Golgi 58K-like protein in pollens and pollen tubes of Lilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.     

Golgi 58K-like protein in pollens and pollen tubes ofLilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.     

Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana

Using the cryo-fixation/freeze-substitution method, we studied the ultrastructural changes and behavior of vacuoles and related organelles (rER and Golgi bodies) during microspore and pollen development, and pollen maturation of Arabidopsis thaliana. In young microspores forming exine (pollen outer cell wall), vacuoles looked like those of somatic cells. In microspores during the formation of intine (inner cell wall), a large vacuole appeared which was made by fusion of pre-existing vacuoles and probably absorption of solutions. In the young pollen grain after the first mitosis, a large vacuole was divided into small vacuoles. The manner of division was not by binary fission and centripetally, but by the invagination of tonoplasts from one side to the opposite side of a vacuole. After the second mitosis, somatic type vacuoles disappeared. In mature pollen grains just before germination, membrane-bound structures containing fine fibrillar substances (MBFs) appeared. The MBFs were considered to be storage vacuoles. In pollen grains from flowers in bloom, MBFs changed to lysosomal structures with acid phosphatases (lytic vacuole). They gradually increased in number and volume, and decomposed the cytoplasm. The autolysis of pollen grains is the first finding in this study, which may contribute to the loss of ability of pollen germination after anthesis.     

Studies on the Development of Glandular Hairs in Nymphoides peltatum and the Ultrastructure During Their Secretory P

Each glandular hair of Nyrnphoides peltaturn (Gmel.) O. Kuntz consisted of only one row of cylindar cells with secretory function. The hairs originated from the protoderm cells on the adaxial surface of the second leaf primordium from the shoot apex. Cells of the glandular hairs prossessed dense cytoplast during the secretory period, but the vacuoles were very small. There were not only abundant mitochondria, Golgi bodies and endoplasmic reticulum in the glandular hair cells, but also many plasmodesmata. The authors' research indicated that the mucilage was carried to the edge of the cells by the membranous multilamellar bodies and the vesicles from both Golgi bodies and endoplasmic reticulum. The mucilage was secreted extracellularly by either exocytosis or ecrine secretion. The side walls of the glandular hairs swelled because of mucilage mass accumulation in the walls. The mucilage, being tested to be composed of polysaccharides and a trace of protein, played an important role in protecting the development of the vegetative buds of N. peltatum.     

Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis

Yeast cells have been shown to internalize lucifer yellow CH by endocytosis. Internalization of the fluorescent dye is time-, temperature-, and energy-dependent, it is not saturable, and the dye is accumulated in the vacuole. Some of the yeast secretory mutants that accumulate endoplasmic reticulum or Golgi bodies are defective for endocytosis at restrictive temperature, while others are not. All of the mutants that accumulate secretory vesicles are defective for endocytosis. These results suggest that efficient transport of proteins from the endoplasmic reticulum to the Golgi apparatus and from the Golgi to secretory vesicles is not necessary for endocytosis. In contrast, endocytosis may be obligatorily coupled with the latest steps of secretion.     

ENDOCYTOSIS OF LANTHANUM NITRATE IN THE ORGANIC ACID-SECRETING TRICHOMES OF CHICKPEA (CICER ARIETINUM L.)

The organic acid-secreting trichomes of chickpea (Cicer arietinum L.) were exposed to 2.5 mm lanthanum nitrate for 24 hr, and this concentration did not inhibit trichome secretion compared with that of controls. We subsequently used this nontoxic concentration of lanthanum to examine endocytosis. In the stalk cells of these secretory trichomes, exogenously applied lanthanum nitrate was present in cell walls and vacuoles, as well as within both invaginations in the plasma membrane and vesicles in the peripheral cytoplasm between the plasma membrane and the tonoplast. In the head cells, lanthanum nitrate was present in cell walls and in vesicles that form a layer in the cytoplasm around the edge of the head cells, but was not present in vacuoles. We propose that fluid phase endocytosis targeted to the vacuole takes place in the stalk cells and that endocytosis occurs in the head cells to remove excess plasma membrane after the fusion of secretory vesicles with the plasma membrane. This is the first demonstration of endocytosis in secretory trichomes.     

The syntaxin homolog AtPEP12p resides on a late post-Golgi compartment in plants.

Soluble proteins are transported to the plant vacuole through the secretory pathway via membrane-bound vesicles. Targeting of vesicles to appropriate organelles requires several membrane-bound and soluble factors that have been characterized in yeast and mammalian systems. For example, the yeast PEP12 protein is a syntaxin homolog that is involved in protein transport to the yeast vacuole. Previously, we isolated an Arabidopsis thaliana homolog of PEP12 by functional complementation of the yeast pep12 mutant. Antibodies raised against the cytoplasmic portion of AtPEP12 have been prepared and used for intracellular localization of this protein. Biochemical analysis indicates that AtPEP12 does not localize to the endoplasmic reticulum, Golgi apparatus, plasma membrane, or tonoplast in Arabidopsis plants; furthermore, based on biochemical and electron microscopy immunogold labeling analyses, AtPEP12 is likely to be localized to a post-Golgi compartment in the vacuolar pathway.     

Evidence for a sorting endosome in Arabidopsis root cells

In eukaryotic cells, the endocytic and secretory pathways are key players in several physiological processes. These pathways are largely inter-connected in animal and yeast cells through organelles named sorting endosomes. Sorting endosomes are multi-vesicular compartments that redirect proteins towards various destinations, such as the lysosomes or vacuoles for degradation, the trans-Golgi network for retrograde transport and the plasma membrane for recycling. In contrast, cross-talk between the endocytic and secretory pathways has not been clearly established in plants, especially in terms of cargo protein trafficking. Here we show by co-localization analyses that endosomes labelled with the AtSORTING NEXIN1 (AtSNX1) protein overlap with the pre-vacuolar compartment in Arabidopsis root cells. In addition, alteration of the routing functions of AtSNX1 endosomes by drug treatments leads to mis-routing of endocytic and secretory cargo proteins. Based on these results, we propose that the AtSNX1 endosomal compartment represents a sorting endosome in root cells, and that this specialized organelle is conserved throughout eukaryotes.     

Cell cycle-dependent changes in Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells of Arabidopsis thaliana: A quantitative and spatial analysis

Cytokinesis in plants involves both the formation of a new wall and the partitioning of organelles between the daughter cells. To characterize the cellular changes that accompany the latter process, we have quantitatively analyzed the cell cycle-dependent changes in cell architecture of shoot apical meristem cells of Arabidopsis thaliana. For this analysis, the cells were preserved by high-pressure freezing and freeze-substitution techniques, and their Golgi stacks, multivesicular bodies, vacuoles and clathrin-coated vesicles (CCVs) characterized by means of serial thin section reconstructions, stereology and electron tomography techniques. Interphase cells possess ∼35 Golgi stacks, and this number doubles during G2 immediately prior to mitosis. At the onset of cytokinesis, the stacks concentrate around the periphery of the growing cell plate, but do not orient towards the cell plate. Interphase cells contain ∼18 multivesicular bodies, most of which are located close to a Golgi stack. During late cytokinesis, the appearance of a second group of cell plate-associated multivesicular bodies coincides with the onset of CCV formation at the cell plate. During this period a 4× increase in CCVs is paralleled by a doubling in number and a 4× increase in multivesicular bodies volume. The vacuole system also undergoes major changes in organization, size, and volume, with the most notable change seen during early telophase cytokinesis. In particular, the vacuoles form sausage-like tubular compartments with a 50% reduced surface area and an 80% reduced volume compared to prometaphase cells. We postulate that this transient reduction in vacuole volume during early telophase provides a means for increasing the volume of the cytosol to accommodate the forming phragmoplast microtubule array and associated cell plate-forming structures.     

The proteolytic processing of seed storage proteins in Arabidopsis embryo cells starts in the multivesicular bodies

We have investigated the transport of storage proteins, their processing proteases, and the Vacuolar Sorting Receptor-1/Epidermal Growth Factor Receptor-Like Protein1 (VSR-1/ATELP1) receptor during the formation of protein storage vacuoles in Arabidopsis thaliana embryos by means of high-pressure freezing/freeze substitution, electron tomography, immunolabeling techniques, and subcellular fractionation. The storage proteins and their processing proteases are segregated from each other within the Golgi cisternae and packaged into separate vesicles. The storage protein-containing vesicles but not the processing enzyme-containing vesicles carry the VSR-1/ATELP1 receptor. Both types of secretory vesicles appear to fuse into a type of prevacuolar multivesicular body (MVB). We have also determined that the proteolytic processing of the 2S albumins starts in the MVBs. We hypothesize that the compartmentalized processing of storage proteins in the MVBs may allow for the sequential activation of processing proteases as the MVB lumen gradually acidifies.     

Testosterone interferes with the kinetics of endocytosis in the hamster seminal vesicle

Endocytosis was studied in the seminal vesicle secretory cells of castrated and control hamsters in order to investigate the effect of testosterone withdrawal in the endocytic activity of these cells. Horseradish peroxidase was injected into the glands lumen after removal of their contents, and tracer distribution was qualitatively studied, and the number of labeled endocytic vesicles quantitatively analyzed, following 5, 20, 40 and 60 min incubation. The following compartments are labeled both in castrate and control cells: 1), endocytic vesicles; 2), vacuoles with or without secretory material; 3), multivesicular bodies; 4), Golgi cisternae; 5), intercellular spaces; 6), sub-epithelial space. The pattern of labeling is lighter in castrate than in control cells and the labeling of Golgi cisternae, which correlates with a significant peak in the number of endocytic vesicles, is observed later in castrated animals than in controls: 40 min vs 20 min. Exocytosis, as evaluated through the fraction of secretory protein released in vitro, decreases following castration. Endocytosis performed in castrated, pilocarpine treated animals shows that the Golgi labeling, coinciding with numerous labeled endocytic vesicles, is advanced from 40 to 20 min after stimulation of exocytosis. The results show that, in the seminal vesicle secretory cells a) the endocytic pathway does not depend on testosterone; b) testosterone withdrawal decreases endocytosis and delays the kinetics of labeling and; c) endocytosis couples to exocytosis, probably so regulating the apical cell membrane area.     

A proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis

Plants degrade cellular materials during senescence and under various stresses. We report that the precursors of two stress-inducible cysteine proteinases, RD21 and a vacuolar processing enzyme (VPE), are specifically accumulated in approximately 0.5 microm diameter x approximately 5 microm long bodies in Arabidopsis thaliana. Such bodies have previously been observed in Arabidopsis but their function was not known. They are surrounded with ribosomes and thus are assumed to be directly derived from the endoplasmic reticulum (ER). Therefore, we propose to call them the ER bodies. The ER bodies are observed specifically in the epidermal cells of healthy seedlings. These cells are easily wounded and stressed by the external environment. When the seedlings are stressed with a concentrated salt solution, leading to death of the epidermal cells, the ER bodies start to fuse with each other and with the vacuoles, thereby mediating the delivery of the precursors directly to the vacuoles. This regulated, direct pathway differs from the usual case in which proteinases are transported constitutively from the ER to the Golgi complex and then to vacuoles, with intervention of vesicle-transport machinery, such as a vacuolar-sorting receptor or a syntaxin of the SNARE family. Thus, the ER bodies appear to be a novel proteinase-storing system that assists in cell death under stressed conditions.     

Native and Artificial Reticuloplasmins Co-Accumulate in Distinct Domains of the Endoplasmic Reticulum and in Post-Endoplasmic Reticulum Compartments

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.     

Overexpression of the Arabidopsis syntaxin PEP12/SYP21 inhibits transport from the prevacuolar compartment to the lytic vacuole in vivo

Golgi-mediated transport to the lytic vacuole involves passage through the prevacuolar compartment (PVC), but little is known about how vacuolar proteins exit the PVC. We show that this last step is inhibited by overexpression of Arabidopsis thaliana syntaxin PEP12/SYP21, causing an accumulation of soluble and membrane cargo and the plant vacuolar sorting receptor BP80 in the PVC. Anterograde transport proceeds normally from the endoplasmic reticulum to the Golgi and the PVC, although export from the PVC appears to be compromised, affecting both anterograde membrane flow to the vacuole and the recycling route of BP80 to the Golgi. However, Golgi-mediated transport of soluble and membrane cargo toward the plasma membrane is not affected, but a soluble BP80 ligand is partially mis-sorted to the culture medium. We also observe clustering of individual PVC bodies that move together and possibly fuse with each other, forming enlarged compartments. We conclude that PEP12/SYP21 overexpression specifically inhibits export from the PVC without affecting the Golgi complex or compromising the secretory branch of the endomembrane system. The results provide a functional in vivo assay that confirms PEP12/SYP21 involvement in vacuolar sorting and indicates that excess of this syntaxin in the PVC can be detrimental for further transport from this organelle.     

High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana

Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles.     

菱的腺毛发育及分泌活动的超微结构研究

菱的腺毛由单列圆筒状细胞组成,具有短暂的分泌功能。它们起源于叶片远轴面、叶柄的表皮细胞以及苗端茎轴、花柄的表皮细胞。处于分泌期的腺毛细胞其胞质浓厚,液泡化程度小,细胞具丰富的线粒体、高尔基体。腺毛丧失了分泌功能后即发育成为表皮毛。粘液物质由高尔基体分泌小泡携带至腺毛细胞侧壁,经胞吐与渗透结合的方式分泌至细胞外,粘液的化学成分主要为多糖。     

The function of the golgi complex in transitional epithelium. Synthesis of the thick cell membrane

The superficial squamous cells of rat transitional epithelium are limited, on their luminal face, by an asymmetrically thickened membrane. Patches of similar thick membrane are found in the walls of the Golgi cisternae and it is suggested that the Golgi system is the site of assembly of the thick plasma membrane. This implies membrane flow from the Golgi apparatus to the cell surface, and there is indirect evidence that the membrane is transported in the form of fusiform vacuoles, derived from the Golgi cisternae, which fuse with, and become part of, the free cell membrane. Uptake of injected Imferon shows that similar, large, thick-walled vacuoles may be formed by invagination of the free cell surface. Some of these vacuoles are subsequently transformed into multivesicular bodies and autophagic vacuoles. The formation of other large heterogeneous bodies is described, and some of these are shown to have acid phosphatase activity.     

vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting.

We have isolated four yeast mutants that are unable to partition maternal vacuoles into growing buds. Three of these vacuole segregation (vac) mutants also mislocalize the vacuolar protease carboxypeptidase Y (CPY) to the cell surface, a phenotype previously reported for vac strains. A fourth mutant, vac2-1, exhibits a temperature-sensitive defect in vacuole segregation but does not show a defect in protein targeting from the Golgi apparatus to the vacuole. Haploid vac2-1 cells grown at the non-permissive temperature do not secrete CPY or a second vacuolar protease, proteinase A (PrA). Furthermore, newly synthesized precursors of CPY are converted to mature forms with similar kinetics in both vac2-1 and wild-type cells. In addition, invertase is secreted normally from vac2-1 cells, indicating that post-Golgi steps in the secretory pathway are not blocked in this mutant. These results suggest that VAC2 function is necessary for vacuole division and segregation in yeast but is not involved in vacuole protein sorting events at the Golgi apparatus.     

Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii

A number of cysteine and serine protease inhibitors blocked the intracellular growth and replication of Toxoplasma gondii tachyzoites. Most of these inhibitors caused only minor alterations to parasite morphology irrespective of the effects on the host cells. However, three, cathepsin inhibitor III, TPCK and subtilisin inhibitor III, caused extensive swelling of the secretory pathway of the parasite (i.e. the ER, nuclear envelope, and Golgi complex), caused the breakdown of the parasite surface membrane, and disrupted rhoptry formation. The disruption of the secretory pathway is consistent with the post-translational processing of secretory proteins in Toxoplasma, and with the role of proteases in the maturation/activation of secreted proteins in general. Interestingly, while all parasites in an individual vacuole (the clonal progeny of a single invading parasite) were similarly affected, parasites in different vacuoles in the same host cell showed different responses to these inhibitors. Such observations imply that there are major differences in the biochemistry/physiology between tachyzoites within different vacuoles and argue that adverse effects on the host cell are not always responsible for changes in the parasite. Treatment of established parasites also leads to an accumulation of abnormal materials in the parasitophorous vacuole implying that materials deposited into the vacuole normally undergo proteolytic modification or degradation. Despite the often extensive morphological changes, nothing resembling lysosomal bodies was seen in any treated parasites, consistent with previous observations showing that mother cell organelles are not recycled by any form of autophagic-lysosomal degradation, although the question of how the parasite recycles these organelles remains unanswered.     

Actin filaments play a critical role in vacuolar trafficking at the Golgi complex in plant cells

Actin filaments are thought to play an important role in intracellular trafficking in various eukaryotic cells. However, their involvement in intracellular trafficking in plant cells has not been clearly demonstrated. Here, we investigated the roles actin filaments play in intracellular trafficking in plant cells using latrunculin B (Lat B), an inhibitor of actin filament assembly, or actin mutants that disrupt actin filaments when overexpressed. Lat B and actin2 mutant overexpression inhibited the trafficking of two vacuolar reporter proteins, sporamin:green fluorescent protein (GFP) and Arabidopsis thaliana aleurain-like protein:GFP, to the central vacuole; instead, a punctate staining pattern was observed. Colocalization experiments with various marker proteins indicated that these punctate stains corresponded to the Golgi complex. The A. thaliana vacuolar sorting receptor VSR-At, which mainly localizes to the prevacuolar compartment, also accumulated at the Golgi complex in the presence of Lat B. However, Lat B had no effect on the endoplasmic reticulum (ER) to Golgi trafficking of sialyltransferase or retrograde Golgi to ER trafficking. Lat B also failed to influence the Golgi to plasma membrane trafficking of H+-ATPase:GFP or the secretion of invertase:GFP. Based on these observations, we propose that actin filaments play a critical role in the trafficking of proteins from the Golgi complex to the central vacuole.