Small Peripheral Vesicles Research Articles

A relapse of pemphigus vulgaris in pemphigus herpetiformis or a phenotypic “switch”

Sir, Pemphigus herpetiformis (PH) was originally described by Jablonska et al. in 1975. Clinically, PH presents itself as a herpetiform dermatitis with immunopathological characteristics of pemphigus [1,2]. We report an exceptional case of typical pemphigus vulgaris (PV) relapsing after 36 years in PH. A 65-year-old patient, followed for PV for 36 years and treated with corticosteroid therapy with a remission for more than thirty years, consulted for pruriginous lesions evolving for the previous eight months. A dermatological examination revealed urticariform pruriginous ring lesions surmounted by small peripheral vesicles spread throughout the body (Fig. 1), sparing the mucous membranes, and without Nikolsky’s sign. After two non-specific skin biopsies, the histological examination revealed an intraepidermal bubble with acantholytic cells and eosinophilic spongiosis (Figs. 2a and 2b). Direct immunofluorescence confirmed the diagnosis of pemphigus and indirect immunofluorescence was at the upper limit. The diagnosis of a PV relapse in PH was retained and a dapsone-based treatment was initiated at a dose of 150 mg/day and stopped seven days later when met with hemolytic anemia. Oral corticosteroid therapy involving prednisone at a dose of 1 mg/kg/day was initiated but, given the persistence of the pruritus, the decision was to combine methotrexate at a dose of 12.5 mg/week. A good evolution and a decline within eight months were observed. An improved pruritus and the disappearance of the skin lesions were achieved after one month of treatment. PV and PH are two different anatomical and clinical entities of the autoimmune disease pemphigus, with distinct clinical, histopathological, and immunopathological characteristics [1,2]. Our observation documents a complete phenotypic “switch” of pemphigus with a transition from PV to PH both clinically, histologically, and immunologically. Several rare cases of PV switching to superficial pemphigus (SP) (“phenotypic switch”) have, since 1991, been reported, with a higher frequency this direction than otherwise; the transition period varies from six months to twenty years [3]. To the best of our knowledge, no case has been described of a progression from PV to PH. Having observed one firsthand, we are first to describe the case of a complete phenotypic switch from PV to PH. The mechanism of such a transition remains poorly understood and is often observed during a relapse. Some authors suggest that the effect of immunosuppressants on the desmoglein DSG3 more marked than on DSG1 could explain the relapse of PS in PH [3,4]. Future studies on the immunological factors and predictors of PV relapses after the discontinuation of treatment would be useful to better understand the mechanisms of a relapse in pemphigus, with or without a phenotypic transition.

Clinical, Confocal, and Morphological Investigations on the Cornea in Human Mucopolysaccharidosis IH-S

The aim was to describe the confocal and histological findings of 2 corneas from a patient with an advanced case of type I mucopolysaccharidoses Hurler-Scheie disease (MPS IH-S). Both corneas from an MPS IH-S-affected patient were examined in vivo using confocal microscopy and then removed and processed for evaluation using light microscopy and transmission and scanning electron microscopy. Confocal microscopy evaluation showed basal epithelial cells with either diffuse or granular hyperreflectivity. Keratocytes were highly reflective determining a web-shaped stromal appearance. Endothelial cells were barely visible. The histopathological study demonstrated superficial cells with apical microfolds, small vesicles, and evident intercellular junctions. The wing cells showed either well-evident tonofilaments and small peripheral vesicles, or large paranuclear vesicles. The basal cells showed polygonal shapes, many small vesicles, and enlarged intercellular spaces. The Bowman layer was either normal or thinner and was formed by variably electron dense material. In the stroma, irregularly oriented lamellae, many vesicle-filled keratocytes, and intercellular granular material were present. The Descemet membrane was normal, whereas the corneal endothelium showed marked degenerative changes. The confocal alterations appeared consequent to the anomalous accumulation of material. The histopathological images gave a clue to better understand the corneal changes demonstrated by the confocal studies in MPS IH-S.

Characterization of a tyrosine phosphatase activity in the oogenesis ofPeriplaneta americana

In this work, phosphatase activity was characterized in the ovary and the haemolymph of Periplaneta americana. The optimum pH for these activities was 4.0, and a temperature of 44 degrees C was ideal for the maximal enzyme activity. The phosphatase activities were inhibited by NaF, sodium tartrate, Pi, sodium orthovanadate, and ammonium molybdate. The ovarian phosphatase activity at pH 4.0 was almost exclusive against phosphotyrosine, with little or no effect on the residues of phosphoserine or phosphothreonine. These results indicate that this phosphatase activity is due to the presence of an acid tyrosine phosphatase. The phosphatase activities of acid extracts from P. americana ovaries (OEX) and an acid extract from P. americana haemolymph (HEX) were analyzed in non-denaturant gel electrophoresis using an analog substrate beta-naphtyl phosphate. The gel revealed two bands with phosphatase activity in the ovary and one band in the haemolymph; these bands were excised and submitted to a 10% SDS-PAGE showing a single 70-kDa polypeptide in both samples. Histochemistry of the ovary with alpha-naphtyl phosphate for localization of acid phosphatase activity showed mainly labeling associated to the oocyte peripheral vesicles, basal lamina, and between follicle cells. Electron microscopy analysis showed that acid phosphatase was localized in small peripheral vesicles in the oocyte, but not inside yolk granules. The possible role of this phosphatase during oogenesis and embryogenesis is also discussed in this article.

Akt Activation Is Required at a Late Stage of Insulin-Induced GLUT4 Translocation to the Plasma Membrane

Insulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortex-this process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PM-this process may be the major Akt-dependent step in the insulin regulation of glucose transport.

The endocytic pathway in Entamoeba histolytica.

We studied the endocytic pathway of Entamoeba histolytica using (a) confocal laser scanning microscopy to observe living cells labeled with fluorescent probes and (b) transmission electron microscopy of cells incubated in the presence of gold-labeled proteins or in a cytochemical medium designed for the localization of acid phosphatase activity. Images of acridine-orange-labeled cells showed that most of the intracellular vacuoles were acidic and were also labeled with Lucifer yellow, a fluorescent dye widely used for labeling of compartments of the endocytic pathway. A similar labeling pattern was observed when the cells were incubated in the present of fluorescein-labeled bovine albumin. However, no labeling was observed when fluorescein-labeled transferrin was used. Gold-labeled proteins (albumin, transferrin, horseradish peroxidase, and lactoferrin) were used for further characterization of the endocytic pathway. With the exception of transferrin, all the proteins bound to the protozoan surface and were subsequently internalized, appearing in small peripheral vesicles and some tubular structures. The ingested molecules accumulated in large vesicles located in the more central portion of the cell, which also presented acid phosphatase activity.

ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

Immunocytochemical comparison of peripheral vesicles in zoospores ofPhytophthoraandPythiumspecies

An ultrastructural and immunocytochemical comparison has been made of vesicles in the peripheral cytoplasm of zoospores of Phytophthora cinnamomi and species of the related Oomycete genus, Pythium. Our results give evidence of three morphologically and immunologically distinct vesicle populations in Pythium aphanidermatum and Py. butleri. Large peripheral vesicles can be recognized by their size and morphology, and by labeling with monoclonal antibody, Cpa-2 raised against a P cinnamomi antigen. They occur predominantly on the dorsal surface of the zoospores and are retained within the cell during encystment. After encystment, the large peripheral vesicles move away from the plasma membrane and become distributed throughout the cyst cytoplasm, in a manner similar to that observed in P. cinnamomi. There are also small vesicles in the Pythium zoospore cortex. Some are identified as ventral vesicles through their reaction with monoclonal antibody Vsv-1, raised against a P. cinnamomi antigen. The ventral vesicles are concentrated along the ridges of the groove on the ventral surface of the zoospores, and their contents are secreted during encystment. The secreted material remains localized on the cyst surface and marks the site of germ tube emergence, as it does in P. cinnamomi. Other small peripheral vesicles occur on the dorsal surface of the Pythium zoospores. These vesicles are not labeled by any of the three antibodies tested but disappear during encystment, suggesting that their contents are secreted. Our results give strong support to the idea that three types of peripheral vesicles are a common feature of zoospores throughout the Peronosporales, and that they have similar fates during encystment.

Subcellular distribution of tissue kallikrein and Na,K-ATPase alpha-subunit in rat parotid striated duct cells.

Intracellular protein distribution and sorting were examined in rat parotid striated duct cells, in which tissue kallikrein is apical, and Na,K-ATPase is basolateral. Electron-microscopic immunogold cytochemistry, with both polyclonal and monoclonal antibodies, demonstrated these enzymes at opposite poles of the cells and in distinct intracellular sites. Kallikrein was found within apical secretory granules, whereas Na,K-ATPase was present on basolateral cell membranes. In addition, kallikrein was localized throughout cisternae of all Golgi profiles, whereas Na,K-ATPase (α-subunit) was found only in small peripheral vesicles and/or lateral cisternal extensions of a basal subset of Golgi profiles. These differences in the subcellular distribution of the two marker antigens were most clearly seen with double immunogold labelling. Our results suggest that kallikrein, an apical, regulated secretory protein, and Na,K-ATPase, a basolateral, constitutively transported membrane protein, are segregated at (or prior to) the level of the Golgi apparatus rather than in the trans-Golgi network (TGN), as was expected.

Ultrastructural and Light Microscopic Localization of Carbohydrates and Peroxidase/Catalases in Lagenidium giganteum Zoospores

Zoospores of the oomycete Lagenidium giganteum specifically recognize and then encyst upon mosquito larvae. As a step towards understanding the basis for this specific recognition, we examined the ultrastructure and cytochemistry of the zoospores. Zoospore shape and organellar arrangement are similar in L. giganteum and other secondary-type oomycete zoospores. The ultrastructure of zoospores of L. giganteum differs from other Oomycetes mainly in details of vesicle morphology and mitochondrial arrangement. In our studies, we emphasized the outer surface of the zoospores where recognition molecules are likely to be present. Using silver methenamine and thiosemicarbazide-silver proteinate stain sequences, carbohydrate was found at the plasma membrane of zoospores. Ferritin- and fluorescein-labelled concanavalin A binding patterns indicated α-D-mannosyl and/or α-D-glucosyl residues are exposed on the outer surface of the plasma membrane of the zoospore and the wall of the cyst. Within the zoospores, the membranes of small peripheral vesicles, and the membranes and contents of golgi apparati and peripheral cisternae showed evidence of carbohydrate content with silver staining. We did not find clear evidence of carbohydrates among the contents of the large and small peripheral vesicles. Zoospores treated with diaminobenzidine to visualize peroxidases and catalases exhibited increased light and electron opacity in their mitochondria, as well as a fuzzy coat on the external surfaces of their plasma membranes.

Ultrastructural and Light Microscopic Localization of Carbohydrates and Peroxidase/Catalases inLagenidium GiganteumZoospores

Zoospores ofthe oomycete Lagenidium giganteum specifically recognize and then encyst upon mosquito larvae. As a step towards understanding the basis for this specific recognition, we examined the ultrastructure and cytochemistry of the zoospores. Zoospore shape and organellar arrangement are similar in E giganteum and other secondary-type oomycete zoospores. The ultrastructure of zoospores of L. giganteum differs from other Oomycetes mainly in details of vesicle morphology and mitochondrial arrangement. In our studies, we emphasized the outer surface of the zoospores where recognition molecules are likely to be present. Using silver methenamine and thiosemicarbazide-silver proteinate stain sequences, carbohydrate was found at the plasma membrane of zoospores. Ferritin- and fluorescein-labelled concanavalin A binding patterns indicated a-D-mannosyl and/or a-D-glucosyl residues are exposed on the outer surface of the plasma membrane of the zoospore and the wall of the cyst. Within the zoospores, the membranes of small peripheral vesicles, and the membranes and contents of golgi apparati and peripheral cisternae showed evidence of carbohydrate content with silver staining. We did not find clear evidence of carbohydrates among the contents of the large and small peripheral vesicles. Zoospores treated with diaminobenzidine to visualize peroxidases and catalases exhibited increased light and electron opacity in their mitochondria, as well as a fuzzy coat on the external surfaces

Immunolocalization of Endosomal Cathepsin D in Rabbit Alveolar Macrophages

Intravacuolar proteolysis appears to be an important component of antigen presentation, the activation of peptide hormones, and the conversion of biologically important mediators from inactive precursors. Cathepsin D has been identified in the endosomes of rabbit alveolar macrophages by biochemical analyses [Diment and Stahl, J. Biol. Chem. 260,15311, 1985; Diment et al., J. Biol. Chem. 263,6901, 1988]. Using affinity-purified goat antirabbit cathepsin D IgG, we have localized cathepsin D to the endosomes of rabbit alveolar macrophages. Immunofluorescent staining of frozen sections showed labeling in lysosomes and small vesicles in the periphery of the cell. Label was not seen on the plasma membrane. With immunoperoxidase labeling at the electron microscopic level on cells containing endocytosed mannose-BSA gold, we saw labeling in endosomes and classical lysosomes. When the results were quantitated using immunogold labeling of thin cryosections, we found that the majority of cathepsin D (62.2%) was present in lysosomes, 4.0% in large clear vacuoles, a surprisingly high percentage (29.3%) in small vesicles, 4.9% in endosomes, and none on the plasma membrane. We conclude from this study that, in addition to being present in lysosomes, cathepsin D is present in endosomes and in small peripheral vesicles.

Glycoconjugates on the surface of spores of the pathogenic fungus Phytophthora cinnamomi studied using fluorescence and electron microscopy and flow cytometry

Glycoconjugates on the surface of zoospores and cysts of the pathogenic fungus Phytophthora cinnamomi have been studied using fluorescein isothiocyanate labelled lectins for fluorescence microscopy and flow cytometry, and ferritin- and gold-labelled lectins for ultrastructural analysis. Of the five lectins used, only concanavalin A (ConA) binds to the surface of the zoospores, including the flagella and water expulsion vacuole. This suggests that of accessible saccharides, glucosyl or mannosyl residues predominate on the outer surface of the zoospore plasma membrane. Early in encystment, a system of flat disc-like cisternae, which underlie the zoospore plasma membrane, vesiculate. These and other small peripheral vesicles quickly disappear. After the induction of encystment, ConA is no longer localised close to the plasma membrane but binds to material loosely associated with the cell surface. Quantitative measurements by flow cytometry indicate that the ConA-binding material is gradually lost from the cell surface. The cyst wall is weakly labelled, but the site of germ tube emergence stains intensely. During the first 2 min after the induction of encystment, material that binds soybean agglutinin, Helix pommatia agglutinin, and peanut agglutinin appears on the surface of the fungal cells. The distribution of this material, rich in galactosyl or N-acetyl-D-galactosaminosyl residues, is initially patchy, but by 5 min the material evenly coats most of the cell surface. Labelling of zoospores in which intracellular sites are accessible indicates that the soybean agglutinin binding material is stored in vesicles that lie beneath the plasma membrane. Quantitation of soybean agglutinin labelling shows that maximum binding occurs 2–3 min after the induction of encystment. Key words: cell surface, flow cytometry, lectins, pathogenic fungi, Phytophthora cinnamomi.

Lectin and Antibody Labelling of Surface Components of Spores of Phytophthora cinnamomi

Molecules on the surface of zoospores and cysts of soilborne fungi such as Phytophthora cinnamomi are important mediators in the early stages of infection of host plants. Surface components of P. cinnamomi cells have been analysed ultrastructurally and at the molecular level using lectins and monoclonal antibodies, with a view to localising and characterising molecules involved in the infection process. Staining with ruthenium red and the lectin, concanavalin A, indicates the presence of a glycocalyx over the surface of the zoospores. Localised antibody binding also reveals that the zoospore surface is subdivided into at least three distinct molecular domains. During encystment, the spectrum of molecules on the cell surface changes dramatically with the secretion of material which is rich in N-acetyl-D-galactosamine and which bonds the cells to any adjacent structure. lmmunolabelling shows that this material is stored in small peripheral vesicles and is secreted 1-3 min after encystment is induced. Encystment can be triggered by the binding of a monoclonal antibody to the flagella, suggesting that specific receptors may be localised there. Surface components on the fungal cells display a variety of taxonomic specificities including those for species and genus. Monoclonal antibodies which bind to these components promise to be valuable tools for diagnostic use.

Secretion of adhesive material during encystment of Phytophthora cinnamomi zoospores, characterized by immunogold labelling with monoclonal antibodies to components of peripheral vesicles

ABSTRACT During encystment, zoospores of the dieback fungus Phytophthora cinnamomi attach themselves to the plant root surface by secretion of adhesive material. Previous workers have proposed, on the basis of ultrastructural studies, that the adhesive material is released by rapid exocytosis of large vesicles that underlie the zoospore plasma membrane. In the present study a monoclonal antibody (Cpa-2) that binds to the adhesive material has been used to investigate the sites of storage of this material in the zoospores, and the timing and pattern of its secretion during encystment. Immunolabelling of thin sections of zoospores with Cpa-2 followed by gold-labelled secondary antibodies has revealed that the adhesive material is not stored in the large peripheral vesicles but in a population of small vesicles in the cell periphery. In studies of other Oomycete fungi, these small vesicles have usually been described as microbodies. Another monoclonal antibody (Lpv-1) does bind to the contents of the large peripheral vesicles. Double immunolabelling of zoospores and encysting cells with Cpa-2-Au10 and Lpv-1-Aui8, coupled with morphometric analysis, has shown that 85% of the adhesive material is secreted within the first minute following induction of encystment and that the large peripheral vesicles are not exocytosed during encystment but instead tend to migrate towards the centre of the cell. Immunoblot analysis with Cpa-2 shows the presence of several high molecular weight bands (Mr> 300 000) which also react with the lectin, soybean agglutinin. These high molecular weight components are susceptible to digestion with Pronase. Lpv-1 also reacts with high molecular weight bands but of different mobility from those recognized by Cpa-2. Concanavalin A binds to bands corresponding to those labelled by Lpv-1. We conclude that adhesion of cysts to plant roots involves the secretion of high molecular weight glycoproteins by rapid exocytosis of small peripheral vesicles and that the material contained in the large peripheral vesicles is also glycoprotein in nature but is not secreted during encystment.

Ultrastructure and serial section reconstruction of zoospores of the fungus Phytophthora cinnamomi

Two zoospores of Phytophthora cinnamomi have been reconstructed from serial, 240-nm sections using high voltage electron microscopy. The mitochondrial complement is generally confined to the subperipheral zone of the cytoplasm and consists of 3 or 4 large, reticulate mitochondria and 19–23 small, unbranched mitochondria. In addition to peripheral cisternae and large peripheral vesicles, which also occur in the zoospores of related species, the peripheral cytoplasm of the P. cinnamomi zoopsores contains small, usually spherical vesicles. There are 105 and 121 large peripheral vesicles and 132 and 168 small peripheral vesicles in the two reconstructed cells. The morphology of the small peripheral vesicles is variable, with different forms showing evidence of regional localization. The cells also contain 45 and 84 endoplasmic reticulum-associated microbody-like organelles and 8–11 stacks of Golgi cisternae. These data, along with information on the structure and disposition of other organelles, provide a basis for ultrastructural comparisons of P. cinnamomi zoospores with those of other Oomycetes, and for studies of the structural and biochemical changes that occur during the early stages of infection of host plants.

Immunoelectron-microscopic studies of endoplasmic reticulum-Golgi relationships in the intracellular transport process of lipoprotein particles in rat hepatocytes.

Endoplasmic reticulum (ER)-Golgi relationships in the intracellular transport process of secretory proteins in rat hepatocytes have been studied using lipoprotein particles as a marker for the secretory protein and cytochrome P-450 as a marker enzyme for the ER membranes. Ferritin immunoelectron-microscopic observation revealed that, while almost all the microsomal vesicles derived from ER membranes are heavily labelled with ferritin anti-cytochrome P-450 antibody conjugates, labelling of the small peripheral vesicles containing lipoprotein particles, the stacks of Golgi saccules, especially the outermost saccule which is sometimes fenestrated, condensing vacuoles in the trans-Golgi region and the secretion droplets of lipoprotein were scanty and at the control level. Such a characteristic pattern of labelling was especially evident when these structures were prepared from phenobarbital-treated rats. These findings indicate that the membranes of the small peripheral vesicles do not contain cytochrome P-450 and that the cytochrome is probably not transferred to Golgi saccules in the transport process of lipoprotein from ER to Golgi. It is suggested, therefore, that the small peripheral vesicles are formed by budding of the special regions of ER membrane where microsomal marker proteins such as cytochrome P-450 are excluded and the membrane proteins destined to the Golgi complexes are clustered. It is also shown that lysosomal membranes are not labelled with the anti P-450 antibody conjugates.

The sequence of early cytological changes in Chinese cabbage leaf cells following systemic infection with turnip yellow mosaic virus

The invasion of small systemically infected Chinese cabbage leaves by turnip yellow mosaic virus (TYMV) has been followed by electron microscopy. The presence of small peripheral vesicles in the chloroplasts was used as a marker of infection by the virus. A plasmolysis procedure which induced virus particles to form crystalline arrays with little damage to chloroplast membranes was used to detect the presence of complete virus. Infection spreads out from the small veins of the leaf at about 4 μm/hr. This is equivalent to about 1 cell in 3 hr. Using the vein as a marker for the origin of the infection, we have been able to distinguish seven cytological stages in the establishment of infection. These stages fall into two phases. In the first phase, comprising the first two stages, only scattered peripheral vesicles are present in the chloroplasts. In the second phase the number of vesicles is approximately doubled by the rapid appearance of clustered vesicles. At the beginning of the second phase endoplasmic reticulum (ER) appears closely apposed over parts of the chloroplast surface. Beneath these areas, the clustered vesicles appear. The ER then disappears, and electron lucent zones appear over the clustered vesicles. Arrays of virus particles in plasmolysed tissue are first seen at the fourth stage. At this stage these arrays are always associated with the electron lucent areas, which presumably have some role in virus synthesis or assembly. The last three stages involve clumped chloroplasts. The electron lucent material appears to be involved in the clumping process.

Fine structure of vesicles induced in chloroplasts of Chinese cabbage leaves by infection with turnip yellow mosaic virus.

SUMMARY Examination of chloroplasts from chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV), using freeze-fracture techniques revealed, that the small peripheral vesicles induced by infection were present in clusters near the surface of the chloroplast, often being arranged in arrays that approximate to hexagonal. The small peripheral vesicles were flask shaped with their necks pointing to the chloroplast surface. The structure of the necks, which were only about 15 to 22 nm in diam., suggested but did not prove that they were open to the cytoplasmic space. The inner membrane of the peripheral vesicles is distinguished from other chloroplast membranes in showing no particles on either the A or the B fracture faces. In the regions where the peripheral vesicles were clustered, the number and kind of particles seen on the chloroplast membrane faces were changed compared with healthy chloroplasts or the non-vesicle bearing areas of chloroplasts from infected cells. In particular, the B face of the outer chloroplast membrane in the vesicle-bearing regions had a reduced number of particles 10 nm in diam., but in addition to these contained large numbers of particles about 5 nm in diam. In the chloroplasts of both healthy and infected tissue we frequently observed protuberances of the stroma corresponding to the mobile phase of chloroplasts postulated from light microscopic observations.