preserved the three vegetative profilin genes in the Arabidopsis lineage.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , DNA Bacteriano/genética , DNA de Plantas/genética , Organogênese Vegetal , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , DNA Bacteriano/metabolismo , DNA de Plantas/metabolismo , Técnicas de Silenciamento de Genes , Mutação , Fenótipo , Profilinas/genética , Profilinas/metabolismo , Interferência de RNARESUMO
Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.
Assuntos
Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Archamoebae/genética , Archamoebae/metabolismo , Clorófitas/genética , Clorófitas/metabolismo , Coanoflagelados/genética , Coanoflagelados/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , FilogeniaRESUMO
The mechanisms leading to changes in mesoscale chromatin organization during cellular aging are unknown. Here, we used transcriptional activator-like effectors, RNA-seq and superresolution analysis to determine the effects of genotoxic stress on oocyte chromatin structure. Major satellites are organized into tightly packed globular structures that coalesce into chromocenters and dynamically associate with the nucleolus. Acute irradiation significantly enhanced chromocenter mobility in transcriptionally inactive oocytes. In transcriptionally active oocytes, irradiation induced a striking unfolding of satellite chromatin fibers and enhanced the expression of transcripts required for protection from oxidative stress (Fermt1, Smg1), recovery from DNA damage (Tlk2, Rad54l) and regulation of heterochromatin assembly (Zfp296, Ski-oncogene). Non-irradiated, senescent oocytes exhibit not only high chromocenter mobility and satellite distension but also a high frequency of extra chromosomal satellite DNA. Notably, analysis of biological aging using an oocyte-specific RNA clock revealed cellular communication, posttranslational protein modifications, chromatin and histone dynamics as the top cellular processes that are dysregulated in both senescent and irradiated oocytes. Our results indicate that unfolding of heterochromatin fibers following acute genotoxic stress or cellular aging induced the formation of distended satellites and that abnormal chromatin structure together with increased chromocenter mobility leads to chromosome instability in senescent oocytes.
Assuntos
Heterocromatina , Oócitos , Animais , Heterocromatina/genética , Heterocromatina/metabolismo , Cromatina/genética , Cromatina/metabolismo , Histonas/metabolismo , Montagem e Desmontagem da Cromatina , Mamíferos/genéticaRESUMO
Actin-related protein 5 (ARP5) is a conserved subunit of the INO80 chromatin-remodeling complex in yeast and mammals. We have characterized the expression and subcellular distribution of Arabidopsis thaliana ARP5 and explored its role in the epigenetic control of multicellular development and DNA repair. ARP5-specific monoclonal antibodies localized ARP5 protein to the nucleoplasm of interphase cells in Arabidopsis and Nicotiana tabacum. ARP5 promoter-reporter fusions and the ARP5 protein are ubiquitously expressed. A null mutant and a severe knockdown allele produced moderately dwarfed plants with all organs smaller than the wild type. The small and slightly deformed organs such as leaves and hypocotyls were composed of small-sized cells. The ratio of leaf stomata to epidermal cells was high in the mutant, which also exhibited a delayed stomatal development compared with the wild type. Mutant plants were hypersensitive to DNA-damaging reagents including hydroxyurea, methylmethane sulfonate, and bleocin, demonstrating a role for ARP5 in DNA repair. Interestingly, the hypersensitivity phenotype of ARP5 null allele arp5-1 is stronger than the severe knockdown allele arp5-2. Moreover, a wild-type transgene fully complemented all developmental and DNA repair mutant phenotypes. Despite the common participation of both ARP4 and ARP5 in the INO80 complex, ARP4- and ARP5-deficient plants displayed only a small subset of common phenotypes and each displayed novel phenotypes, suggesting that in Arabidopsis they have both shared and unique functions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Reparo do DNA , Regulação da Expressão Gênica de Plantas , Proteínas dos Microfilamentos/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Bleomicina/farmacologia , Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Técnicas de Silenciamento de Genes , Genes Reporter , Teste de Complementação Genética , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Proteínas dos Microfilamentos/genética , Mutagênicos/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fenótipo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Plântula/anatomia & histologia , Plântula/efeitos dos fármacos , Plântula/metabolismo , Nicotiana/citologia , Nicotiana/metabolismo , TransgenesRESUMO
Centromeres are epigenetically determined nuclear domains strictly required for chromosome segregation and genome stability. However, the mechanisms regulating centromere and kinetochore chromatin modifications are not known. Here, we demonstrate that LSH is enriched at meiotic kinetochores and its targeted deletion induces centromere instability and abnormal chromosome segregation. Superresolution chromatin analysis resolves LSH at the inner centromere and kinetochores during oocyte meiosis. LSH knockout pachytene oocytes exhibit reduced HDAC2 and DNMT-1. Notably, mutant oocytes show a striking increase in histone H3 phosphorylation at threonine 3 (H3T3ph) and accumulation of major satellite transcripts in both prophase-I and metaphase-I chromosomes. Moreover, knockout oocytes exhibit centromere fusions, ectopic kinetochore formation and abnormal exchange of chromatin fibers between paired bivalents and asynapsed chromosomes. Our results indicate that loss of LSH affects the levels and chromosomal localization of H3T3ph and provide evidence that, by maintaining transcriptionally repressive heterochromatin, LSH may be essential to prevent deleterious meiotic recombination events at repetitive centromeric sequences.
Assuntos
DNA Helicases/metabolismo , Meiose/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Animais , Centrômero/genética , Centrômero/metabolismo , DNA Helicases/deficiência , DNA Helicases/genética , Feminino , Histonas/metabolismo , Cinetocoros/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Transcrição GênicaRESUMO
Arabidopsis encodes six nuclear actin-related proteins (ARPs), among them ARP8 is unique in having an F-box domain and an actin homology domain. Analysis of the ARP8 promoter-beta-glucuronidase (GUS) fusion suggests that ARP8 is ubiquitously expressed in all organs and cell types. Immunocytochemical analysis with ARP8-specific monoclonal antibodies revealed that ARP8 protein is localized to the nucleolus in interphase cells and dispersed in the cytoplasm in mitotic cells. The cell cycle-dependent subcellular patterns of distribution of ARP8 are conserved in other members of Brassicaceae. Our findings provide the first insight into the possible contributions of plant ARP8 to nucleolar functions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Nucléolo Celular/metabolismo , Proteínas F-Box/metabolismo , Especificidade de Anticorpos , Proteínas de Arabidopsis/genética , Brassica/metabolismo , Proteínas F-Box/genética , Genes Reporter , Transporte ProteicoRESUMO
Plants encode at least two ancient and divergent classes of actin, reproductive and vegetative, and each class produces several subclasses of actin isovariants. To gain insight into the functional significance of the actin isovariants, we generated transgenic Arabidopsis lines that expressed a reproductive actin, ACT1, under the control of the regulatory sequences of a vegetative actin gene, ACT2. In the wild-type plants, ACT1 is predominantly expressed in the mature pollen, growing pollen tubes, and ovules, whereas ACT2 is constitutively and strongly expressed in all vegetative tissues and organs, but not in pollen. Misexpression of ACT1 in vegetative tissues causes dwarfing of plants and altered morphology of most organs, and the effects are in direct proportion to protein expression levels. Similar overexpression of ACT2 has little effect. Immunolocalization of actin in leaf cells from transgenic plants with highest levels of ACT1 protein revealed massive polymerization, bundling, and reorganization of actin filaments. This phenomenon suggests that misexpression of ACT1 isovariant in vegetative tissues affects the dynamics of actin and actin-associated proteins, in turn disrupting the organization of actin cytoskeleton and normal development of plants.
Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Actinas/imunologia , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Western Blotting , Imunofluorescência , Microscopia Confocal , Microscopia Eletrônica de Varredura , Fenótipo , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Distribuição TecidualRESUMO
Actin-related proteins (ARPs) constitute a family of divergent and evolutionarily ancient eukaryotic proteins whose primary sequences display homology to conventional actins. Whereas actins play well-characterized cytoskeletal roles, the ARPs are implicated in various cellular functions in both the cytoplasm and in the nucleus. Cytoplasmic ARPs, for example, are known to participate in the assembly of branched actin filaments and dynein-mediated movement of vesicles in many eukaryotes. Nuclear ARPs, by contrast, are enigmatic components of various chromatin-modifying complexes involved in transcriptional regulation. Here, we review homologs to several known classes of ARPs and two distinct ARP classes in plants, and summarize recent work elucidating the biological functions of ARPs in eukaryotes.
Assuntos
Actinas/metabolismo , Proteínas de Plantas/metabolismo , Actinas/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Evolução Molecular , FilogeniaRESUMO
BACKGROUND: Obesity-related comorbidities are thought to result from the reprogramming of the epigenome in numerous tissues and cell types, and in particular, mature adipocytes within visceral and subcutaneous adipose tissue, VAT and SAT. The cell-type specific chromatin remodeling of mature adipocytes within VAT and SAT is poorly understood, in part, because of the difficulties of isolating and manipulating large fragile mature adipocyte cells from adipose tissues. METHODS: We constructed MA-INTACT (Mature Adipocyte-Isolation of Nuclei TAgged in specific Cell Types) mice using the adiponectin (ADIPOQ) promoter (ADNp) to tag the surface of mature adipocyte nuclei with a reporter protein. The SUN1mRFP1Flag reporter is comprised of a fragment of the nuclear transmembrane protein SUN1, the fluorescent protein mRFP1, and three copies of the Flag epitope tag. RESULTS: Mature adipocyte nuclei were rapidly and efficiently immuno-captured from VAT and SAT (MVA and MSA nuclei, respectively), of MA-INTACT mice. MVA and MSA nuclei contained 1,000 to 10,000-fold higher levels of adipocyte-specific transcripts, ADIPOQ, PPARg2, EDNRB, and LEP, relative to uncaptured nuclei, while the latter expressed higher levels of leukocyte and endothelial cell markers IKZF1, RETN, SERPINF1, SERPINE1, ILF3, and TNFA. MVA and MSA nuclei differentially expressed several factors linked to adipogenesis or obesity-related health risks including CEBPA, KLF2, RETN, SERPINE1, and TNFA. The various nuclear populations dramatically differentially expressed transcripts encoding chromatin remodeler proteins regulating DNA cytosine methylation and hydroxymethylation (TETs, DNMTs, TDG, GADD45s) and nucleosomal histone modification (ARID1A, KAT2B, KDM4A, PRMT1, PRMT5, PAXIP1). Remarkably, MSA and MVA nuclei expressed 200 to 1000-fold higher levels of thermogenic marker transcripts PRDM16 and UCP1. CONCLUSIONS: The MA-INTACT mouse enables a simple way to perform cell-type specific analysis of highly purified mature adipocyte nuclei from VAT and SAT and increases the statistical significance of data collected on adipocytes. Isolated VAT and SAT adipocyte nuclei expressed distinct patterns of transcripts encoding chromatin remodeling factors and proteins relevant to diabetes, cardiovascular disease, and thermogenesis. The MA-INTACT mouse is an useful model to test the impact of caloric intake, dietary nutrients, exercise, and pharmaceuticals on the epigenome-induced health risks of obesity.
RESUMO
Actin interacts with a wide variety of cytoplasmic and nuclear proteins to support spatial development in nearly all eukaryotes. Null mutations in plant vegetative actins produce dramatically altered cell, tissue, and organ morphologies. Animal cytoplasmic actins (e.g., human HsACTB, HsACTG1) and some ancestral protist actins fully suppress these mutant phenotypes suggesting that some animal, plant, and protist actins share functional competence for spatial development. Considering that fungi have a phylogenetic origin closer to animals than plants, we were interested to explore whether the fungal actins may have this same capacity to function in plants and support development. We ectopically expressed actins from four highly divergent ascomycete fungi in two different Arabidopsis double vegetative actin null mutants. We found that expression of actin from the earliest diverging ascomycete subphyla, the archiascomycete Schizosaccharomyces pombe, qualitatively and quantitatively suppressed the root cell polarity and root organ developmental defects of act8/act7 mutants and the root-hairless cell elongation phenotype of act2/act8 mutants. Interestingly, the actin from the pyrenomycete Neurospora crassa was modestly effective in the suppression of vegetative actin mutant phenotypes. In contrast, actins from the saccharomycetes Saccharomyces cerevisiae and Candida albicans were unable to support any aspect of plant development, and moreover induced severe dwarfism and sterility. These data imply that basal fungi inherited an actin with full competence for spatial development from their protist ancestor and maintained it via non-progressive sequence evolution, while the later more derived fungal species lost these activities.
Assuntos
Actinas/fisiologia , Arabidopsis/crescimento & desenvolvimento , Ascomicetos/fisiologia , Actinas/genética , Actinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , TransgenesRESUMO
Conventional actin has been implicated in various nuclear processes including chromatin remodeling, transcription, nuclear transport, and overall nuclear structure. Moreover, actin has been identified as a component of several chromatin remodeling complexes present in the nucleus. In animal cells, nuclear actin exists as a dynamic equilibrium of monomers and polymers. Actin-binding proteins (ABPs) such as ADF/cofilin and profilin play a role in actin import and export, respectively. However, very little is known about the localization and roles of nuclear actin in plants. In multicellular plants and animals, actin is comprised of an ancient and divergent family of protein variants. Here, we have investigated the presence and differential localization of two ancient subclasses of actin in isolated Arabidopsis nuclei. Although the subclass 1 variants ACT2 and ACT8 and subclass 2 variant ACT7 were found distributed throughout the nucleoplasm, ACT7 was often found more concentrated in nuclear speckles than subclass 1 variants. The nuclei from the act2-1/act8-2 double null mutant and the act7-5 null mutant lacked their corresponding actin variants. In addition, serial sectioning of several independent nuclei revealed that ACT7 was notably more abundant in the nucleolus than the subclass 1 actins. Profilin and ADF proteins were also found in significant levels in plant nuclei. The possible functions of differentially localized nuclear actin variants are discussed.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/metabolismo , Actinas/classificação , Actinas/genética , Sequência de Aminoácidos , Animais , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Destrina/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Sinais de Localização Nuclear , Filogenia , Proteínas de Plantas/genética , Profilinas/metabolismo , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genéticaRESUMO
Nuclear Actin-Related Proteins (ARPs) and actin combine as heterodimers to bind a large helicase subunit and form a core complex essential to the assembly and function of most chromatin remodeling and modifying machines. They are the most common shared subunits of these large and diverse assemblies in eukaryotes. We recently argued that most nuclear ARPs evolved directly from actin prior to the divergence of the eukaryotic kingdoms and did not evolve from pre-existing ARPs.2 Arabidopsis plants defective in nuclear ARP4, ARP5, ARP6, or ARP7 have extreme developmental phenotypes. Our recent publication demonstrates that ARP5-defective plants are not only dwarfed and have aberrant cell sizes, but are also hypersensitive to mutagenic agents that cause double strand DNA breaks.5 In Smith et al.6 we show that ARP6-defective plants, in addition to their extreme developmental phenotypes like small organs and early flowering, present an apparent "Phosphate Starvation Response" with strong morphological and molecular phenotypes. Herein, we interpret our latest data in the light of a hypothesis stating that in addition to their roles in overcoming DNA compaction that affects basal gene expression and silencing, nuclear ARP-containing chromatin complexes exert primary epigenetic control over high-level regulatory factors.
Assuntos
Actinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Epigênese Genética , Proteínas Nucleares/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Epigênese Genética/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Fenótipo , Fosfatos/farmacologia , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimentoRESUMO
The nuclear actin-related proteins (ARPs) share overall structure and low-level sequence homology with conventional actin. They are indispensable subunits of macromolecular machines that control chromatin remodeling and modification leading to dynamic changes in DNA structure, transcription, and DNA repair. Cellular, genetic, and biochemical studies suggest that the nuclear ARPs are essential to the epigenetic control of the cell cycle and cell proliferation in all eukaryotes, while in plants and animals they also exert epigenetic controls over most stages of multicellular development including organ initiation, the switch to reproductive development, and senescence and programmed cell death. A theme emerging from plants and animals is that in addition to their role in controlling the general compaction of DNA and gene silencing, isoforms of nuclear ARP-containing chromatin complexes have evolved to exert dynamic epigenetic control over gene expression and different phases of multicellular development. Herein, we explore this theme by examining nuclear ARP phylogeny, activities of ARP-containing chromatin remodeling complexes that lead to epigenetic control, expanding developmental roles assigned to several animal and plant ARP-containing complexes, the evidence that thousands of ARP complex isoforms may have evolved in concert with multicellular development, and ARPs in human disease.
Assuntos
Actinas/metabolismo , Epigênese Genética , Proteínas Nucleares/metabolismo , Actinas/química , Actinas/genética , Animais , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genéticaRESUMO
The relative significance of gene regulation and protein isovariant differences remains unexplored for most gene families, particularly those participating in multicellular development. Arabidopsis thaliana encodes three vegetative actins, ACT2, ACT7, and ACT8, in two ancient and highly divergent subclasses. Mutations in any of these differentially expressed actins revealed only mild phenotypes. However, double mutants were extremely dwarfed, with altered cell and organ morphology and an aberrant F-actin cytoskeleton (e.g., act2-1 act7-4 and act8-2 act7-4) or totally root-hairless (e.g., act2-1 act8-2). Our studies suggest that the three vegetative actin genes and protein isovariants play distinct subclass-specific roles during plant morphogenesis. For example, during root development, ACT7 was involved in root growth, epidermal cell specification, cell division, and root architecture, and ACT2 and ACT8 were essential for root hair tip growth. Also, genetic complementation revealed that the ACT2 and ACT8 isovariants, but not ACT7, fully rescued the root hair growth defects of single and double mutants. Moreover, we synthesized fully normal plants overexpressing the ACT8 isovariant from multiple actin regulatory sequences as the only vegetative actin in the act2-1 act7-4 background. In summary, it is evident that differences in vegetative actin gene regulation and the diversity in actin isovariant sequences are essential for normal plant development.
Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Isoformas de Proteínas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Actinas/classificação , Actinas/genética , Arabidopsis/anatomia & histologia , Proteínas de Arabidopsis/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Morfogênese , Mutação , Fenótipo , Filogenia , Raízes de Plantas/fisiologia , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igalpha and Igbeta of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igalpha expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).
Assuntos
Anticorpos Monoclonais/biossíntese , Hibridomas/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Fusão Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Hibridomas/imunologia , Camundongos , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
The macroevolution of organs and tissues in higher plants and animals may have been contingent upon the expansion of numerous gene families encoding interacting proteins. For example, there are dozens of gene families encoding actin cytoskeletal proteins that elaborate intercellular structures influencing development. Once gene family members evolve compartmentalized expression, protein isovariants are free to coevolve new interacting partners that may be incompatible with other related protein networks. Ancient classes of actin isovariants and actin-binding proteins are clear examples of such coevolving networks. Ectopic expression and suppression studies were used to dissect these interactions. In higher plants, the ectopic expression of a reproductive actin isovariant in vegetative cell types causes aberrant reorganization of the F-actin cytoskeleton and bizarre development of most organs and tissues. In contrast, overexpression of vegetative actin in vegetative cell types has little effect. The extreme ectopic actin expression phenotypes are suppressed by the coectopic expression of reproductive profilin or actin depolymerizing factor (ADF/cofilin) isovariants, but not by the overexpression of vegetative profilin or ADF. These data provide evidence for the coevolution of organ-specific protein-protein interactions. Thus, understanding the contingent relationships between the evolution of organ-specific isovariant networks and organ origination may be key to explaining multicellular development.
RESUMO
The Actin Depolymerizing Factor (ADF) gene family of Arabidopsis thaliana encodes 11 functional protein isovariants in four ancient subclasses. We report the characterization of the tissue-specific and developmental expression of all Arabidopsis ADF genes and the subcellular localization of several protein isovariants. The four subclasses exhibited distinct expression patterns as examined by qRT-PCR and histochemical assays of a GUS reporter gene under the control of individual ADF regulatory sequences. Subclass I ADFs were expressed strongly and constitutively in all vegetative and reproductive tissues except pollen. Subclass II ADFs were expressed specifically in mature pollen and pollen tubes or root epidermal trichoblast cells and root hairs, and these patterns evolved from an ancient dual expression pattern comprised of both polar tip growth cell types, still observed in the monocot Oryza sativa. Subclass III ADFs were expressed weakly in vegetative tissues, but were strongest in fast growing and/or differentiating cells including callus, emerging leaves, and meristem regions. The single subclass IV ADF was constitutively expressed at moderate levels in all tissues, including pollen. Immunocytochemical analysis with subclass-specific monoclonal antibodies demonstrated that subclass I isovariants localize to both the cytoplasm and the nucleus of leaf cells, while subclass II isovariants predominantly localize to the cytoplasm at the tip region of elongating root hairs and pollen tubes. The distinct expression patterns of the ADF subclasses support a model of ADF s co-evolving with the ancient and divergent actin isovariants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Destrina/genética , Arabidopsis/classificação , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Destrina/química , Destrina/isolamento & purificação , Destrina/metabolismo , Regulação da Expressão Gênica de Plantas , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Pólen/genética , Pólen/metabolismo , Alinhamento de SequênciaRESUMO
Two ancient and highly divergent actin-based cytoskeletal systems have evolved in angiosperms. Plant genomes encode complex actin and actin binding protein (ABP) gene families, most of which are phylogenetically grouped into gene classes with distinct vegetative or constitutive and reproductive expression patterns. In Arabidopsis thaliana, ectopic expression of high levels of a reproductive class actin, ACT1, in vegetative tissues causes severe dwarfing of plants with aberrant organization of most plant organs and cell types due to a severely altered actin cytoskeletal architecture. Overexpression of the vegetative class actin ACT2 to similar levels, however, produces insignificant phenotypic changes. We proposed that the misexpression of the pollen-specific ACT1 in vegetative cell types affects the dynamics of actin due to its inappropriate interaction with endogenous vegetative ABPs. To examine the functionally distinct interactions among the major classes of actins and ABPs, we ectopically coexpressed reproductive profilin (PRF4) or actin-depolymerizing factor (ADF) isovariants (e.g., ADF7) with ACT1. Our results demonstrated that the coexpression of these reproductive, but not vegetative, ABP isovariants suppressed the ectopic ACT1 expression phenotypes and restored wild-type stature and normal actin cytoskeletal architecture to the double transgenic plants. Thus, the actins and ABPs appear to have evolved class-specific, protein-protein interactions that are essential to the normal regulation of plant growth and development.
Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Destrina/metabolismo , Profilinas/metabolismo , Actinas/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Western Blotting , Citoesqueleto/metabolismo , Destrina/genética , Imunofluorescência , Regulação da Expressão Gênica de Plantas , Microscopia Confocal , Fenótipo , Filogenia , Plantas Geneticamente Modificadas , Profilinas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Leveduras/genética , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismoRESUMO
Regulating developmental transitions, cell proliferation and cell death through differential gene expression is essential to the ontogeny of all multicellular organisms. Chromatin remodeling is an active process that is necessary for managing the genome-wide suppression of gene activities resulting from DNA compaction. Recent data in plants suggest a general theme, whereby chromatin remodeling complexes containing nuclear actin-related proteins (ARPs) potentiate the activities of crucial regulatory genes involved in plant growth and development, in addition to their basal activities on a much larger set of genes.