Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean

Vacuolar compartments associated with leaf senescence and the subcellular localization of the senescence-specific cysteine-protease SAG12 (senescence-associated gene 12) were studied using specific fluorescent markers, the expression of reporter genes, and the analysis of high-pressure frozen/freeze-substituted samples. Senescence-associated vacuoles (SAVs) with intense proteolytic activity develop in the peripheral cytoplasm of mesophyll and guard cells in Arabidopsis and soybean. The vacuolar identity of these compartments was confirmed by immunolabeling with specific antibody markers. SAVs and the central vacuole differ in their acidity and tonoplast composition: SAVs are more acidic than the central vacuole and, whereas the tonoplast of central vacuoles is highly enriched in gamma-TIP (tonoplast intrinsic protein), the tonoplast of SAVs lacks this aquaporin. The expression of a SAG12-GFP fusion protein in transgenic Arabidopsis plants shows that SAG12 localizes to SAVs. The analysis of Pro(SAG12):GUS transgenic plants indicates that SAG12 expression in senescing leaves is restricted to SAV-containing cells, for example, mesophyll and guard cells. A homozygous sag12 Arabidopsis mutant develops SAVs and does not show any visually detectable phenotypical alteration during senescence, indicating that SAG12 is not required either for SAV formation or for progression of visual symptoms of senescence. The presence of two types of vacuoles in senescing leaves could provide different lytic compartments for the dismantling of specific cellular components. The possible origin and functions of SAVs during leaf senescence are discussed.     

Endosomal proteases facilitate the fusion of endosomes with vacuoles at the final step of the endocytotic pathway

The mechanism by which plasma membrane proteins are transported to vacuoles for degradation has not been well characterized in plants. To clarify how plasma membrane proteins are degraded, we monitored the endocytotic pathway in tobacco suspension-cultured BY-2 cells with a fluorescent endocytosis marker, FM4-64. Because of the efficient and rapid delivery of endosomes to the vacuoles, endosomes were scarcely detectable. Interestingly, we found that E-64d, an inhibitor of papain family proteases, caused the accumulation of a large number of endosomes in the cells under the sucrose-starved condition. This result indicates that E-64d attenuates the fusion of endosomes with vacuoles. We identified two papain homologues, which are localized in the endosomes, with a biotinylated inhibitor. We designated them as endosome-localized papains (ENPs). Immunofluorescent analysis revealed that vacuolar sorting receptor, a marker of prevacuolar compartment (PVC), was localized in the endosomes. This result and their acidic nature show that the endosomes correspond to PVC. These results suggest that ENPs facilitate the final step in the vacuolar trafficking pathway under the sucrose-starved condition. We further examined the effects of E-64d on two transgenic Arabidopsis plants that constitutively express a fusion protein composed of green fluorescent protein (GFP) and a plasma membrane protein (GFP-PIP2a or GFP-LTI6b). GFP fluorescence was observed on the plasma membrane of root cells in these transgenic plants. Treatment with E-64d induced the accumulation of GFP-fluorescent endosomes and inhibited the degradation of these fusion proteins. No GFP fluorescence was observed in vacuoles in E-64d-treated transgenic plants. Taken together, these results suggest that endosomal proteases are required for the fusion of endosomes with vacuoles at the final step in the endocytotic pathway for degradation of plasma membrane proteins in plants.     

Selective targeting of breast cancer cells through ROS-mediated mechanisms potentiates the lethality of paclitaxel by a novel diterpene, gelomulide K

Defects in apoptotic pathways confer resistance to tubulin-binding agents via downregulation of caspases or overexpression of antiapoptotic factors, urging the need for novel agents acting on an alternative pathway. The purpose of this study was to investigate whether induction of ROS can induce caspase-independent cell death in breast cancer cells and thereby enhance the activity of paclitaxel. Here, we report that gelomulide K acts as a caspase-independent cell death-inducing agent that synergizes with paclitaxel in breast cancer cells and has low toxicity in normal cells. Treatment with gelomulide K induced PARP-1 hyperactivation, AIF nuclear translocation, and cytoprotective autophagy. These effects were associated with increased ROS production and a decrease in cellular GSH levels in cancer cells. Furthermore, pretreatment with NAC, a precursor of intracellular GSH, effectively abrogated gelomulide K-induced caspase-independent cell death and autophagy, suggesting that ROS-mediated downstream signaling is essential to the anticancer effects of gelomulide K. Additionally, in a xenograft model, gelomulide K induced PARP-1 activation and reduced tumor growth. In terms of structure-activity relationships, analysis not only showed a correlation between ROS levels and drug activity but also highlighted the importance of the 8,14-epoxy group. Taken together, our results show that enhancement of paclitaxel activity can be achieved with gelomulide K and that the structurally relevant pharmacophore provides important insight into the development of new caspase-independent cell death-inducing agents.     

The vacuolated morphology of chordoma cells is dependent on cytokeratin intermediate filaments

Notochordal cells (NCs), characterized by their vacuolated morphology and coexpression of cytokeratin and vimentin intermediate filaments (IFs), form the immature nucleus pulposus (NP) of the intervertebral disc. As humans age, NCs give way to mature NP cells, which do not possess a vacuolated morphology and typically only express vimentin IFs. In light of their concomitant loss, we investigated the relationship between cytosolic vacuoles and cytokeratin IFs, specifically those containing cytokeratin-8 proteins, using a human chordoma cell line as a model for NCs. We demonstrate that the chemical disruption of IFs with acrylamide, F-actin with cytochalasin-D, and microtubules with nocodazole all result in a significant (p < 0.001) decrease in vacuolation. However, vacuole loss was the greatest in acrylamide-treated cells. Examination of the individual roles of vimentin and cytokeratin-8 IFs in the existence of vacuoles was accomplished using small interfering RNA–mediated RNA interference to knock down either vimentin or cytokeratin-8 expression. Reduction of cytokeratin-8 expression was associated with a less-vacuolated cell morphology. These data demonstrate that cytokeratin-8 IFs are involved in stabilizing vacuoles and that their diminished expression could play a role in the loss of vacuolation in NCs during aging. A better understanding of the NCs may assist in preservation of this cell type for NP maintenance and regeneration.     

Inactivation of the PPN1 gene exerts different effects on the metabolism of inorganic polyphosphates in the cytosol and the vacuoles of the yeast Saccharomyces cerevisiae

Inactivation of the PPN1 gene, encoding one of the enzymes involved in polyphosphate metabolism in the yeast Saccharomyces cerevisiae, was found to decrease exopolyphosphatase activity in the cytosol and vacuoles. This effect was more pronounced in the stationary growth phase than in the phase of active growth. The gene inactivation resulted in elimination of a 440-kDa exopolyphosphatase in the vacuoles but did not influence a previously unknown vacuolar exopolyphosphatase with a molecular mass of >1000 kDa, which differed from the former enzyme in the requirement for bivalent cations and sensitivity to heparin. Inactivation of the PPN1 gene did not influence the level of polyphosphates in the cytosol but increased it more than twofold in the vacuoles. In this case, the polyphosphate chain length in the cytosol increased from 10–15 to 130 phosphate residues both in the stationary and active growth phases. In the vacuoles, the polyphosphate length increased only in the stationary growth phase. A conclusion can be made that the PPN1 gene product has different effects on polyphosphate metabolism in the cytosol and the vacuoles.     

鱼腥藻7120三类液泡三种内含物的比较分析

分别从培养4d,24d和KCl诱导的材料分离液泡,对这3种液泡进行了蛋白质、还原糖和藻青蛋白测定,结果表明,3种物质含量呈现规律性变化。培养4d的材料液泡中各物质含量低,培养24d的材料液泡中物质含量升高,KCl诱导的液泡中含量下降,液泡中各种物质的相对含量在3种液泡中依次升高。这一结果说明,培养4d,液泡处于初期阶段,培养24d,液泡处于充分发育阶段,KCl诱导液泡为衰老阶段。随着细胞发育,液泡的生理作用提高。     

鱼腥藻7120细胞液泡内含物的初步测定

对鱼腥藻7120细胞液泡内含物中4种水溶性的物质进行了测定,液泡中4种物质占整个细胞中4种物质的比例分别为:蛋白质:14.1％;还原糖:34.4％;核酸:28.5％;藻青蛋白12.1％。     

'Senescence-associated vacuoles' are involved in the degradation of chloroplast proteins in tobacco leaves

Massive degradation of photosynthetic proteins is the hallmark of leaf senescence; however the mechanism involved in chloroplast protein breakdown is not completely understood. As small 'senescence-associated vacuoles' (SAVs) with intense proteolytic activity accumulate in senescing leaves of soybean and Arabidopsis, the main goal of this work was to determine whether SAVs are involved in the degradation of chloroplastic components. SAVs with protease activity were readily detected through confocal microscopy of naturally senescing leaves of tobacco (Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of the chloroplast degradation rate by ethylene treatment correlated with a twofold increase in the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, GFP was re-located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast-targeted GFP and the chloroplast stromal proteins Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and glutamine synthetase, but lacked the thylakoid proteins D1 and light-harvesting complex II of the photosystem II reaction center and photosystem II antenna, respectively. In SAVs incubated at 30 degrees C, there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. These results indicate that SAVs are involved in degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves.     

Morphology and phylogeny of a new wall‐less freshwater volvocalean flagellate,Hapalochloris nozakii gen. et sp. nov. (Volvocales,Chlorophyceae)

New strains of a wall‐less unicellular volvocalean flagellate were isolated from a freshwater environment in Japan. Observations of the alga, described here as Hapalochloris nozakii Nakada, gen. et sp. nov., were made using light, fluorescence, and electron microscopy. Each vegetative cell had two flagella, four contractile vacuoles, and a spirally furrowed cup‐shaped chloroplast with an axial pyrenoid, and mitochondria located in the furrows. Based on the morphology, H. nozakii was distinguished from other known wall‐less volvocalean flagellates. Under electron microscopy, fibrous material, instead of a cell wall and dense cortical microtubules, was observed outside and inside the cell membrane, respectively. Based on the phylogenetic analyses of 18S rRNA gene sequences, H. nozakii was found to be closely related to Asterococcus, Oogamochlamys, Rhysamphichloris, and “Dunaliella” lateralis and was separated from other known wall‐less flagellate volvocaleans, indicating independent secondary loss of the cell wall in H. nozakii. In the combined 18S rRNA and chloroplast gene tree, H. nozakii was sister to Lobochlamys.     

Theonellamide F, a Bicyclic Peptide Marine Toxin, Induces Formation of Vacuoles in 3Y1 Rat Embryonic Fibroblast

The effect of a marine bicyclic peptide, theonellamide F, on vacuole formation in exponentially growing 3Y1 rat embryonic fibroblasts was studied in comparison with the effect of monensin. Many abnormally large vacuoles appeared around the nucleus in the cells treated with 6 μM theonellamide F within 24 hours. Following prolonged treatment with this agent, the number of enlarged vacuoles increased. Such vacuoles accumulated many granules that showed Brownian movement. The effect of theonellamide F on the cells was more drastic in an amino-acid-deficient medium, in which all cells died within 1 hour at a 3-μM concentration of the agent. Theonellamide F probably affects cellular autophagy, inhibiting the degradation of the organelles and turnover of proteins. Monensin, a well-known Na⁺ ionophore that disintegrates the Golgi apparatus, induced similar types of vacuole formation, although the vacuoles were localized in a region slightly distant from the nucleus. Monensin readily affected cell morphology, resulting in cell death. We propose that theonellamide F, like monensin, is a useful agent for investigating membrane structures in cells. Received November 25, 1998; accepted February 10, 1999