Isolation and structure of the lipid envelopes from the nitrogen-fixing vesicles of Frankia sp. strain CpI1.

Frankia vesicles are differentiated during nitrogen starvation; they contain nitrogenase whether produced by free-living frankiae or by frankiae in actinorhizal root nodules. Vesicles are surrounded by envelopes of several monolayers of uncharacterized lipid. It has been suggested that the envelope limits diffusion of O2 into the vesicle cytoplasm, thereby preventing inactivation of nitrogenase. Whole vesicles were prepared on sucrose gradients and sonicated, and vesicle envelopes were isolated on top of a cushion of 40% sucrose. Transmission electron microscopy of potassium permanganate-fixed envelopes confirmed the purity of these preparations. Only the outer and inner envelope layers were visible in permanganate-fixed intact vesicles; the laminae were not visible in aldehyde-osmium-fixed, lead citrate-uranyl acetate-stained whole vesicles. However, the laminated nature of the envelope was clearly evident in sonicated vesicles and in envelope fragments fixed with KMnO4. The observations indicate that partial disruption of the vesicle envelope enables its visualization with permanganate fixation, and these observations open the way for further studies on the relationship of the vesicle surface to environmental conditions.     

Is Protein Import into Plastids with Four‐Membrane Envelopes Dependent on Two Toc Systems Operating in Tandem?

Abstract: Plastids with four‐membrane envelopes have evolved by several independent endosymbioses involving a eukaryotic alga as the endosymbiont and a protozoan predator as the host. It is assumed that their outermost membrane is derived from the phagosomal membrane of the host and that protein targeting to and across this membrane proceeds co‐translationally, including ER and the Golgi apparatus (e.g., chlorarachniophytes) or only ER (e.g., heterokonts). Since the two inner membranes (or the plastid envelope) of such a complex plastid are derived from the endosymbiont plastid, they are probably provided with Toc and Tic systems, enabling post‐translational passage of the imported proteins into the stroma. The third envelope membrane, or the periplastid one, originates from the endosymbiont plasmalemma, but what import apparatus operates in it remains enigmatic. Recently, Cavalier‐Smith (1999^[5]) has proposed that the Toc system, pre‐existing in the endosymbiont plastid, has been relocated to the periplastid membrane, and that plastids having four envelope membranes contain two Toc systems operating in tandem and requiring the same targeting sequence, i.e., the transit peptide. Although this model is parsimonious, it encounters several serious obstacles, the most serious one resulting from the complex biogenesis of Toc75 which forms a translocation pore. In contrast to most proteins targeted to the outer membrane of the plastid envelope, this protein carries a complex transit peptide, indicating that a successful integration of the Toc system into the periplastid membrane would have to be accompanied by relocation of the stromal processing peptidase to the endosymbiont cytosol. However, such a relocation would be catastrophic because this enzyme would cleave the transit peptide off all plastid‐destined proteins, thus disabling biogenesis of the plastid compartment. Considering these difficulties, I suggest that in periplastid membranes two Toc‐independent translocation apparatuses have evolved: a porin‐like channel in chlorarachniophytes and cryptophytes, and a vesicular pathway in heterokonts and haptophytes. Since simultaneous evolution of a new transport system in the periplastid membrane and in the phagosomal one would be complicated, it is argued that plastids with four‐membrane envelopes have evolved by replacement of plastids with three‐membrane envelopes. I suggest that during the first round of secondary endosymbioses (resulting in plastids surrounded by three membranes), myzocytotically‐engulfed eukaryotic alga developed a Golgi‐mediated targeting pathway which was added to the Toc/Tic‐based apparatus of the endosymbiont plastid. During the second round of secondary endosymbioses (resulting in plastids surrounded by four membranes), phagocytotically‐engulfed eukaryotic alga exploited the Golgi pathway of the original plastid, and a new translocation system had to originate only in the periplastid membrane, although its emergence probably resulted in modification of the import machinery pre‐existing in the endosymbiont plastid.     

ON THE FINE STRUCTURE AND COMPOSITION OF THE NUCLEAR ENVELOPE

Nuclei from nearly ripe eggs of Rana pipiens were isolated and cleaned in 0.1 M KCl. The whole nucleus was then digested to various degrees with ribonuclease or trypsin, followed by washing and fixation in either osmium tetroxide or potassium permanganate. The nuclear envelope was dissected off, placed on a grid, air dried, and compared with undigested controls in the electron microscope. Some envelopes were dehydrated, embedded in methacrylate, and sectioned. Annuli around "pores" are composed of a substance or substances, at least partially fibrillar, which is preserved by osmium but lost during permanganate fixation. Material within the "pores" is also preserved by osmium but partially lost after permanganate. No evidence of granules or tubules in the annuli was found in air dried mounts although a granular appearance could be seen in tangentially oriented thin sections. Thin sections of isolated envelopes give evidence of diffuse material within the "pores" as well as a more condensed diaphragm across their waists. In whole mounts of the envelope the total density within "pores" is relatively constant from "pore" to "pore." All material within "pores," including the condensed diaphragm, is removable by trypsin digestion. Wispy material from the "pore" structure projects into the nucleus and annular material extends into the cytoplasm. Both annular and diaphragm materials remain with the envelope when it is isolated and are thus considered a part of its structure, not merely evidences of material passing through. There is no evidence of ribonuclease-removable material in any part of the "pore" complex.     

A role for lipid trafficking in chloroplast biogenesis

Chloroplasts are the defining plant organelle carrying out photosynthesis. Photosynthetic complexes are embedded into the thylakoid membrane which forms an intricate system of membrane lamellae and cisternae. The chloroplast boundary consists of two envelope membranes controlling the exchange of metabolites between the plastid and the extraplastidic compartments of the cell. The plastid internal matrix (stroma) is the primary location for fatty acid biosynthesis in plants. Fatty acids can be assembled into glycerolipids at the envelope membranes of plastids or they can be exported and assembled into lipids at the endoplasmic reticulum (ER) to provide building blocks for extraplastidic membranes. Some of these glycerolipids, assembled at the ER, return to the plastid where they are remodeled into the plastid typical glycerolipids. As a result of this cooperation of different subcellular membrane systems, a rich complement of lipid trafficking phenomena contributes to the biogenesis of chloroplasts. Considerable progress has been made in recent years towards a better mechanistic understanding of lipid transport across plastid envelopes. Lipid transporters of bacteria and plants have been discovered and their study begins to provide detailed mechanistic insights into lipid trafficking phenomena relevant to chloroplast biogenesis.     

Lipid composition of envelopes,prolamellar bodies and other plastid membranes in etiolated,green and greening wheat leaves

Summary Comparative studies of lipid composition were made on prolamellar bodies, envelopes and other plastid membranes separately extracted from etiolated, green or greening (intermittent or continuous light) wheat (Triticum sativum L.) leaves. The different membrane fractions were examined by electron microscopy.The major lipid was digalactosyldiglyceride in the envelopes and prolamellar bodies and monogalactosyldiglyceride in stroma lamellae and grana. Phosphatidylcholine represented 60% of total phospholipids in the envelopes, 30% in prolamellar bodies and 14% in grana. All types of envelopes had the same lipid proportions.For all lipids the lowest fatty acid unsaturation was always found in the envelope membranes. The relative amount of {ie193-1} acid in the phosphatidylglycerol of envelopes increased from 4% (etioplasts) to an average of 15% (etiochloroplasts and chloroplasts).Abbreviations DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SL sulfolipid     

Protein import: the hitchhikers guide into chloroplasts

Plastids originated from an endosymbiotic event between an early eukaryotic host cell and an ancestor of today's cyanobacteria. During the events by which the engulfed endosymbiont was transformed into a permanent organelle, many genes were transferred from the plastidal genome to the nucleus of the host cell. Proteins encoded by these genes are synthesised in the cytosol and subsequently translocated into the plastid. Therefore they contain an N-terminal cleavable transit sequence that is necessary for translocation. The sequence is plastid-specific, thus preventing mistargeting into other organelles. Receptors embedded into the outer envelope of the plastid recognise the transit sequences, and precursor proteins are translocated into the chloroplast by a proteinaceous import machinery located in both the outer and inner envelopes. Inside the stroma the transit sequences are cleaved off and the proteins are further routed to their final locations within the plastid.     

Egg envelopes in diplopods, a comparative ultrastructural study

By means of electron microscopy two types of egg envelope have been described in representatives of two diplopod subclasses, the Chilognatha and the Pselaphognatha. The vitelline envelope appears on the oocyte surface in early previtellogenesis and persists till ovulation. In its thin and filamentous structure it resembles basement membranes. During vitellogenesis electron dense material is deposited on filamentous scaffolding which fills the space between the oolemma and the vitelline envelope. As a result, the thick and spongy or filamentous chorion is formed. In the present study it has been shown that regardless of the type of oogenesis (solitary-the Chilognatha, or follicular-the Pselaphognatha) both envelopes in diplopods are produced by the oocyte itself, and although completely different in structure and time of appearance, they must be both considered as primary.     

The origins of plastids

A new theory of plastid origins is presented in which only two symbiotic events are needed to explain the origin of the six fundamentally different types of plastid, which all probably originated in anteriorly biciliated phagotrophic cells. Four of them can be derived directly from a single endosymbiotic cyanophyte by the independent loss of different cyanophyte characters and the evolution of new characters in the immediate descendants of this primary endosymbiosis. Retention of the phagosomal membrane as well as the prokaryotic plasma and outer membrane could produce the dinozoan and euglenid plastids with three envelope membranes, whereas the loss of the phagosomal membrane could produce the two-membraned envelopes characteristic of the Biliphyta and Verdiplantae*. The phycobilins were retained essentially unaltered in the Biliphyta, but are modified or lost in the other lines. In the ancestor of the Euglenozoa and Verdiplantae they were replaced by chlorophyll b. In the ancestor of algae possessing chlorophyll c they were modified to the cryptophyte type, concomitantly with the evolution of chlorophyll c₂: one line of descent from this ancestor produced the dinozoan plastid by the complete loss of phycobilins, while the other was incorporated by endosymbiosis into another phagotrophic bibiliate to produce the cryptophyte plastid. The latter evolved into the chromophyte plastid by the loss of phycobilins and the evolution of chlorophyll c₂. The conversion of the endosymbiont into a plastid depended on the evolution of a system to transport proteins into it. I argue that this occurred by the modification of the pre-existing mitochondrial transport system, and that the major modifications needed to adjust this to plastids with more than two envelope membranes led to evolution of a new tubular or disc-like morphology for the mitochondrial cristae of these groups. This new cristal morphology is maintained by stabilizing selection even in species that have secondarily lost plastids.     

Fine Structure and Biomineralization of the Mucilage in Envelopes of Trachelomonas lefevrei (Euglenophyceae)1

Envelope development in Trachelomonas lefevrei (Deflandre) begins with the production of short, coarse mucilaginous strands, morphologically similar to the mucilage produced by non-enveloped euglenoids. These initial mucilaginous secretions subsequently become impregnated with manganese (Mn) and/or iron (Fe). Continued mucilage secretion and mineralization results in a mature envelope that is characteristic for the species. When these mature envelopes are treated with oxalic acid to remove the Mn and Fe, the envelopes collapse and are composed only of short, coarse mucilaginous strands similar to those present during early stages of development, prior to their mineralization. Brief treatment with 10 mM EDTA renders dark envelopes colorless, and our EM-EDS analyses show that this corresponds to a loss of Mn from the envelope; however, if Fe is present in the envelope, it is unaffected by the brief treatment. The mucilage present during early stages of envelope development and that remaining after complete demineralization is also morphologically similar to the mucilage in the plug at the anterior end of the envelope.     

ORGANIZATION AND ACTIVITY IN THE PRE- AND POSTOVULATORY FOLLICLE OF NECTURUS MACULOSUS

The established follicle envelope of Necturus maculosus consists of a layer of follicle cells (granulosa) surrounding the developing oocyte, a layer of theca comprised of connective tissue cells, fibers, and matrix, and a layer of serosal cells. The changes in shape and fine structure of these layers during differentiation accompanying oogenesis are described. The cells and capillaries of the follicle envelope are engaged in an extensive pinocytotic activity, the details of which are described. We used cytochemical techniques to analyze the activity of the follicle envelope with respect to lipid accumulation and alkaline phosphatase activity. Radioautographic results indicate that cells of the follicle envelope are capable of incorporating tritium-labeled uridine and amino acids at certain times during oocyte growth. A comparative analysis was made of the soluble proteins in follicle envelopes isolated from immature oocytes and of those in follicle envelopes isolated from nearly mature oocytes and in postovulatory follicles. After the oocyte is ovulated, the cells of the follicle envelope are converted into a postovulatory follicle. The cells of the postovulatory follicle undergo further differentiation resulting in their becoming actively engaged in the formation of a secretion, the details of which are described at the electron microscope level. Analysis of the postovulatory follicle by thin-layer chromatography and cytochemistry demonstrated the presence of a wide variety of lipid substances and the possible presence of steroid. That the postovulatory follicle may be engaged in steroid biosynthesis is also suggested by studies involving the demonstration of 3 β-hydroxysteroid dehydrogenase activity with cytochemical techniques applied to frozen sections and to soluble proteins separated by gel electrophoresis.     

The vitelline envelope-to-fertilization envelope transformation in the toad Bufo arenarum.

An investigation of some changes associated with the transformation of the vitelline envelope into the fertilization envelope in the egg of the toad Bufo arenarum is reported here. In most of the experiments described, the parameter used to demonstrate these changes was the stability of structural integrity of isolated envelopes when submitted to different agents and conditions. The envelopes used for this purpose exhibited a high degree of purity and remained apparently unaltered by the isolation procedure. As a quantitative method to ascertain their solubility rate, the release of uv-absorbing materials into solution was determined. Compared to the vitelline envelope, the fertilization envelope has proven to be less soluble in water, more stable in the presence of the chaotropic ion thiocyanate, and less susceptible to digestion in the presence of sperm lysin, trypsin, and pronase. In Bufo arenarum, as in other species, the vitelline envelope appears to be composed of glycoproteins. In contrast to previous results, however, disulfide bonds do not seem to be involved in their structural integrity. Thus, experiments carried out with isolated envelopes as well as with envelopes in situ have demonstrated a lack of effect of disulfide bond breaking agents on envelope stability. Evidence is presented suggesting that the solubility of envelopes in mercaptan solutions, as reported by other laboratories, is likely to be the expression of artifactual results.     

Changes in the protein components of vitelline envelope during oogenesis of a tubiculous polychaete,Schizobranchia insignis

In sabellid polychaetes the vitelline envelopes, in which microvilli with glycocalyx structures at the tips are invested, change in structure during oogenesis. Vitelline envelopes isolated from Schizobranchia oocytes 25–100 μ m and 160–185 μ m in diameter, were analyzed in protein components by iodination, electrophoresis, Western blotting and radioactive labeling technique. The observations demonstrate that the membrane proteins of the vitelline envelopes are not consistent but variable in components during oogenesis. Most of these proteins, particularly the high molecular weight proteins, are PAS-positive glycoproteins, which may have specific carbohydrate residues binding to wheat germ agglutinin. The proteins could be labeled with [³H]valine within 36 h by incubating the whole oocytes in sea water to a high level, indicating that the proteins are actively synthesized by the growing oocytes. Synthetic rates of the proteins differ from each other at one stage and are higher in the small than in the large oocytes in general, suggesting that the membrane proteins are involved in the function of the vitelline envelopes during oogenesis.     

Structural modifications and δ-aminolevulinate dehydratase synthesis/transport in cotyledons of far-red irradiated radish

δ-Aminolevulinate dehydratase (ALAD) is synthesized in the cytoplasm and subsequently transported into the plastids in cotyledons of tar-red irradiated radish ( Raphanus sativus L. ev. Longue Rave Saumonée). The intra-cellular localization of ALAD activity was determined under far-red irradiation and darkness. After 60 h far-red, ALAD was essentially eytoplasmic; 96 h far-red corresponded to a peak in total ALAD activity, as well as the onset of massive ALAD transfer into the plastids, and after 120 h a maximum enzyme activity was located in the plastids. Under dark conditions, essentially no transport of ALAD was noted.
Structural modifications of plastid-eytoplasmic relationships were analysed at the three developmental periods under dark and far-red conditions. 60 h far-red induced a change in the "elasticity" of plastid envelopes leading to envelope proliferations and amoeboid shaped plastids. After 96 h tar-red, membrane proliferations formed a highly evaginated plastid periphery. It is suggested that these membranes form a transport network between the two compartments. Also at 96 h, numerous vesicles, which appear to originate from the endoplasmic reticulum (ER), surround the plastid profile. On the basis of the structural observations, it is proposed that ALAD would be synthesized within the ER and transported toward the plastids via ER extensions, which take the form of smooth vesicles. Our hypothesis is that the transition vesicles and proliferations of the outer envelope membrane undergo a fusion process emptying the content of the vesicle into the intermembrane space of the envelope.     

Regulation by vitamin A of envelope cross-linking in cultured keratinocytes derived from different human epithelia.

When keratinocytes derived from different squamous epithelia are cultured in the absence of vitamin A, they form cross-linked envelopes during the last stage of terminal differentiation. Addition of the vitamin inhibits envelope formation, but the degree of inhibition is not the same for different keratinocyte subtypes. In the presence of low concentrations of retinyl acetate, conjunctival keratinocytes form virtually no cross-linked envelopes; esophageal and vaginal keratinocytes are less sensitive to the vitamin, and epidermal keratinocytes are the least sensitive. The suppression of cross-linked envelope formation is not associated with a proportional decrease in the concentration of involucrin, a precursor of the envelope, but occurs at the level of cross-linking itself, a process dependent on an increase in the intracellular concentration of calcium ions. Keratinocytes in which spontaneous envelope cross-linking has been prevented by retinyl acetate promptly form cross-linked envelopes if Ca2+ is introduced into the cytoplasm.     

Deletion of core components of the plastid protein import machinery causes differential arrest of embryo development in Arabidopsis thaliana

Among the genes that have recently been pinpointed to be essential for plant embryo development a large number encodes plastid proteins suggesting that embryogenesis is linked to plastid localized processes. However, nuclear encoded plastid proteins are synthesized as precursors in the cytosol and subsequently have to be transported across the plastid envelopes by a complex import machinery. We supposed that deletion of components of this machinery should allow a more general assessment of the role of plastids in embryogenesis since it will not only affect single proteins but instead inhibit the accumulation of most plastid proteins. Here we have characterized three Arabidopsis thaliana mutants lacking core components of the Toc complex, the protein translocase in the outer plastid envelope membrane, which indeed show embryo lethal phenotypes. Remarkably, embryo development in the atToc75-III mutant, lacking the pore forming component of the translocase, was arrested extremely early at the two-cell stage. In contrast, despite the complete or almost complete lack of the import receptors Toc34 and Toc159, embryo development in the a tToc33/34 and atToc132/159 mutants proceeded slowly and was arrested later at the transition to the globular and the heart stage, respectively. These data demonstrate a strict dependence of cell division and embryo development on functional plastids as well as specific functions of plastids at different stages of embryogenesis. In addition, our analysis suggest that not all components of the translocase are equally essential for plastid protein import in vivo.     

南方鲶卵巢滤泡细胞和卵膜生成的组织学研究

南方鲶的卵巢滤泡细胞源于卵巢基质细胞,从发生到退化分为零散卵泡膜细胞期、单层扁平泡膜细胞期、多层扁平卵泡膜细胞期、立方形颗粒细胞期柱状颗粒细胞期、颗粒细胞分泌期和颗粒细胞退化期。精孔细胞中发育中滤泡细胞分化形成。初级卵精源于卵母细胞,次级卵膜由晚期滤泡细胞分泌形成。本文还对滤泡细胞和卵膜的作用进行了阐述。     

Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell

Stromules are motile extensions of the plastid envelope membrane, whose roles are not fully understood. They are present on all plastid types but are more common and extensive on non-green plastids that are sparsely distributed within the cell. During tomato fruit ripening, chloroplasts in the mesocarp tissue differentiate into chromoplasts and undergo major shifts in morphology. In order to understand what factors regulate stromule formation, we analysed stromule biogenesis in tobacco hypocotyls and in two distinct plastid populations in tomato mesocarp. We show that increases in stromule length and frequency are correlated with chromoplast differentiation, but only in one plastid population where the plastids are larger and less numerous. We used tobacco hypocotyls to confirm that stromule length increases as plastids become further apart, suggesting that stromules optimize the plastid-cytoplasm contact area. Furthermore, we demonstrate that ectopic chloroplast components decrease stromule formation on tomato fruit chromoplasts, whereas preventing chloroplast development leads to increased numbers of stromules. Inhibition of fruit ripening has a dramatic impact on plastid and stromule morphology, underlining that plastid differentiation status, and not cell type, is a significant factor in determining the extent of plastid stromules. By modifying the plastid surface area, we propose that stromules enhance the specific metabolic activities of plastids.     

Cation interactions and biochemical composition of the cell envelope of a marine bacterium

Envelopes of a marine isolate, c-A1, and of a terrestrial isolate, 121, were compared for their susceptibility to disintegration in distilled water after exposure to 0.05 m MgCl(2) and to 0.1 and 1.0 m NaCl. After exposure to MgCl(2) alone, both types of envelopes remained intact in distilled water. Envelopes of marine isolate c-A1, but not of the terrestrial isolate, fragmented in distilled water after exposure to 1.0 m NaCl. Partial reaggregation of the c-A1 envelope fragments occurred on addition of MgCl(2). In cation-exchange experiments, bound Mg(++) in the envelopes of both organisms was displaced by Na(+). The envelopes of c-A1 were found to contain lipopolysaccharide, muramic acid, and a variety of phospholipids, of which the major component was phosphatidylethanolamine, accompanied by lesser amounts of phosphatidic acid, diphosphatidylglycerol, and phosphatidylserine. Analyses of envelope acid hydrolysates revealed a similar amino acid distribution in the marine and terrestrial isolates, but envelopes of c-A1 had less than half the total amino acid content of envelopes of 121 per envelope dry weight. Possible relationships between cations and biochemical components of the envelopes are considered in terms of differences in behavior of the two organisms in low ionic environments.     

Dynamics of the nuclear envelope and of nuclear pore complexes during mitosis in the Drosophila embryo

Early embryonic development in Drosophila melanogaster is marked by a series of thirteen very rapid (10-15 min) and highly synchronous nuclear divisions, the last four of which occur just beneath the embryo surface. A total of some 6000 blastoderm nuclei result, which are subsequently enclosed by furrow membranes to form the cellular blastoderm. We have examined the fine structure of nuclear division in late syncytial embryos. The mitotic spindle forms adjacent to the nuclear envelope on the side facing the embryo surface. During prophase, astral microtubules deform the nuclear envelope which then ruptures at the poles at the onset of prometaphase. The nuclear envelope remains essentially intact elsewhere throughout mitosis. A second envelope begins to form around the nuclear envelope in prometaphase and is completed by metaphase; the entire double layered structure, referred to as the spindle envelope, persists through early in the ensuing interphase. Pole cell spindles are enclosed by identical spindle envelopes. Interphase and prophase nuclei contain nuclear pore complexes (PCs) of standard dimensions and morphology. In prometaphase PCs become much less electron-dense, although they retain their former size and shape. By metaphase, no semblance of PC structure remains, and instead, both layers of the spindle envelope are interrupted by numerous irregular fenestrae. PCs are presumably disassembled into their component parts during mitosis, and reassembled subsequently. Yolk nuclei remain among the central yolk mass when most nuclei migrate to the surface, cease to divide, yet become polyploid. These nuclei nonetheless lose and regain PCs in synchrony with the dividing blastoderm nuclei. In addition, they gain and lose a second fenestrated membrane layer with the same timing. Cytoplasmic membranes containing PCs (annulate lamellae) also lose and regain pores in synchrony with the two classes of nuclear envelopes. The factors that affect the integrity of PCs in dividing blastoderm nuclei appear to affect those in other membrane systems to an equivalent degree and with identical timing.