Cellular localization of steroid hormone-regulated proteins during sexual development in Achlya

In the fungus Achlya ambisexualis sexual development in the male strain E87 is controlled by the steroid hormone antheridiol. To investigate the effects of antheridiol on the synthesis and/or accumulation of specific cellular proteins we have analysed [35S]methionine-labeled proteins from control and hormone-treated cells using both one-dimensional (1D) and two-dimensional (2D) PAGE. Since in a total cell extract, hormone-induced changes in specific proteins might not be apparent against a background of more abundant proteins, cells were fractionated prior to protein isolation. It was also necessary to establish a concentration of hormone carrier, in this case methanol, which by itself did not alter the pattern of protein synthesis. Using these approaches the addition of the hormone antheridiol to vegetatively growing cells of Achlya E87 was found to result in changes in the synthesis and/or accumulation of at least 16 specific proteins, which could be localized to the cytoplasmic, nuclear or cell wall/cell membrane fractions. The most prominent changes observed in the hormone-treated cells included the appearance in the cytoplasmic fraction of labeled proteins at 28.4 and 24.3 kD which were not detectable in control cells, and a significant enrichment in the labeling of a 24.3 kD protein in the cell wall/cell membrane fraction. A marked increase in the labeling of 85, 63 and 47 kD proteins in the nuclear fraction from hormone-treated cells was also noted. The molecular weight (MW) and the behavior on 2D gels of the 85 kD hormone-induced protein appeared very similar to that of the 85 kD heat-shock protein reported in Achlya. Quantitive changes in the [35S]methionine labeling of several other proteins were noted in all three cell fractions.     

Cellular localization of the signaling components of Arabidopsis His-to-Asp phosphorelay.

In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.     

Tissue-specific localization of heat-stress proteins during embryo development

We report on the stress-independent, tissue-specific expression of the heat-stress protein HSP17 in developing seeds of different plant species and on its intracellular localization. Though HSP17 expression during seed development seems to be a general phenomenon, the isoform patterns, the relative amounts in embryonic tissues and the intracellular localization show species-specific variations. In contrast to the results on the stressinduced protein forming large cytoplasmic aggregates (heat stress granules) the developmentally expressed HSP17 is mainly found in nuclei. But in addition, a considerable part is also detected in protein bodies of mature seeds of Lycopersicon esculentum and Vicia faba, but not of Zea mays. The mechanism of this transition into the vacuolar compartment remains to be investigated.Abbreviations 2D two-dimensional - HSE heat shock elements - HSP heat stress protein     

Cellular and subcellular localization of stathmin during oocyte and preimplantation embryo development

Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT‐PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two‐cell and later four‐ to eight‐cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation. Mol. Reprod. Dev. 53:306–317, 1999. © 1999 Wiley‐Liss, Inc.     

Subcellular localization of ubiquitin and ubiquitinated proteins in Arabidopsis thaliana.

Ubiquitin is a highly conserved, 76-amino acid, eukaryotic protein. Its widely accepted role as a proteolytic cofactor depends on its unique ability to covalently ligate to other cellular proteins. While there is good evidence for the existence of such ubiquitinated proteins in the cytosolic and nuclear compartments, relatively little is known about the presence of free ubiquitin and ubiquitinated proteins in other subcellular compartments. This is especially true of higher plants, which have not previously been the subject of extensive biochemical subcellular localizations of ubiquitinated proteins. We extracted cell wall proteins and purified nuclei, vacuoles, chloroplasts, and microsomes from chlorophyllous tissues of Arabidopsis. Immunoblot analyses were used to compare the profiles of ubiquitinated proteins from purified subcellular fractions to those from unfractionated extracts. Purified nuclei contained, in addition to a complex mixture of high molecular mass ubiquitinated proteins, a strongly immunoreactive 28-kDa protein. In the apoplastic extract, we did not detect any ubiquitinated proteins enriched above the background level of those due to cytosolic contamination. Vacuoles appeared to contribute significantly to the ubiquitinated proteins present in the whole protoplast extract. At least three high molecular mass ubiquitinated proteins were unique to the vacuolar extract. Chloroplast stromal proteins did not react specifically with anti-ubiquitin antibodies. When microsomal ubiquitinated proteins were compared to those found in a whole protoplast extract, a distinct pattern was evident. Microsomal ubiquitinated proteins were not visible in the 10,000 x g supernatant used to prepare the 100,000 x g pellet, indicating that they were probably low abundance proteins in the protoplast extract.     

Cellular localization and characterization of proteins that bind high density lipoprotein.

High density lipoprotein (HDL) stimulates excretion of excess intracellular cholesterol from cells, presumably by interacting with a cell-surface receptor. A 110 kDa membrane protein that is a candidate for the HDL receptor has been identified by ligand blot analysis. In this study we determined the cellular localization of this and other HDL-binding proteins and characterized their properties. The plasma membranes (PM) of cultured bovine aortic endothelial cells were labeled with trace amounts of [3H]cholesterol, and cell homogenates were fractionated on sucrose and Percoll gradients. Ligand blot analysis of homogenates of cultured bovine aortic endothelial cells demonstrated that cells contain multiple proteins that bind HDL3, including a major membrane protein with an apparent M(r) of 110 kDa and two minor ones with M(r) of 105 and 130 kDa. The gradient distribution of the 105, 110, and 130 kDa HDL-binding proteins mirrored that of labeled cholesterol and 5'-nucleotidase, both PM markers. Treatment of intact cells with the water-soluble cross-linker bis(sulfosuccinimidyl)suberate abolished the HDL binding activity of the 110 and 130 kDa proteins but not that of the 105 kDa protein. These findings suggest that the 105, 110, and 130 kDa HDL-binding proteins are localized to the PM and that at least two of these proteins are exposed to the extracellular fluid. Solubilized 110 and 130 kDa proteins were retained on wheat-germ agglutinin and abrin lectin columns, showing that they are glycoproteins. The cellular localization and physical properties of the 110 and 130 kDa proteins suggest that they may play a role in binding of HDL to the cell surface.     

Cellular localization of ROS and NO in olive reproductive tissues during flower development

Background  

Recent studies have shown that reactive oxygen species (ROS) and nitric oxide (NO) are involved in the signalling processes taking place during the interactions pollen-pistil in several plants. The olive tree (Olea europaea L.) is an important crop in Mediterranean countries. It is a dicotyledonous species, with a certain level of self-incompatibility, fertilisation preferentially allogamous, and with an incompatibility system of the gametophytic type not well determined yet. The purpose of the present study was to determine whether relevant ROS and NO are present in the stigmatic surface and other reproductive tissues in the olive over different key developmental stages of the reproductive process. This is a first approach to find out the putative function of these signalling molecules in the regulation of the interaction pollen-stigma.     

Cellular localization of HSP23 during Drosophila development and following subsequent heat shock

The low-molecular-weight heat-shock protein HSP23 is synthesized in the absence of heat shock during Drosophila development. Here, I present a quantitative analysis of this phenomenon and describe the cellular localization of this protein during normal development and after a subsequent heat shock. HSP23 is first detected in the late third instar larvae and continues to accumulate reaching a maximum level in late pupae. In a 1-week-old adult, HSP23 can no longer be detected. Following lysis of whole pupae, HSP23 is found in the soluble lysate fraction in a form which sediments between 10 and 20 S. Exposure of larvae, pupae, and the adult fly to heat stress (37 degrees C) results in an increased amount of HSP23 which, however, is recovered in an insoluble particulate form following insect lysis. During recovery from heat shock, HSP23 is again found in the soluble 10- to 20-S lysate fraction. In pupae which are exposed to a severe heat stress (41 degrees C) HSP23 remains in the pellet fraction after the heat stress and no pupae are able to emerge as adult flies. However, when pupae are first exposed to a mild heat-shock treatment prior to the 41 degrees C stress, the thermotolerance process is induced and HSP23 is again rapidly found in the soluble lysate fraction during the recovery from heat shock. These observations suggest a possible correlation between the survival of pupae after heat shock and the recovery of HSP23 in the soluble lysate fraction as 10- to 20-S structures after the heat shock.     

Pine xyloglucan. Occurrence, localization and interaction with cellulose

The occurrence, localization, and properties of xyloglucan in the cell walls of growing regions of Pinus pinaster hypocotyls have been studied. Xyloglucan was released from the cell wall with alkali solutions, the concentration increasing from 4 through 35%; KOH. In vitro experiments showed that xyloglucan and cellulose can interact, forming a macromolecular complex. Electron microscope observations showed that the cell wall material extracted with 35%; KOH, which contained some amount of xyloglucan, was enough to cover and join the cellulose microfibrils.     

Cellular interactions during cartilage and bone development.

Both interactions between like cells, as between chondrogenic cells in a developing cartilaginous rudiment, and between unlike cells, as in epithelial-mesenchymal interactions, are dealt with in this review. Such interactions may involve direct apposition of cell membranes or may be mediated via interaction with peri- or extracellular matrices. An ontogenetic approach is taken in which cellular interactions involved in five processes of the development of cartilage and bone are discussed, the five being (1) origin of the cells, (2) migration of the cells within the embryo, (3) localization of the cells at their final embryonic site, (4) differentiation, and (5) morphogenesis. Some emphasis is placed on interactions affecting neural crest-derived cells both before and during their migration and on interactions, especially epithelial-mesenchymal interactions, that precede cytodifferentiation of chondroblasts or osteoblasts. Whether epithelial or mesenchymal specificity is required for such interactions to occur is discussed with reference to the otic vesicle-otic mesenchyme interaction that leads to differentiation and morphogenesis of the cartilaginous otic capsule.     

Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis

The expression of nitrilase in Arabidopsis during the development of the clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-beta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant (nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development is discussed.     

Mitochondrial localization of Arabidopsis thaliana Isu Fe-S scaffold proteins

Isu are scaffold proteins involved in iron-sulfur cluster biogenesis and playing a key role in yeast mitochondria and Escherichia coli. In this work, we have characterized the Arabidopsis thaliana Isu gene family. AtIsu1,2,3 genes encode polypeptides closely related to their bacterial and eukaryotic counterparts. AtIsu expression in a Saccharomyces cerevisiae Deltaisu1Deltanfu1 thermosensitive mutant led to the growth restoration of this strain at 37 degrees C. Using Isu-GFP fusions expressed in leaf protoplasts and immunodetection in organelle extracts, we have shown that Arabidopsis Isu proteins are located only into mitochondria, supporting the existence of an Isu-independent Fe-S assembly machinery in plant plastids.     

Cellular localization of ovarian proopiomelanocortin messenger RNA during follicular and luteal development in the rat

Opioid peptides are expressed in the reproductive system and have been reported to regulate reproductive function. The present study used in situ hybridization to selectively localize ovarian cells containing high levels of proopiomelanocortin (POMC) mRNA, an opioid precursor, during different stages of ovarian development. Prepubertal rats were primed with PMSG to stimulate follicular development, followed by hCG to induce ovulation. Treatment groups consisted of control (no treatment), PMSG (2 days post-PMSG), 1 day corpus luteum (CL; 1 day post-hCG), and 8 day CL (8 days post-hCG). POMC mRNA-containing cells were present in antral follicles, CL, and the interstitial compartment. With gonadotropin treatment, the percentage of follicles containing heavily labeled cells increased in the PMSG and 1 day CL groups. The number of POMC mRNA-containing cells per follicle also increased in the 1 day CL group. In the CL, no difference was observed in the percentage of CL exhibiting labeled cells between the 1 day CL and 8 day CL groups; however, more labeled luteal cells per CL were present in the 1 day CL group. A marked increase in POMC mRNA-containing cells was observed in the interstitial compartment of the 1 day CL group. These results indicate that the number of POMC mRNA-containing cells increases with follicular development and CL formation; however, the ovarian distribution suggests that the labeled cells could be nonendocrine cells, possibly white blood cells. The in situ hybridization findings are indicative of low total concentrations of ovarian POMC mRNA, suggesting mainly an autocrine or paracrine role for POMC or POMC-derived peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular localization of Arabidopsis EARLY FLOWERING3 is responsive to light quality

Circadian clocks facilitate the coordination of physiological and developmental processes to changing daily and seasonal cycles. A hub for environmental signaling pathways in the Arabidopsis (Arabidopsis thaliana) circadian clock is the evening complex (EC), a protein complex composed of EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRYTHMO (LUX). Formation of the EC depends on ELF3, a scaffold protein that recruits the other components of the EC and chromatin remodeling enzymes to repress gene expression. Regulating the cellular distribution of ELF3 is thus an important mechanism in controlling its activity. Here, we determined that the cellular and sub-nuclear localization of ELF3 is responsive to red (RL) and blue light and that these two wavelengths have apparently competitive effects on where in the cell ELF3 localizes. We further characterized the RL response, revealing that at least two RL pathways influence the cellular localization of ELF3. One of these depends on the RL photoreceptor phytochrome B (phyB), while the second is at least partially independent of phyB activity. Finally, we investigated how changes in the cellular localization of ELF3 are associated with repression of EC target-gene expression. Our analyses revealed a complex effect whereby ELF3 is required for controlling RL sensitivity of morning-phased genes, but not evening-phased genes. Together, our findings establish a previously unknown mechanism through which light signaling influences ELF3 activity.

Light signaling pathways converge on the evening complex scaffold protein EARLY FLOWERING3 by controlling its cellular and sub-cellular localization and regulating circadian clock gene expression.