Transport of 1-kestose across the tonoplast of Jerusalem artichoke tubers

The capacity for 1-kestose uptake into the vacuole of fructan storing Jerusalem artichoke tubers was investigated. 1-kestose serves both as building block for fructan initiation and as a fructose donor for chain elongation. Tonoplast vesicles were isolated from actively storing tubers, and their vesicles were capable of transporting sucrose in a manner indicative of a sucrose/H(+) antiport. Under similar conditions, 1-kestose was not taken up by vesicles energized by either a pH jump or in the presence of ATP. When added together at 2 mM, sucrose uptake was not affected by the presence of 1-kestose. The data argues against the possible synthesis of 1-kestose in the cytosol and subsequent transport to the vacuole. The data also presents definite evidence for the existence a mechanism for sucrose accumulation in fructan storing vacuoles.     

Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent citrate transport across the tonoplast of citrus juice cells

Citrus juice cells accumulate high levels of citric acid in their vacuoles when compared to other organic ions including malate. Uptake of citrate into tonoplast vesicles from Citrus juice cells was investigated in the presence of malate, and after incubation with antibodies raised against the vacuolar malate-specific channel of Kalancho? diagremontiana leaves. Antibodies against the vacuolar malate channel immunoreacted with a protein of similar size in tonoplast extracts from three Citrus varieties differing in citric acid content. Malate channel antibodies inhibited both delta MicroH(+)-dependent and delta MicroH(+)-independent ATP-dependent citrate transport, indicating common domains in both transport systems and to the malate-specific channel of Kalancho? diagremontiana leaves. Malate strongly inhibited electrogenic citrate transport, whereas ATP-dependent citrate uptake was less affected. Kinetic analysis of citrate transport in the presence of malate confirmed the existence of two citrate transport mechanisms and indicated that both citrate and malate share a common transport channel across the tonoplast of Citrus juice cells.     

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

Heteromers of Kv7.2/Kv7.3 subunits constitute the main substrate of the neuronal M-current that limits neuronal hyper-excitability and firing frequency. Calmodulin (CaM) binding is essential for surface expression of Kv7 channels, and disruption of this interaction leads to diseases ranging from mild epilepsy to early onset encephalopathy. In this study, we addressed the impact of a charge neutralizing mutation located at the periphery of helix B (K526N). We found that, CaM binding and surface expression was impaired, although current amplitude was not altered. Currents were reduced at a faster rate after activation of a voltage-dependent phosphatase, suggesting that phosphatidylinositol-4,5-bisphosphate (PIP₂) binding was weaker. In contrast, a charge neutralizing mutation located at the periphery of helix A (R333Q) did not affect CaM binding, but impaired trafficking and led to a reduction in current amplitude. Taken together, these results suggest that disruption of CaM-dependent or CaM-independent trafficking of Kv7.2/Kv7.3 channels can lead to pathology regardless of the consequences on the macroscopic ionic flow through the channel.     

Evidence for two endocytic transport pathways in plant cells

Intracellular trafficking of endocytic vesicles in eukaryotes varies with the nature of the cargo molecules and the targeted organelle, and proceeds through an intricate network of internal endosomal compartments. However, the path for fluid-phase endocytosis (FPE), the internalization of external solutes from the apoplast via plasmalemma generated vesicles, remains unresolved despite some indication of a direct transport route to the vacuole. To test this hypothesis, we made use of the membrane-impermeable Na-dependent fluorescent marker Coro-Na in combination with the fluorescent membrane marker FM 4-64 and confocal laser scanning microscopy. When protoplasts from sweet lime juice cells were incubated in Na-free solution, FM 4-64, Coro-Na, and 200 mM sucrose, two distinct types of labeled vesicles were evident. A set of vesicles (1 μm in diameter) was intensely labeled with Coro-Na and to a lesser extent with FM 4-64, whereas the second type of 1–7 μm structures appeared exclusively labeled with FM 4-64. These data demonstrate the parallel functioning of two endocytic pathways in plant cells. In one system, a set of small endocytic vesicles merge with the endosome, whereas a separate set of vesicles fuse to form larger vesicles independent from the endosome. Although it is likely that both vesicle systems eventually contribute to solutes reaching the vacuole, given their size (1–7 μm), and based on previous observations of endocytic vesicle formation protruding from the plasmalemma and merging with the vacuole, we conclude that these latter vesicles constitute the primary FPE vesicle system.     

Fluid Phase Endocytic Uptake of Artificial Nano-Spheres and Fluorescent Quantum Dots by Sycamore Cultured Cells: Evidence for the Distribution of Solutes to Different Intracellular Compartments

Fluid phase endocytic uptake of external solutes in plant cells was further substantiated using artificial polystyrene nano-spheres (40 nm) and CdSe/ZnS quantum dots (20 nm). Both types of artificial nano-particles were taken up by sycamore-cultured cells. However, whereas polystyrene nano-spheres were delivered to the central vacuole, CdSe/ZnS nano-dots were sequestered into cytoplasmic vesicular structures. Using dextran-Texas Red (m.w. 3,000; d-TR) as additional marker, confocal micrographs confirmed the distinct topographic distribution of CdSe/ZnS quantum dots within the cell. Initially, d-TR and CdSe/ZnS quantum dots colocalized within cytoplasmic vesicles. After 18 h incubation, d-TR was distinctly localized in the vacuole whereas CdSe/ZnS quantum dots remained sequestered in cytoplasmic membranous compartments. The data provide a first evidence for the rapid distribution of solutes taken up by endocytosis to distinct intracellular compartments.Key Words: apoplast, assimilate partitioning, endocytosis, protoplasts, sucrose transport, sucrose uptake, vacuole     

Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells

The implied existence of two mechanisms for glucose uptake into heterotrophic plant cells was investigated using the fluorescent glucose derivative 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), two membrane impermeable fluorescent markers (3000 mol. wt. dextran-Texas Red (d-TR) and Alexa-488), hexose carrier and endocytic inhibitors (phloridzin and wortmannin-A, respectively), and fluorescent and confocal microscopy. Both phloridzin and wortmannin-A significantly reduced the uptake of 2-NBDG into sycamore cultured cells, which was confirmed by fluorescent microscopy. Phloridzin prevented 2-NBDG uptake exclusively into the cytosol, whereas the wortmannin-A effect was more general, with 2-NBDG uptake into the vacuole being the more affected. Simultaneous incubation of cells in the membrane-impermeable fluorescent probes Alexa-488 and d-TR for 24 h resulted in co-localization of the labelling in the central vacuole and other endosomal compartments. Cytoplasts, cells devoid of vacuoles, were instrumental in demonstrating the transport of 2-NBDG by separate uptake mechanisms. In cytoplasts incubated simultaneously in 2-NBDG and d-TR for 2 h, a green fluorescent cytosol was indicative of transport of hexoses across the plasmalemma, while the co-localization of 2-NBDG and d-TR in internal vesicles demonstrated transport via an endocytic system. The absence of vesicles when cytoplasts were pre-incubated in wortmannin-A authenticated the endocytic vesicular nature of the co-shared 2-NBDG and d-TR fluorescent structures. In summary, uptake of 2-NBDG occurs by two separate mechanisms: (i) a plasmalemma-bound carrier-mediated system that facilitates 2-NBDG transport into the cytosol, and (ii) an endocytic system that transports most of 2-NBDG directly into the vacuole.     

Architectural remodeling of the tonoplast during fluid-phase endocytosis

During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration.