Functional organisation of the endomembrane network in the digestive gland of the Venus flytrap: revisiting an old story with a new microscopy toolbox.

Up-to-date imaging approaches were used to address the spatiotemporal organisation of the endomembrane system in secretory cells of Dionaea muscipula. Different 'slice and view' methodologies were performed on resin-embedded samples to finally achieve a 3D reconstruction of the cell architecture, using ultrastructural tomography, array tomography, serial block face-scanning electron microscopy (SBF-SEM), correlation, and volume rendering at the light microscopy level. Observations of cryo-fixed samples by high-pressure freezing revealed changes of the endomembrane system that occur after trap activation and prey digestion. They provide evidence for an original strategy that adapts the secretory machinery to a specific and unique case of stimulated exocytosis in plant cells. A first secretion peak is part of a rapid response to deliver digestive fluids to the cell surface, which delivers the needed stock of digestive materials 'on site'. The second peak of activity could then be associated with the reconstruction of the Golgi apparatus (GA), endoplasmic reticulum (ER) and vacuolar machinery, in order to prepare for a subsequent round of prey capture. Tubular continuum between ER and Golgi stacks observed on ZIO-impregnated tissues may correspond to an efficient transfer mechanism for lipids and/or proteins, especially for use in rapidly resetting the molecular GA machinery. The occurrence of one vacuolar continuum may permit continuous adjustment of cell homeostasy. The subcellular features of the secretory cells of Dionaea muscipula outline key innovations in the organisation of plant cell compartmentalisation that are used to cope with specific cell needs such as the full use of the GA as a protein factory, and the ability to create protein reservoirs in the periplasmic space. Shape-derived forces of the pleiomorphic vacuole may act as signals to accompany the sorting and entering flows of the cell.

Characterisation of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize (Zea mays L.).

Aerenchyma is a tissue type characterised by prominent intercellular spaces which enhance flooding tolerance in some plant species by facilitating gas diffusion between roots and the aerial environment. Aerenchyma in maize roots forms by collapse and death of some of the cortical cells in a process that can be promoted by imposing oxygen shortage or by ethylene treatment. Maize roots grown hydroponically in 3% oxygen, 1 microl x l(-1) ethylene or 21% oxygen (control) were analysed by a combination of light and electron microscopy. Use of in-situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) suggested internucleosomal cleavage of DNA. However, chromatin condensation detectable by electron microscopy was preceded by cytoplasmic changes including plasma membrane invagination and the formation of vesicles, in contrast to mammalian apoptosis in which chromatin condensation is the first detectable event. Later, cellular condensation, condensation of chromatin and the presence of intact organelles surrounded by membrane resembling apoptotic bodies were observed. All these events were complete before cell wall degradation was apparent. Therefore, aerenchyma formation initiated by hypoxia or ethylene appears to be a form of programmed cell death that shows characteristics in part resembling both apoptosis and cytoplasmic cell death in animal cells.

Organization of transport from endoplasmic reticulum to Golgi in higher plants.

In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endomembrane trafficking in plant cells.

Endomembranes and vesicle trafficking.

Over the past year extensive analyses of the accumulated data on the structural and functional organisation of the endomembrane system and vesicular trafficking in higher plants have shown it to be far more complex than previously anticipated. The availability of molecular tools combined with new opportunities to visualise endomembrane dynamics in vivo will allow better understanding of the fundamental processes underlying the complexity of endomembrane behaviour and vesicular trafficking.

[3H]ATPA: a high affinity ligand for GluR5 kainate receptors.

The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum.

Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period.

PP7, a plant phosphatase representing a novel evolutionary branch of eukaryotic protein Ser/Thr phosphatases.

We describe a novel protein Ser/Thr phosphatase from Arabidopsis thaliana, PP7, which is only 27-32% identical in amino acid sequence to the known phosphatases and is the most divergent member of the PPP (PP1/2A/2B) family for today. Some structural features suggest more close relationship of PP7 to the PP5/rdgC subfamily. PP7 contains all of the residues essential for the phosphatase activity and possesses three major insertions in its presumable C-terminal subdomain, which suggest its unique regulation and/or optimisation of its structure for interaction with specific substrates or regulators. A phosphatase structurally related to PP7 is expressed in rice. PP7 conservation between mono- and dicotyledonous plants may point to its essential role in the plant cell.

Proteins involved in membrane transport between the ER and the Golgi apparatus: 21 putative plant homologues revealed by dbEST searching.

Numerous proteins have been identified in yeast and mammalian cells which are involved in trafficking between the endoplasmic reticulum and the Golgi apparatus. A great number of partial cDNA sequences now available from the two major plant model species, Arabidopsis thaliana and Oryza sativa, makes it possible to identify putative plant homologues of known genes/proteins from non-plant species. The authors used this approach to screen the database of Expressed Sequence Tags (dbEST) in order to detect plant homologues of proteins involved in membrane transport between ER and Golgi. Availability of these partial sequences will facilitate the screening of cDNA and genomic libraries otherwise performed using heterologous probes derived from animal and yeast genes. As the plant Golgi complex differs in many respects from its mammalian and yeast counterparts, the dbEST clones found can be directly used for various functional assays (immunoprecipitation, two-hybrid analysis, transgenic plants etc.) to test the exact roles of the encoded proteins and identify their functional partners, some of which may be specific for plants.

[Genes of plant Ser/Thr protein phosphatases: detection of sequences related to PPT/rdgC]. / Geny Ser/Thr-fosfataz rasteni'i: detektsiia posledovatel'noste'i, rodstvennykh PPT/rdgC.

An unknown sequence that may encode a fragment of the Ser/Thr protein phosphatase (designated PP6Zm) related to PPT/rdgC phosphatases was identified using PCR on maize genomic DNA. A dbEST search using a partial amino acid sequence of PP6Zm revealed a putative homolog of PP6Zm expressed in Arabidopsis thaliana (EMBL AT6726). A search of the SwissProt database indicated that the partial amino acid sequence of AT6726 has the highest identity (54.3%) to the rdgC phosphatase from Drosophila melanogaster. The maize phosphatase PP1Zm6, described previously as a PP1 isoform (EMBO J., 1993, vol. 12, p. 3497), was found by us to be plant homolog of mammalian PPT. In addition, six fragments of new (pseudo) genes homologous to the phosphatase genes encoding PP1, PP2A, and PPX isoforms were detected in the maize genome. The existence in maize of a multigene PP2A family, reported only for dicotyledons, and of a PP1 multigene family, found earlier in both di- and monocotyledons, was shown.

[Cloning and expression of cDNA for tobacco rab1, and structural-functional analysis of the protein]. / Klonirovanie i ékspressiia kDNK rab1 tabaka i strukturno-funktsional'nyi analiz belka.

rab1 cDNA coding for a small GTP binding protein Rab1 was isolated from cDNA library of tobacco (Nicotiana tabacum) leaves. The primary structure of this protein was deduced from the rab1 structure. Tobacco rab1 cDNA was expressed in Escherichia coli, and the product was purified and shown to exhibit GTPase activity. A set of Rab1 mutants with altered GTP binding and/or GTPase activities was obtained. Polyclonal antipeptide antibodies were raised against a sequence in the C-terminal region of the tobacco Rab1 capable of recognizing this protein.

Retention of maize auxin-binding protein in the endoplasmic reticulum: quantifying escape and the role of auxin.

The localisation of maize (Zea mays L.) auxin-binding protein (ABP1) has been studied using a variety of techniques. At the whole-tissue level, tissue printing indicated that ABP1 is expressed to similar levels in all cells of the maize coleoptile and in the enclosed leaf roll. Within cells, the signals from immunofluorescence and immunogold labelling of ultrathin sections both indicated that ABP1 is confined to the endoplasmic reticulum (ER), none being detected in either Golgi apparatus or cell wall. This distribution is consistent with targeting motifs in its sequence. These observations are discussed with reference to the various reports which place a population of ABP1 on the outer face of the plasma membrane, including those suggesting that it is necessary on the cell surface for rapid, auxin-mediated protoplast hyperpolarisation. We have tested the ER, namely that auxin binding induces a conformational change in ABP1 leading to concealment of the KDEL retention motif. Using double-label immunofluorescence the characteristic auxin-induced rise in Golgi-apparatus signal was found, yet no change in the distribution of the ABP1 signal was detected. Maize suspension cultures were used to assay for auxin-promoted secretions of ABP1 into the medium, but secretion was below the limit of detection. This can be ascribed at least partly to the very active acidification of the medium by these cells and the instability of ABP 1 in solution below pH 5.0. In the insect-baculovirus expression system, in which cell cultures maintain pH 6.2, a small amount of ABP1 secretion, less than 1% of the total, was detected under all conditions, Insect cells were shown to take up auxin and no inactivation of added auxin was detected, but auxin did not affect the level of ABP1 in the medium. Consequently, no evidence was found to support the model for auxin promotion of ABP1 secretion. Finally, quantitative glycan analysis was used to determine what proportion of ABP1 might reach the plasma membrane in maize coleoptile tissue. The results suggest that less than 15% of ABP1 ever escapes from the ER as far as the cis-Golgi and less than 2% passes further through the secretory pathway. Such leakage rates probably do not require a specialised mechanism allowing ABP1 past the KDEL retrieval pathway, but we are not able to rule out the possibility that some ABP1 is carried through associated with other proteins. The data are consistent with the presence of ABP1 both on the plasma membrane and in the ER. The relative sizes of the two pools explain the results obtained with immunofluorescence and immunogold labelling and illustrate the high efficiency of ER retention in plants.

Protein retention in the endoplasmic reticulum of insect cells is not compromised by baculovirus infection.

High level expression of the major auxin-binding protein (ABP1) from maize (Zea mays L.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immunolocalization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.

Stable expression of maize auxin-binding protein in insect cell lines.

To achieve continuous expression of the major maize auxin-binding protein (ABP1) in insect cells, the ABP1 gene coding region was placed under control of a baculovirus immediate-early gene promoter and transfected into Spodoptera frugiperda Sf9 cells. The ABP1 gene was detected in twelve cell lines, one of which was selected for detailed analysis. Immunolocalisation demonstrated that ABP1 was targeted to and retained in the endoplasmic reticulum (ER), in accordance with its signal peptide and carboxy-terminal KDEL ER-retention signal. We discuss the advantages of stable-transformation over transient expression systems for characterising proteins targeted to the secretory system of insect cells.

Efficient membrane targeting of the chick nicotinic acetylcholine receptor α-subunit in stable insect cell lines.

We have synthesised the α-subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChRα, encoding the α-subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR α-subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.

Assessment of a computerized system for the diagnosis of iron deficiency.

The aim of this study was to develop a computer expert system that could reproduce a pathologist's diagnosis of iron deficiency from the data obtained from blood tests. 275 cases were collected for construction and testing of the expert system. The expert system used a combination of fuzzy set logic and cut-off points from 14 parameters to arrive at one of 5 diagnostic categories graded from "iron deficient" to "no evidence of iron deficiency". The agreement between pathologist and expert system was 0.91 (Spearman rank correlation coefficient) in the learning population; this dropped to 0.79 in the test population. Absolute agreement on diagnostic category was reached in 71% of cases. In no case was there disagreement by more than 3 grades.

Elevated levels of acute phase plasma proteins in major depression.

Levels of acute phase and other plasma proteins were measured in 21 men with major depression, 28 men with alcohol dependence, and 12 men who acted as controls. The depressed men had significantly elevated levels of the acute phase proteins, haptoglobin and alpha-1-antichymotrypsin, and of immunoglobulin G. The elevations in haptoglobin and alpha-1-antichymotrypsin were highly correlated with each other, and were correlated with the severity of depression and negatively correlated with the thyroid stimulating hormone response to thyrotropin. The alcoholic men had elevated haptoglobin levels, but significantly decreased levels of immunoglobulin G. These findings provide further evidence for an inflammatory response during depression.

Antibodies to brain clathrin recognise plant coated vesicles.

Coated vesicles isolated from carrot suspension culture cells were immune-blotted against four antibodies to porcine brain clathrin. Positive cross-reaction was obtained with three antibodies. Two of these cross-reacted with both the heavy clathrin chain and the putative light chains. Three out of five antibodies immunofluorescently stained permeabilised carrot suspension culture cells.

Structure of amyloplasts and endoplasmic reticulum in the root caps of Lepidium sativum and Zea mays observed after selective membrane staining and by high-voltage electron microscopy.

The structure of plastids in the root cap of cress and maize was studied by low- and high-voltage electron microscopy after staining their membranes with a mixture of zinc iodide and osmium tetroxide. In plastids of both species electron-opaque membranes were found in the plastid interior while membranes of lesser electron-opacity comprised the outer envelope and vesicles and cisternae underlying it. Electron-opaque tubules, often in groups attached to the inner membrane of the amyloplast envelope, were found in cress but not in maize. The internal, less-opaque membranes were often found associated with the starch grains. No specific association could be seen between amyloplasts and endoplasmic reticulum (ER); their surfaces showed no regular contact or connexion, though the amyloplasts clearly indented the underlying ER. The ER in statocytes was predominantly tubular in cress but predominantly cisternal in maize.