RESUMO
The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.
Assuntos
Ciclo Celular , Núcleo Celular/metabolismo , Proteínas Contráteis/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Tradescantia/metabolismo , Actinas/metabolismo , Animais , Núcleo Celular/ultraestrutura , Células Cultivadas , Galinhas , Proteínas Contráteis/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Células Ciliadas Auditivas/ultraestrutura , Masculino , Proteínas dos Microfilamentos/ultraestrutura , Microinjeções , Microscopia Confocal , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/ultraestrutura , Pólen/metabolismo , Profilinas , Tradescantia/ultraestruturaRESUMO
Actin microfilaments (MFs) are essential for the growth of the pollen tube. Although it is well known that MFs, together with myosin, deliver the vesicles required for cell elongation, it is becoming evident that the polymerization of new actin MFs, in a process that is independent of actomyosin-dependent vesicle translocation, is also necessary for cell elongation. Herein we review the recent literature that focuses on this subject, including brief discussions of the actin-binding proteins in pollen, and their possible role in regulating actin MF activity. We promote the view that polymerization of new actin MFs polarizes the cytoplasm at the apex of the tube. This process is regulated in part by the apical calcium gradient and by different actin-binding proteins. For example, profilin binds actin monomers and gives the cell control over the initiation of polymerization. A more recently discovered actin-binding protein, villin, stimulates the formation of unipolar bundles of MFs. Villin may also respond to the apical calcium gradient, fragmenting MFs, and thus locally facilitating actin remodeling. While much remains to be discovered, it is nevertheless apparent that actin MFs play a fundamental role in controlling apical cell growth in pollen tubes.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Estruturas Vegetais/crescimento & desenvolvimento , Pólen , Polaridade Celular , Lilium/crescimento & desenvolvimento , Lilium/fisiologia , Proteínas dos Microfilamentos/metabolismo , Estruturas Vegetais/metabolismo , Estruturas Vegetais/ultraestruturaRESUMO
Pollen tubes and root hairs are highly elongated, cylindrically shaped cells whose polarized growth permits them to explore the environment for the benefit of the entire plant. Root hairs create an enormous surface area for the uptake of water and nutrients, whereas pollen tubes deliver the sperm cells to the ovule for fertilization. These cells grow exclusively at the apex and at prodigious rates (in excess of 200 nm/s for pollen tubes). Underlying this rapid growth are polarized ion gradients and fluxes, turnover of cytoskeletal elements (actin microfilaments), and exocytosis and endocytosis of membrane vesicles. Intracellular gradients of calcium and protons are spatially localized at the growing apex; inward fluxes of these ions are apically directed. These gradients and fluxes oscillate with the same frequency as the oscillations in growth rate but not with the same phase. Actin microfilaments, which together with myosin generate reverse fountain streaming, undergo rapid turnover in the apical domain, possibly being regulated by key actin-binding proteins, e.g., profilin, villin, and ADF/cofilin, in concert with the ion gradients. Exocytosis of vesicles at the apex, also dependent on the ion gradients, provides precursor material for the continuously expanding cell wall of the growing cell. Elucidation of the interactions and of the dynamics of these different components is providing unique insight into the mechanisms of polarized growth.
Assuntos
Polaridade Celular , Citoesqueleto/metabolismo , Desenvolvimento Vegetal , Raízes de Plantas/fisiologia , Pólen/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cálcio/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/química , Microtúbulos/fisiologia , Células Vegetais , Raízes de Plantas/citologia , Pólen/crescimento & desenvolvimentoRESUMO
Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Proteínas Contráteis , Proteínas de Plantas/metabolismo , Estruturas Vegetais/metabolismo , Pólen/metabolismo , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Cultivadas , Citocalasina D/farmacologia , Corrente Citoplasmática , Desoxirribonuclease I/farmacologia , Relação Dose-Resposta a Droga , Humanos , Lilium , Proteínas dos Microfilamentos/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Estruturas Vegetais/efeitos dos fármacos , Estruturas Vegetais/ultraestrutura , Polímeros/metabolismo , Profilinas , Tiazóis/farmacologia , TiazolidinasRESUMO
In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells.
Assuntos
Actinas/metabolismo , Corrente Citoplasmática/fisiologia , Proteínas dos Microfilamentos/fisiologia , Raízes de Plantas/metabolismo , Vacúolos/metabolismo , Actinas/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Soros Imunes/administração & dosagem , Immunoblotting , Proteínas dos Microfilamentos/imunologia , Microinjeções , Microscopia de Fluorescência , Raízes de Plantas/citologia , Raízes de Plantas/ultraestrutura , Vacúolos/efeitos dos fármacosRESUMO
Actin-binding proteins such as profilins participate in the restructuration of the actin cytoskeleton in plant cells. Profilins are ubiquitous actin-, polyproline-, and inositol phospholipid-binding proteins, which in plants are encoded by multigene families. By 2D-PAGE and immunoblotting, we detected as much as five profilin isoforms in crude extracts from nodules of Phaseolus vulgaris. However, by immunoprecipitation and gel electrophoresis of in vitro translation products from nodule RNA, only the most basic isoform of those found in nodule extracts, was detected. Furthermore, a bean profilin cDNA probe hybridised to genomic DNA digested with different restriction enzymes, showed either a single or two bands. These data indicate that profilin in P. vulgaris is encoded by only two genes. In root nodules only one gene is expressed, and a single profilin transcript gives rise to multiple profilin isoforms by post-translational modifications of the protein. By in vivo 32P-labelling and immunoprecipitation with both, antiprofilin and antiphosphotyrosine-specific antibodies, we found that profilin is phosphorylated on tyrosine residues. Since chemical (TLC) and immunological analyses, as well as plant tyrosine phosphatase (AtPTP1) treatments of profilin indicated that tyrosine residues were phosphorylated, we concluded that tyrosine kinases must exist in plants. This finding will focus research on tyrosine kinases/tyrosine phosphatases that could participate in novel regulatory functions/pathways, involving not only this multifunctional cytoskeletal protein, but other plant proteins.
Assuntos
Proteínas Contráteis , Fabaceae/genética , Proteínas dos Microfilamentos/genética , Plantas Medicinais , Tirosina/metabolismo , Western Blotting , Fabaceae/metabolismo , Proteínas dos Microfilamentos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Profilinas , Biossíntese de Proteínas , RNA Mensageiro/metabolismoRESUMO
The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.
RESUMO
Pollen tubes show a rapid and dramatically polarized growth in which the actin cytoskeleton appears to play a central role. In order to understand the regulation of actin we characterized its associated protein, profilin, in pollen tubes of Lilium longiflorum. By using purified polyclonal antibodies prepared against bean root profilin [Vidali et al., 1995: Plant Physiol. 108:115-123] we detected in pollen grains and tubes two profilin polypeptides with molecular masses of 14.4 and 13.4 KDa, and an identical isoelectric point of 5.05. Profilin comprises approximately 0.47% of the total grain protein, with actin being approximately 1.4%. We were unable to detect a statistically significant profilin increase after germination, while the actin increased approximately 68%. We also spatially localized the distribution of profilin using immunocytochemistry of fixed cells at both the light and electron microscope level, and by fluorescent analog cytochemistry on live cells. The results show that profilin is evenly distributed throughout the cytoplasm and does not specifically associate with any cellular structure.
Assuntos
Proteínas Contráteis/biossíntese , Proteínas dos Microfilamentos/biossíntese , Fenômenos Fisiológicos Vegetais , Actinas/análise , Anticorpos , Anticorpos Monoclonais , Western Blotting , Cromatografia em Gel , Proteínas Contráteis/análise , Eletroforese em Gel de Poliacrilamida , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/química , Modelos Estruturais , Peso Molecular , Proteínas de Plantas/análise , Proteínas de Plantas/biossíntese , Pólen/fisiologia , Pólen/ultraestrutura , Profilinas , Estrutura Secundária de Proteína , Zea maysRESUMO
Profilin from common bean (Phaseolus vulgaris L.) was purified to homogeneity by poly-L-Pro affinity chromatography and gel filtration. The hypocotyl and symbiotic root nodule protein was detected as a single isoform with a 14.4-kD molecular mass and an isoelectric point of 5.3. Partial amino acid and DNA sequencing of a full-length cDNA clone confirmed its identity as profilin. An antibody generated against the purified protein binds to a protein with the same molecular mass in leaves and nodules. Immunolocalization of the protein showed a diffuse distribution in the cytoplasm of hypocotyls and nodules but enhanced staining at the vascular bundles. The strong identity of the sequence among the profilins of birch, maize, and bean suggests that it may play an important role in the signal transduction mechanism of plant cells and plant-bacterial symbioses.