Plants are able to sense the magnitude and direction of gravity. This capacity is thought to reside in selected cell types within the plant body that are equipped with specialized organelles called statoliths. However, most plant cells do not possess statoliths, yet they respond to changes in gravitational acceleration. To understand the effect of gravity on the metabolism and cellular functioning of non-specialized plant cells, we investigated a rapidly growing plant cell devoid of known statoliths and without gravitropic behavior, the pollen tube. The effects of hyper-gravity and omnidirectional exposure to gravity on intracellular trafficking and on cell wall assembly were assessed in Camellia pollen tubes, a model system with highly reproducible growth behavior in vitro. Using an epi-fluorescence microscope mounted on the Large Diameter Centrifuge at the European Space Agency, we were able to demonstrate that vesicular trafficking is reduced under hyper-gravity conditions. Immuno-cytochemistry confirmed that both in hyper and omnidirectional gravity conditions, the characteristic spatial profiles of cellulose and callose distribution in the pollen tube wall were altered, in accordance with a dose-dependent effect on pollen tube diameter. Our findings suggest that in response to gravity induced stress, the pollen tube responds by modifying cell wall assembly to compensate for the altered mechanical load. The effect was reversible within few minutes demonstrating that the pollen tube is able to quickly adapt to changing stress conditions.
Computational modeling of growing plant tissues raises two basic questions about plant cell division: when does a cell decide to divide and where is the new wall placed? Although biologists and modelers commonly assume that a cell divides after it reaches a threshold size, two recent experiments show that models with variable division sizes better replicate the tissue. Similarly, comparing model predictions with living plant cells reveals that the choice of division plane is variable, although the shortest path dividing a cell in half (i.e. the minimal surface area) is the most probable division plane.
Catharanthus roseus is one of the most studied medicinal plants due to the interest of their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine. Although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. Such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aiming at increasing TIA production. In the present study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP-dependent and this transport activity was strongly inhibited by NH4(+) and CCCP, and insensitive to the ABC transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate a H+ gradient pre-established across the tonoplast by either V-H(+)-ATPase or V-H(+)-PPase. The initial rates of H+gradient dissipation followed a Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, results strongly support that TIAs are actively uptaken by C. roseus mesophyll vacuoles through a specific H(+) antiport system, and not by an ion trap mechanism or ABC transporters.
Here, we sought to investigate the vacuole-targeting fungicidal activity of amphotericin B (AmB) in parent strain and AmB-resistant mutant of Saccharomyces cerevisiae, and elucidate the mechanisms involved in this process. Our data demonstrated that the vacuole-targeting fungicidal activity of AmB was markedly enhanced by N-methyl-N'‘-dodecylguanidine (MC12), a synthetic analog of the alkyl side chain in niphimycin, as represented by the synergy in their antifungal activities against parent cells of S. cerevisiae. Indifference was observed only with erg3 cells, indicating that the replacement of ergosterol with episterol facilitated their resistance to the combined lethal actions of AmB and MC12. Dansyl-labeled amphotericin B (AmB-Ds) was concentrated into normal rounded vacuoles when parent cells were treated with AmB-Ds alone, even at a nonlethal concentration. The additional supplementation of MC12 resulted in a marked loss of cell viability and vacuole disruption, as judged by the fluorescence from AmB-Ds scattered throughout the cytoplasm. In erg3 cells, AmB-Ds was scarcely detected in the cytoplasm, even with the addition of MC12, reflecting its failure to normally incorporate across the plasma membrane into the vacuole. Thus, this study supported the hypothesis that ergosterol is involved in the mobilization of AmB into the vacuolar membrane so that AmB-dependent vacuole disruption can be fully enhanced by cotreatment with MC12.
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 5 years ago
Membrane fusion along the endocytic pathway occurs in a sequence of tethering, docking, and fusion. At endosomes and vacuoles, the CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) tethering complexes require their organelle-specific Rabs for localization and function. Until now, despite the absence of experimental evidence, it has been assumed that CORVET is a membrane-tethering factor. To test this theory and understand the mechanistic analogies with the HOPS complex, we set up an in vitro system, and establish CORVET as a bona-fide tether for Vps21-positive endosome/vacuole membranes. Purified CORVET binds to SNAREs and Rab5/Vps21-GTP. We then demonstrate that purified CORVET can specifically tether Vps21-positive membranes. Tethering via CORVET is dose-dependent, stimulated by the GEF Vps9, and inhibited by Msb3, the Vps21-GAP. Moreover, CORVET supports fusion of isolated membranes containing Vps21. In agreement with its role as a tether, overexpressed CORVET drives Vps21, but not the HOPS-specific Ypt7 into contact sites between vacuoles, which likely represent vacuole-associated endosomes. We therefore conclude that CORVET is a tethering complex that promotes fusion of Rab5-positive membranes and thus facilitates receptor down-regulation and recycling at the late endosome.
Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured-cells (BY-2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY-2 cells harboring a vSPO-cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N-terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
Vacuolar programmed cell death (PCD) is indispensable for plant development and is accompanied by a dramatic growth of lytic vacuoles, which gradually digest cytoplasmic content leading to self-clearance of dying cells. Our recent data demonstrate that vacuolar PCD critically requires autophagy and its upstream regulator, a caspase-fold protease metacaspase. Furthermore, both components lie downstream of the point of no return in the cell-death pathway. Here we consider the possibilities that i) autophagy could have both cytotoxic and cytoprotective roles in the vacuolar PCD, and ii) metacaspase could augment autophagic flux through targeting an as yet unknown autophagy repressor.
Stomatal pores are formed between a pair of guard cells, and allow plant uptake of CO2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K+ and Mal2-. Efflux of Mal2- from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here we report the identification of ALMT4 (ALUMINIUM ACTIVATED MALATE TRANSPORTER 4) as an Arabidopsis thaliana ion channel that can mediate Mal2- release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knock-out mutants showed impaired stomatal closure in response to the drought-stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal2- efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal2- efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein (MAP) kinases in vitro. In brief, ALMT4 likely mediates Mal2- efflux during ABA-induced stomatal closure and its activity depends on phosphorylation.
Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFNγ) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFNγ-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of type II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that type II, but not type I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFNγ-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the type II vacuole in distinct, overlapping microdomains with a loss of IFNγ-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to <10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where type II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.
Organelles are distributed to daughter cells, via inheritance pathways. However, it is unclear whether there are mechanisms beyond inheritance, which ensure that organelles are present in all cells. Here we present the unexpected finding that the yeast vacuole plays a positive essential role in initiation of the cell-cycle. When inheritance fails, a new vacuole is generated. We show that this occurs prior to the next cell-cycle, and gain insight into this alternative pathway. Moreover, we find that a combination of a defect in inheritance with an acute block in the vacuole biogenesis results in the loss of a functional vacuole and a specific arrest of cells in early G1 phase. Furthermore, this role for the vacuole in cell-cycle progression requires an intact TORC1-SCH9 pathway that can only signal from a mature vacuole. These mechanisms may serve as a checkpoint for the presence of the vacuole/lysosome.