RESUMO
During larva-to-pupa metamorphosis Drosophila salivary glands undergo programmed cell death by autophagocytosis. Although ultrastructure of Drosophila salivary glands has been extensively studied in the past, little is known about mechanism of programmed cell death, especially the role of the cytoskeleton. In this paper we describe changes in microtubule and actin filament network compared to the progress of DNA fragmentation and redistribution of acid phosphatase. In feeding and wandering larvae microtubules and actin filaments form regular networks localized mostly along the plasma membrane. The first major rearrangement of microtubules and actin filaments occurred when larvae everted spiracles and the glands shifted their secretion from saliva to mucoprotein glue (stage L1). Microtubule cytoskeleton became denser and actin filaments concentrated along cell boundaries. At the same time nuclei flattened and migrated into the microtubule-rich layer near the basal membrane. In late prepupae (8-10 h after P1) the microtubule network became fainter, and actin filaments appeared frequently deeper in cytoplasm, gradually concentrating around nuclei. Simultaneously large patches of acid phosphatase activity surrounded nuclei and shortly thereafter chromosomal DNA began to fragment. During the final collapse of the gland (early pupae, 13.5 h after formation of white puparium) cellular fragments and autophagic vacuoles contained a continuous F-actin lining and the microtubule network displayed signs of extensive degradation. The results are consistent with the hypothesis that, in Drosophila salivary glands, extensive autophagic activities target nuclei for degradation; that this process occurs late in the course of programmed cell death; and that it directly involves cytoskeletal structures which are altered far earlier during the course of cell death.
RESUMO
In Schizosaccharomyces pombe, nitrogen starvation induces transient acceleration of cell division and reduction in cell size with a final arrest in G1. The division size control appears to be impaired by mutations in cdr1/nim1 and cdr2, genes that encode protein kinases mediating nutritional control over the mitotic cycle. cdr- cells arrest after fewer rounds of division and are larger than the wild type. Recent work suggests that long-term nitrogen starvation causes S. pombe wild-type cells to become spherical, which suggests loss of cell polarity. cdr mutants retain the elongated shape, indicating a potential difference in cell polarity control relative to the wild type. We examined several markers related to maintenance of cell polarity in S. pombe following nitrogen starvation including cell division scar pattern and actin and microtubule cytoskeleton. Wild-type cells as well as cdr mutants maintained a normal cell division scar pattern throughout nitrogen starvation but cells dividing under these conditions developed a wall malformation in the center of the septum. In cells arrested by nitrogen starvation, actin patches, normally associated with sites of cell wall deposition, were larger and distributed randomly along the cell surface. Cytoplasmic arrays of microtubules, which are thought to be involved in control of the polarity signal, were not visibly affected. The effects were similar in wild-type cells and in cdr- mutants. Upon refeeding, the new growth always reoccurred at the tip zones and there were only small deviations of its direction from the original axis. The results indicate that cell polarity is preserved both in wild-type cells, which arrest in G1 and appear spherical, and in cdr1/nim1 and cdr2 mutants, which arrest in G2 and appear polarized throughout the starvation period.
Assuntos
Polaridade Celular , Proteínas de Membrana Transportadoras , Nitrogênio/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/citologia , Transportadores de Cassetes de Ligação de ATP/genética , Actinas/análise , Biomarcadores/análise , Divisão Celular , Polaridade Celular/genética , Tamanho Celular , Proteínas Fúngicas/genética , Fase G2/genética , Microscopia Eletrônica , Microtúbulos/química , Microtúbulos/ultraestrutura , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/ultraestruturaRESUMO
Labial glands of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingiidae, homologues of Drosophila salivary glands, undergo programmed cell death (PCD) in a 4-day period during larva-to-pupa metamorphosis. The programmed death of the labial gland was examined by electron microscopy and measurement of protein synthesis as well as measurement of DNA synthesis, end-labeling of single strand breaks, and pulsed-field gel electrophoresis. One of the earliest changes observed is a sharp drop in synthesis of most proteins, coupled with synthesis of a glycine-rich protein, reminiscent of silk-like proteins. From a morphological standpoint, during the earliest phases the most prominent changes are the formation of small autophagic vacuoles containing ribosomes and an apparent focal dissolution of the membranes of the endoplasmic reticulum, whereas later changes include differing destruction at the lumenal and basal surfaces of the cell and erosion of the basement membrane. By the fourth day of metamorphosis, individual cells become rapidly vacuolated in a cell-independent manner. In the vacuolated cells on day 3, chromatin begins to coalesce. It is at this period that unequivocal nucleosomal ladders are seen and end-labeling in situ or electrophoretic techniques document single on double-strand breaks, respectively. DNA synthesis ceases shortly after the molt to the fifth instar, as detected by incorporation of tritiated thymidine and weak TUNEL labeling. Large size fragments of DNA are seen shortly after DNA synthesis ceases and thence throughout the instor, raising the possibility of potential limitations built into the cells before their final collapse.
Assuntos
Apoptose , DNA/biossíntese , Biossíntese de Proteínas , Glândulas Salivares/patologia , Animais , Manduca , Metamorfose Biológica , Glândulas Salivares/metabolismoRESUMO
We investigated the nuclear transport of a fusion protein consisting of a nuclear localization signal linked to beta-galactosidase, normally a cytoplasmic protein. We microinjected the radiolabeled fusion protein into the cytoplasm of living Xenopus oocytes or supplied it directly to the surface of the oil-isolated oocyte nucleus and measured its transport into the nucleus. Our data confirm that a nuclear localization signal is sufficient to entrain a protein's facilitated transport through the nuclear pore complex and its subsequent nuclear accumulation. Moreover, nuclear envelope micropuncture experiments determine that the fusion protein's accumulation results from its intranuclear binding, demonstrating that no specific region of a transported protein--other than the nuclear localization signal itself--is required for facilitated transport and intranuclear binding. Finally, we present evidence that the intranuclear binding of a transported protein requires not only its nuclear localization signal, but also its prior facilitated transport through the nuclear pore complex.
Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas , Sinais Direcionadores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Escherichia coli/genética , Feminino , Dados de Sequência Molecular , Nucleoplasminas , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevis , beta-Galactosidase/metabolismoRESUMO
Using the monoclonal antibody MA-01, which recognizes a 210-kDa protein in cell-free extracts, spindle and cytoplasmic microtubules were visualized in budding yeast, Saccharomyces cerevisiae. In additional, a spot-like staining was found beneath the plasma membrane, revealing in part correlation with F-actin distribution. This pattern was common for cells of all cell-cycle stages. The interaction of the protein recognized by MA-01 with microtubules was confirmed in the double labeling with a polyclonal antitubulin antibody and by the sensitivity of intranuclear structures stained by MA-01 to the microtubule disrupting drug nocodazole.
Assuntos
Proteínas Associadas aos Microtúbulos/isolamento & purificação , Saccharomyces cerevisiae/química , Actinas/análise , Anticorpos Monoclonais , Citoplasma/química , Citoplasma/imunologia , Imunofluorescência , Proteínas Associadas aos Microtúbulos/imunologia , Microtúbulos/química , Microtúbulos/imunologia , Saccharomyces cerevisiae/imunologia , Fuso Acromático/química , Fuso Acromático/imunologiaRESUMO
By rhodamine-phalloidin fluorescence, distinct continuous F-actin rings were visualized in 18-20% of the protoplasts of Schizosaccharomyces pombe and S. japonicus var. versatilis, in addition to randomly distributed F-actin dots. Whereas the reversion of ring-lacking protoplasts coincided with the polarization of the dotted F-actin pattern, the ring-containing protoplasts became furrowed as the F-actin rings constricted. The furrowing was more conspicuous in S. japonicus var. versatilis than in S. pombe protoplasts and it was blocked when the reversion was inhibited by Novozyme 234 indicating that the cell wall formation is essential for the F-actin ring constriction.