Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion trans...Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of -31 inV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2＋ sensor R-GECO1, rapid elevation of the cytosolic Ca^2＋ concentration was observed, after impalement with microelectrodes, or injection of the Ca^2＋ chelator BAPTA. Elevation of the cytosolic Ca^2＋ level stimulated the activity of voltage- independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca^2＋ level in cells injected with fluorescent Ca^2＋ indicator FURA-2. These data thus show that cytosolic Ca^2＋ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.展开更多
Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles...Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.展开更多
基金This work was supported by a grant from the Deutsche Forschungsgemeinschaft to M.R.G.R. （GK 1342, Project B5）, grants from the NSFC of China （No. 31270306） and the ＂111＂ Project of China （No. B06003）, grants from the Deutsche Forschungsgemeinschaft （FOR 964） to K.S., and by grants from the National Institutes of Health （GM060396） and National Science Foundation （MCB1414339） to Julian Schroeder （University of California, San Diego, USA） for the generation of the R-GECO1 plasmids and initial Ca^2＋ imaging experiments in the Schroeder lab by R.W.We thank Tracey Ann Cuin （University of Wurzburg） for help with preparation of the manuscript. No conflict of interest declared.
文摘Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of -31 inV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2＋ sensor R-GECO1, rapid elevation of the cytosolic Ca^2＋ concentration was observed, after impalement with microelectrodes, or injection of the Ca^2＋ chelator BAPTA. Elevation of the cytosolic Ca^2＋ level stimulated the activity of voltage- independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca^2＋ level in cells injected with fluorescent Ca^2＋ indicator FURA-2. These data thus show that cytosolic Ca^2＋ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.
文摘Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.
