Multivesicular compartments proliferate in susceptible and resistant MLA12-barley leaves in response to infection by the biotrophic powdery mildew fungus

There is growing evidence that multivesicular bodies and cell wall-associated paramural bodies participate in the enhanced vesicle trafficking induced by pathogen attack. Here, we performed transmission electron microscopy in combination with cytochemical localization of H2O2 to investigate multivesicular compartments during establishment of compatible interaction in susceptible barley (Hordeum vulgare) and during hypersensitive response in resistant MLA12-barley infected by the barley powdery mildew fungus (Blumeria graminis f. sp. hordei). Multivesicular bodies, intravacuolar vesicle aggregates and paramural bodies proliferated in the penetrated epidermal cell during development of the fungal haustorium. These vesicular structures also proliferated at the periphery of intact cells, which were adjacent to the hypersensitive dying cells and deposited cell wall appositions associated with H2O2 accumulation. All plasmodesmata between intact cells and hypersensitive cells were constricted or blocked by cell wall appositions. These results suggest that multivesicular compartments participate in secretion of building blocks for cell wall appositions not only to arrest fungal penetration but also to contain hypersensitive cell death through blocking plasmodesmata. They may also participate in internalization of damaged membranes, deleterious materials, nutrients, elicitors and elicitor receptors.     

Paramural Bodies in Callus Beside the Isolation Layer of the Graft Union

The isolation layer of the graft union is a changeable component. It is formed and thickened during the early stage (Fig. 6) and disrupted, thinned and even disappeared durmg tile later stage of development of the graft (Fig. 1, 2, 14). A number of vesicles, paramarat bodies (Fig. 3, 5, 8, 9, 11, 13, 19), multivesicular bodies (Fig. 4, 15, 16, 18, 20) and concentric membrane bodies (Fig. 7) are observed in callus 'beside the isolation layer during both stages of development based on transmission electron microscopy. The paramural body comprises invagination of plasmalemma containing numerous vesicles and/or tubules situated between the cell wall and the plasmalemma. The multivesicular body is a organelle about 0.5-μm in diameter with a single membrane surrounding several smaller single vesicles. It is possible that multivesicular body and/or single iesicles transverse the plasmalemma to produce paramural body. The figures show that the paramural bodies appear always beside the isolalion layer at the different stages of development of the graft union and the multivesicular bodies appear mostly near the region where plasmodesmata are secondarily formed between the stock and the scion (Fig. 15, 17, 20). This may reflect that paramural body and multivesi- cular body, as well as single vesicles, are capable of performing vesicular transport. The deposition and reabsorption of material of the isolation layer occur due to vesicular transport. All the above facts seem to indicate that paramural body, multivesicular body and single vesicles can be both endocytotic and exocytotic. The present study supports the theory of vesicular transport, and authors suggest that transcellular cytosis occurs not only through plasmodesmata but also through plasmalemma in oapoplast. The single vesicles, paramural bodies and multivesicular bodies take an active part in the transport process of symplast-apoplast-symplast.     

Endocytosis of 1,3-β-glucans by broad bean cells at the penetration site of the cowpea rust fungus (haploid stage)

Te penetration hypha of basidiospore-derived infection structures of the cowpea rust fungus (Uromyces vignae Barclay) in epidermal cells of the nonhost, broad bean (Vicia faba L.), was studied with the electron microscope after high-pressure freezing and freeze substitution. After fungal invasion of the epidermis, a plug in the penetration hypha separated the infection structures on the cuticle from the intraepidermal vesicle of the fungus. The plug and the fungal cell wall reacted with a polyclonal 1,3-β-glucan antibody. The plug in the haploid stage seems to have a task similar to the septum formed in the diploid stage of the fungus. Around the penetration hypha, the plant wall stained darkly and a papilla was deposited by the plant. In the papilla, 1,3-β-glucans were labelled by a monoclonal and a polyclonal antibody. In the infected epidermal cell, clathrin-coated pits, coated vesicles, partially coated reticula and multivesicular bodies were found. The contents of the coated pits, coated vesicles, partially coated reticula and multivesicular bodies bound to monoclonal and polyclonal 1,3-β-glucan antibodies. Accumulation and uptake of this paramural material into the plant cell by endocytosis is concentrated at the fungal penetration site. It may influence the host-parasite interaction.     

Do Plant Cells Secrete Exosomes Derived from Multivesicular Bodies?

Multivesicular bodies (MVBs) are spherical endosomal organelles containing small vesicles formed by inward budding of the limiting membrane into the endosomal lumen. In mammalian red cells and cells of immune system, MVBs fuse with the plasma membrane in an exocytic manner, leading to release their contents including internal vesicles into the extracellular space. These released vesicles are termed exosomes. Transmission electron microscopy studies have shown that paramural vesicles situated between the plasma membrane and the cell wall occur in various cell wall-associated processes and are similar to exosomes both in location and in morphology. Our recent studies have revealed that MVBs and paramural vesicles proliferate when cell wall appositions are rapidly deposited beneath fungal penetration attempts or during plugging of plasmodesmata between hypersensitive cells and their intact neighboring cells. This indicates a potential secretion of exosome-like vesicles into the extracellular space by fusion of MVBs with the plasma membrane. This MVB-mediated secretion pathway was proposed on the basis of pioneer studies of MVBs and paramural vesicles in plants some forty years ago. Here, we recall the attention to the occurrence of MVB-mediated secretion of exosomes in plants.Key Words: cell wall, endocytosis, endosome, exocytosis, exosome, multivesicular body, paramural bodyMultivesicular bodies (MVBs) are spherical endosomal organelles containing a number of small vesicles formed by inward budding of the limiting membrane into the endosomal lumen.¹ MVBs contain endocytosed cargoes and deliver them into lysosomal/vacuolar compartments for degradation. They also incorporate newly synthesized proteins destined for lysosomal/vacuolar compartments.² In mammalian cells of hematopoietic origin, endosomal MVBs function in removal of endocytosed surface proteins in an exocytic manner. They are redirected to the plasma membrane, where they release their contents including internal vesicles into the extracellular space by membrane fusion. The released vesicles are termed exosomes.³ During reticulocyte maturation to erythrocyte, a group of surface proteins, such as the transferrin receptor, become obsolete and are discarded via MVB-mediated secretion.³ Time-course transmission electron microscopy (TEM) first revealed that colloidal gold-transferrin was internalized into MVBs via receptor-mediated endocytosis and then transferrin together with its receptor were delivered into the extracellular space via the fusion of MVBs with the plasma membrane of reticulocytes.⁴ Some other cell types of hematopoietic origin, such as activated platelets, cytotoxic T cells and antigen-presenting cells, also secrete exosomes. Exosomes thus may play a role in various physiological processes other than discarding obsolete proteins.³Our recent TEM studies provided ultrastructural evidence on the enhanced vesicle trafficking in barley leaf cells attacked by the biotrophic powdery mildew fungus. Multivesicular compartments including MVBs, intravacuolar MVBs, and paramural bodies turned out to proliferate in intact host cells during formation of cell wall appositions (papilla response), in the hypersensitive response, and during accommodation of haustoria.^5,6 MVBs proliferated in the cytoplasm of haustorium-containing epidermal cells during compatible interactions and near sites of cell wall-associated oxidative microburst either during the papilla response or during the hypersensitive response. Because MVBs in plant cells have been demonstrated to be endosomal compartments,^7–9 they may participate in internalization of nutrients from the apoplast of intact haustorium-containing epidermal cells and sequestration of damaged membranes and deleterious materials originating from the oxidative microburst.^5,6 The presence of intravacuolar MVBs with double limiting membranes (Fig. 1A) indicates an engulfment of MVBs by the tonoplast and a vacuole-mediated autophagy of MVBs.^5,6 MVBs, as prevacuolar compartments in plant cells,⁹ thus probably deliver their contents into the central vacuole via both the fusion with the tonoplast and the engulfment by the tonoplast (Fig. 2A and B). On the other hand, paramural bodies, in which small vesicles are situated between the cell wall and the plasma membrane, were associated with cell wall appositions deposited beneath fungal penetration attempts (Fig. 1B) or around hypersensitive cells including sites of plugged plasmodesmata (Fig. 1C and D).^5,6 Because paramural vesicles are similar to exosomes both in location and in morphology, we speculated that MVBs fuse with the plasma membrane in an exocytic manner to form paramural bodies.^5,6 Endocytosed cell surface materials in endosomal MVBs may be reused and delivered together with newly synthesized materials in Golgi apparatus-derived vesicles to cell wall appositions, which are deposited rapidly to prevent fungal penetration (Fig. 2A) or to contain hypersensitive cell death (Fig. 2B). MVBs thus may be driven along two distinct pathways to deliver their contents into either central vacuole or extracellular space.Open in a separate windowFigure 1Multivesicular compartments in intact cells in barley leaves attacked by the barley powdery mildew fungus. (A) An intravacuolar multivesicular body (MVB) with double limiting membranes in an intact epidermal cell (EC) adjacent to a hypersensitive epidermal cell (EC*). The arrows point to the outer limiting membrane, which is seemingly derived from the tonoplast. Note that neighboring intravacuolar vesicles (in between two arrowheads) may result from degradation of double limiting membranes of intravacuolar MVBs or may be delivered into the vacuole by MVB-fusion with the tonoplast. (B) Paramural vesicles (arrowheads) in a paramural body associated with cell wall appositions (asterisk) deposited by an intact epidermal cell. (C) A multivesicular body (MVB) in contact with a paramural body (PMB) (a nonmedian section) associated with cell wall appositions (asterisk) deposited by an intact mesophyll cell adjacent to a hypersensitive mesophyll cell. Note that cell wall appositions deposit beside an intercellular space (IS). The arrows point to the tonoplast. (D) A paramural body (PMB) associated with cell wall appositions (asterisks) blocking plasmodesmata (in between two arrowheads) at the side of an intact mesophyll cell (MC) underlying a hypersensitive epidermal cell (EC*). The arrows point to the tonoplast. CV, central vacuole; CW, cell wall; MB, microbody. Bars, 1µm.Open in a separate windowFigure 2Hypothetical diagram of delivery of endocytosed cell surface materials via MVBs into the central vacuole or the extracellular space where intact barley cells deposit cell wall appositions. (A) Deposition of cell wall appositions (asterisk) beneath powdery mildew penetration attempts. AGT, appressorial germ tube; PP, penetration peg. (B) Deposition of cell wall appositions (asterisks) against constricted plasmodesmata (PD) between a hypersensitive epidermal cell (EC) penetrated by the powdery mildew fungus and an underlying mesophyll cell (MC). H, haustorium. Arrows and numbers show pathways of vesicle trafficking. 1, Secretion of Golgi-derived vesicles containing newly synthesized materials; G, Golgi body; TGN, trans-Golgi network; 2, Endocytosis of cell surface materials from coated pits (coated open circles) via coated vesicles (coated circles) to multivesicular bodies (MVB); 3, Delivery of endocytosed materials for degradation inside the central vacuole (CV) via membrane fusion between MVBs and the tonoplast (T); small broken circles, vesicles in degradation; 4, Delivery of endocytosed materials for degradation inside the central vacuole via engulfment of MVBs by the tonoplast; large broken circles; MVB limiting membranes in degradation; 5, delivery of endocytosed materials into the extracellular space for deposition of cell wall appositions (asterisks) via membrane fusion between MVBs and the plasma membrane (PM). CW, cell wall; PMB, paramural body. PD0, 1, 2, 3 and 4 represent stages of plugging plasmodesmata. PD0, open plasmodesmata between two intact mesophyll cells (MC) subjacent to the hypersensitive epidermal cell (EC); PD1, constriction of plasmodesmata by callose (grey dots) deposition at plasmodesmal neck region; PD2, constricted plasmodesmata associated with plasmodesma-targeted secretion; PD3, further blocking of plasmodesmata by deposition of cell wall appositions; PD4, completely blocked plasmodesmata.Earlier than the discovery in animal cell systems,⁴ it was proposed in two independent papers in 1967 that the fusion of MVBs with the plasma membrane might result in the release of small vesicles into the extracellular space in fungi and in higher plants.^10,11 Several lines of evidence support the occurrence of MVB-mediated secretion of exosome-like vesicles in plants. First, vesicles of the same morphology as MVB internal vesicles have been observed in extracellular spaces or paramural spaces in various types of plant cells in various plant species by TEM.¹² An early study on endocytosis by soybean protoplasts also showed small extracellular vesicles attaching on the plasma membrane.⁸ Second, cooccurrence of MVBs and paramural vesicles has been observed in processes of cell proliferation, cell differentiation, and cell response to abiotic and biotic stress. Examples are cell plate formation,^13,14 secondary wall thickening,^15,16 cold hardness,^17,18 and deposition of cell wall appositions upon pathogen attack.^5,6,19–21 Third, identical molecular components, such as arabinogalactan proteins^22,23 and peroxidases,⁶ have been immunolocalized in both MVBs and paramural bodies. Despite these pieces of evidence, a conclusive demonstration of MVB-mediated secretion of exosomes in plants requires further exploration.The presently available experimental systems, approaches, and membrane markers may allow future demonstration of MVB-mediated secretion of exosomes in plants. Recent in vivo real-time observation and colocalization of cell surface and endosomal markers have already revealed that endosomes filled with endocytosed preexisting cell wall and plasma membrane materials are rapidly delivered to cytokinetic spaces to form cell plates in dividing tobacco, Arabidopsis, and maize cells.²⁴ Because TEM observed paramural bodies attaching to cell plates¹³ and MVBs in the vicinity of cell plates during all stages of cell plate formation,^14,25,26 MVBs and paramural bodies may participate in delivery of endocytosed building blocks to cell plates. Jiang''s and Robinson''s labs together developed a transgenic tobacco BY-2 cell line stably expressing a YFP-labeled vacuolar sorting receptor protein and antibodies against the vacuolar sorting receptor protein localized to the limiting membrane of MVBs.⁹ These tools together with live cell imaging and immunoelectron microscopy may allow visualization of MVB-fusion to the new plasma membrane, of vacuolar sorting receptors in both the limiting membrane of MVBs and the new plasma membrane, and of identical cell plate components in both internal vesicles of MVBs and paramural vesicles.In spite of obvious differences in plant and animal cytokinesis, the generation of cell plates by cell-plate-directed fusion of endosomes resembles the plugging of midbody canals by midbody-directed endosomes to separate daughter cells at the terminal phase of animal cytokinesis.²⁷ Likely, functional similarities of the fusion between endosomal MVBs and the plasma membrane to eliminate unwanted cell contents may also exist in maturation of mammalian red blood cells and plant sieve elements in the sense that the fusion of MVBs with the plasma membrane may occur during maturation of the latter.²⁸ On the other hand, although plant cells may secrete MVB-derived exosomes in defense response upon pathogen attack,^5,6 plant cell walls rule out the direct intercellular communication during the immune response mediated by exosomes in the circulation of mammals.³ In contrast, plasmodesma-directed secretion of exosomes would block the cell-to-cell communication between hypersensitive cells and their neighboring cells during hypersensitive response.⁵ Further exploration will lead us to a better understanding of similarities and differences of exosome secretion between plants and animals.     

Secretory Activity Is Rapidly Induced in Stigmatic Papillae by Compatible Pollen,but Inhibited for Self-Incompatible Pollen in the Brassicaceae

[In the Brassicaceae, targeted exocytosis to the stigmatic papillar plasma membrane under the compatible pollen grain is hypothesized to be essential for pollen hydration and pollen tube penetration. In contrast, polarized secretion is proposed to be inhibited in the stigmatic papillae during the rejection of self-incompatible pollen. Using transmission electron microscopy (TEM), we performed a detailed time-course of post-pollination events to view the cytological responses of the stigmatic papillae to compatible and self-incompatible pollinations. For compatible pollinations in Arabidopsis thaliana and Arabidopsis lyrata, vesicle secretion was observed at the stigmatic papillar plasma membrane under the pollen grain while Brassica napus stigmatic papillae appeared to use multivesicular bodies (MVBs) for secretion. Exo70A1, a component of the exocyst complex, has been previously implicated in the compatible pollen responses, and disruption of Exo70A1 in both A. thaliana and B. napus resulted in a loss of secretory vesicles/MVBs at the stigmatic papillar plasma membrane. Similarly, for self-incompatible pollinations, secretory vesicles/MVBs were absent from the stigmatic papillar plasma membrane in A. lyrata and B. napus; and furthermore, autophagy appeared to be induced to direct vesicles/MVBs to the vacuole for degradation. Thus, these findings support a model where the basal pollen recognition pathway in the stigmatic papilla promotes exocytosis to accept compatible pollen, and the basal pollen recognition pathway is overridden by the self-incompatibility pathway to prevent exocytosis and reject self-pollen.     

Biogenesis of a specialized plant-fungal interface during host cell internalization of Golovinomyces orontii haustoria

Powdery mildew fungi are biotrophic pathogens that require living plant cells for their growth and reproduction. Elaboration of a specialized cell called a haustorium is essential for their pathogenesis, providing a portal into host cells for nutrient uptake and delivery of virulence effectors. Haustoria are enveloped by a modified plant plasma membrane, the extrahaustorial membrane (EHM), and an extrahaustorial matrix (EHMx), across which molecular exchange must occur, but the origin and composition of this interfacial zone remains obscure. Here we present a method for isolating Golovinomyces orontii haustoria from Arabidopsis leaves and an ultrastructural characterization of the haustorial interface. Haustoria were progressively encased by deposits of plant cell wall polymers, delivered by secretory vesicles and multivesicular bodies (MVBs) that ultimately become entrapped within the encasement. The EHM and EHMx were not labelled by antibodies recognizing eight plant cell wall and plasma membrane antigens. However, plant resistance protein RPW8.2 was specifically recruited to the EHMs of mature haustoria. Fungal cell wall-associated molecular patterns such as chitin and β-1,3-glucans were exposed at the surface of haustoria. Fungal MVBs were abundant in haustoria and putative exosome vesicles were detected in the paramural space and EHMx, suggesting the existence of an exosome-mediated secretion pathway.     

Multivesicular bodies in the transitional epithelium of the neonatal mouse urinary border.

The origin of late endosomes - multivesicular bodies (MVBs) in the superficial cells of 16 and 17 embryonic old transitional epithelium of mouse urinary bladder was studied by electron microscopy, lectin labelling and HRP tracing. Analysis of hexagonally structured membrane particles, WGA, and RCA I binding sites revealed structural similarity between plasmalemma, fusiform vesicles and multivesicular bodies. Early endosomes are lined by symmetric unit membrane as well as by asymmetric thickened membrane regions. Multivesicular bodies and fusiform vesicles have asymmetric unit membranes. MVBs may be derived from primary endosomes as well as from fusiform vesicles in the cytoplasm.     

The multivesicular body-localized GTPase ARFA1b/1c is important for callose deposition and ROR2 syntaxin-dependent preinvasive basal defense in barley

Host cell vesicle traffic is essential for the interplay between plants and microbes. ADP-ribosylation factor (ARF) GTPases are required for vesicle budding, and we studied the role of these enzymes to identify important vesicle transport pathways in the plant-powdery mildew interaction. A combination of transient-induced gene silencing and transient expression of inactive forms of ARF GTPases provided evidence that barley (Hordeum vulgare) ARFA1b/1c function is important for preinvasive penetration resistance against powdery mildew, manifested by formation of a cell wall apposition, named a papilla. Mutant studies indicated that the plasma membrane-localized REQUIRED FOR MLO-SPECIFIED RESISTANCE2 (ROR2) syntaxin, also important for penetration resistance, and ARFA1b/1c function in the same vesicle transport pathway. This was substantiated by a requirement of ARFA1b/1c for ROR2 accumulation in the papilla. ARFA1b/1c is localized to multivesicular bodies, providing a functional link between ROR2 and these organelles in penetration resistance. During Blumeria graminis f sp hordei penetration attempts, ARFA1b/1c-positive multivesicular bodies assemble near the penetration site hours prior to the earliest detection of callose in papillae. Moreover, we showed that ARFA1b/1c is required for callose deposition in papillae and that the papilla structure is established independently of ARFA1b/1c. This raises the possibility that callose is loaded into papillae via multivesicular bodies, rather than being synthesized directly into this cell wall apposition.     

Fine Structure of Cryptococcus neoformans Grown In Vivo as Observed by Freeze-Etching

Cryptococcus neoformans grown in the parasitic state was observed by the freeze-etching technique and was compared with that grown on culture media. Unlike other yeasts, this organism grown in vivo is very often devoid of the "ordinary" invaginations. The membrane of the cell grown in vivo was almost free from concavity and convexity except for many round depressions which represent the surface view of paramural bodies. Some of the paramural bodies were found to be multivesicular systems. Most were spherical invaginations containing a single vesicle or its ghost remaining after secretion of the vesicles. In clear contrast to the cell grown in vitro, the in vivo cell contained a great number of vesicles in the cytoplasm. These seemed to show high-secretion activity in C. neoformans grown in the parasitic state. On transfer from in vitro to in vivo, this organism enlarged the cell wall, capsule, and cell body. The appearance of a large vacuole, accumulation of storage organelles, and the existence of rodlike structures, seemingly lipid deposits, were also noted in the cytoplasm of the cell grown in vivo. the meaning of these results as well as the mode of capsular production are discussed.     

Fine Structure of Cryptococcus neoformans Grown In Vitro as Observed by Freeze-Etching

Cryptococcus neoformans grown on culture media was observed by the freeze-etching technique. In the capsule, short fibrils were seen when freezeetched. This organism was unique in the appearance of the cell wall, which showed two strata. The outer one was dense with particles of about 20 nm in diameter, whereas the inner one was sparse in particles. The appearance of the cell membrane of this organism differed distinctly depending on the culture media. When grown on glycerol medium, the cell membrane possessed, as do other yeasts, clear but somewhat longer and curved invaginations. The membrane of cells grown on nonglycerol medium exhibited, however, only a few invaginations of irregular shape. Instead, characteristically of this organism, the cell membrane showed round depressions of 40 to 200 nm in diameter which were the surface view of the paramural bodies. In cross-fractured cells, both types of paramural bodies were found. Some of them contained a single vesicle of about 50 nm in diameter. These seem to play a role in secreting the cytoplasmic vesicles. Data suggesting the existence of multivesicular bodies in the cytoplasm and multivesicular lomasomes were also obtained. Some of the baglike paramural bodies showed multilayered membrane. These are thought to be plasmalemmasomes. This organism was similar to other yeasts reported in other respects.     

Paramural bodies of Albugo candida

The paramural bodies of Albugo candida were formed solely by elaboration of the plasmalemma. Two major forms were recognized: one consisting of plasmalemmal invaginations projecting into the cytoplasm; the other appearing like a pocket containing a number of vesicles and tubules. It is suggested that the first is the basic form of paramural body. In sporangia the paramural bodies break away from the plasmalemma and undergo autodigestion while in vegetative hyphae their tubules and lamellae break up into vesicles that are finally sequestered into the wall.     

Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

The matrix protein of Marburg virus is transported to the plasma membrane along cellular membranes: exploiting the retrograde late endosomal pathway

VP40, the matrix protein of Marburg virus, is a peripheral membrane protein that has been shown to associate with membranes of multivesicular bodies (MVBs) (L. Kolesnikova, H. Bugany, H.-D. Klenk, and S. Becker, J. Virol. 76:1825-1838, 2002). The present study revealed that VP40 is bound to cellular membranes rapidly after synthesis. Time course studies were performed to trace the distribution of VP40 during the course of expression. First, VP40 was homogenously distributed throughout the cytoplasm, although the majority of protein (70%) was already membrane associated. Next, VP40 accumulated in MVBs and in tubular protrusions emerging from MVBs. Finally, VP40 appeared in a patch-like pattern beneath the plasma membrane. These morphological results were supported by iodixanol density gradient analyses. The majority of VP40-positive membranes were first detected comigrating with small vesicles. VP40 was then shifted to fractions containing endosomal marker proteins, and later, to fractions containing plasma membrane marker proteins. Blocking of protein synthesis by use of cycloheximide at the time when VP40 was mainly associated with the small vesicles did not prevent the redistribution of VP40 to the late endosomes and further to the plasma membrane. The inhibition of intracellular vesicular trafficking by monensin significantly reduced the appearance of VP40 at the plasma membrane. In conclusion, we suggest that the transport of the Marburg virus matrix protein VP40 involves its accumulation in MVBs followed by the redistribution of VP40-enriched membrane clusters to the plasma membrane.     

The role of EGF receptor ubiquitination in regulating its intracellular traffic

Progression of activated EGF receptor (EGFR) through the endocytic pathway regulates EGFR signaling. Here we show that a non-ubiquitinated EGFR mutant, unable to bind the endosomal-sorting complex required for transport (ESCRT) component, Hrs, is not efficiently targeted onto intraluminal vesicles (ILVs) of multivesicular endosomes/bodies (MVBs). Moreover, ubiquitination and ESCRT engagement of activated EGFR are required for EGF-stimulated ILV formation. Non-ubiquitinated EGFRs enter clathrin-coated tubules emanating from MVBs and show enhanced recycling to the plasma membrane, compared to wild-type EGFR.     

内吞体分选转运复合体 (ESCRT) 及其在包膜病毒出芽中的作用

内吞体分选转运复合体(Endosomal sorting complex required for transport,ESCRT)主要识别泛素化修饰的膜蛋白,介导内吞小泡出芽和多泡体(Multivesicular bodies,MVBs)的形成。此外,以类似的拓扑方式,ESCRT也参与胞质分裂、自体吞噬、以及包膜病毒的出芽等过程。已有的研究表明,大量的反转录病毒和RNA病毒含有晚期结构域(Late-domains),该结构域与ESCRT组分相互作用,将ESCRT-Ⅲ和VPS4等募集在病毒组装与出芽区域,并利用ESCRT-Ⅲ使病毒粒子得以释放。最近,有研究发现,一些DNA包膜病毒、如乙肝病毒、疱疹病毒和杆状病毒等的出芽释放也依赖于宿主细胞ESCRT系统,但其机理尚需深入研究。     

Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes

Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here, we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmunoelectron microscopy showed that these corresponded to MVBs. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal-transduction pathway.     

Lamellar bodies of rat alveolar type 2 cells have late endosomal marker proteins on their limiting membranes

Alveolar type 2 cells are known to take up surfactant phospholipids and proteins from the alveolar space and recycle them into secretory organelles via a receptor-mediated endocytic pathway. To clarify the intracellular route(s) through which materials ingested by the cells are processed, we examined the immunocytochemical localization of late endosomal and lysosomal membrane markers, rab 7 and lamp 1 proteins, within rat alveolar type 2 cells. The limiting membranes of lamellar bodies (LBs) showed positive immunoreactivity for both proteins, whereas multivesicular bodies (MVBs) exhibited positive immunoreactivity only for lamp 1 protein on free vesicles in the MVB lumen. From these findings, it is suggested that LBs are not only secretory granules, but also constitute one of the late endosomal compartments of the cells and that MVBs of this cell type may be targeted to cell organelle(s) other than lysosomes.     

Both clathrin-positive and -negative coats are involved in endosomal sorting of the EGF receptor

Sorting of endocytosed EGF receptor (EGFR) to internal vesicles of multivesicular bodies (MVBs) depends on sustained activation and ubiquitination of the EGFR. Ubiquitination of EGFR is mediated by the ubiquitin ligase Cbl, being recruited to the EGFR both directly and indirectly through association with Grb2. Endosomal sorting of ubiquitinated proteins further depends on interaction with ubiquitin binding adaptors like Hrs. Hrs localizes to flat, clathrin-coated domains on the limiting membrane of endosomes. In the present study, we have investigated the localization of EGFR, Cbl and Grb2 with respect to coated and non-coated domains of the endosomal membrane and to vesicles within MVBs. Both EGFR, Grb2, and Cbl were concentrated in coated domains of the limiting membrane before translocation to inner vesicles of MVBs. While almost all Hrs was in clathrin-positive coats, EGFR and Grb2 in coated domains only partially colocalized with Hrs and clathrin. The extent of colocalization of EGFR and Grb2 with Hrs and clathrin varied with time of incubation with EGF. These results demonstrate that both clathrin-positive and clathrin-negative electron dense coats exist on endosomes and are involved in endosomal sorting of the EGFR.     

浙贝母鳞片细胞休眠解除前后超微结构的变化

浙贝母休眠解除后,鳞片近轴面表皮附近的几层细胞首先降解。本文观察了这些细胞在休眠解除前后超微结构的变化。与休眠状态时的细胞相比,解除休眠后的细胞中颗粒和丝状物的数量明显增加,一些细胞中出现了复杂的膜结构。线粒体的数目增加,常聚集在细胞核和细胞壁旁。胞间连丝的直径略有增加。休眠解除前后的细胞中,常可见到各类囊泡、多泡体与壁旁体,一些小囊泡正在进入细胞中。表明两种时期的细胞间都发生着物质和信息的交换