To follow endocytosis in BY-2 cells we made use of fluorescent nano beads. Beads with 20nm diameter were internalised rapidly and accumulated partially compartments also labelled by the endocytic marker FM4-64. Studies protoplasts revealed that larger beads (100nm) excluded from uptake into turgescent plasmolysed while able to internalise a up 1000nm. Endocytosis was only inhibited clathrin-specific inhibitor Ikarugamycin strongly blocked wortmannin. These results imply involves...

Summary The relevance of endocytosis in plants against high turgor pressure has frequently been questioned on the basis energetic considerations. Here, we examine dynamics plasma membrane (PM) turgid guard cells Vicia faba by monitoring with confocal microscopy fate fluorescent styryl dyes (FM1‐43, FM2‐10 and FM4‐64). As a second marker, also observe retrieval chimaera K + ‐inward rectifying channel from Arabidopsis thaliana green protein (KAT1::GFP). Analysis cytoplasmic structures, which...

In eukaryotic cells, several pathways exist for the internalization of plasma membrane proteins and extracellular cargo molecules. These endocytic can be divided into clathrin-dependent clathrin-independent pathways. While are known to involved in a variety cellular processes plants, have so far only been identified animal yeast cells. Here we show that fluorescent glucose BY-2 cells leads accumulation sugar compartments pathway. This uptake was not blocked by ikarugamycin, an inhibitor...

Summary Trafficking of K + inward (K in ) rectifying channels was analyzed guard cells Vicia faba transfected with the rectifier from Arabidopsis thaliana KAT1 fused to green fluorescent protein (GFP). Confocal images and whole‐cell patch‐clamp measurements confirmed incorporation active into plasma membrane cell protoplasts. The current density much larger protoplasts than wild‐type (wt) This shows a coupling between channel synthesis membrane. Pressure‐driven increase decrease surface area...

• Salinity tolerance in plants involves controlled Na+ transport at the site of accumulation and intracellular compartmentation. The focus this study was identification analysis expression Na+/H+ antiporters response to NaCl stress one particular plant, facultative halophyte Mesembryanthemum crystallinum M. were cloned by RACE-PCR from total mRNA leaf mesophyll cells. Functional complementation Saccharomyces cerevisiae Escherichia coli mutants performed. kinetics changes quantified real-time...

For a number of mammalian ion channels, trafficking to the plasma membrane was found be controlled by intrinsic sequence motifs. Among these sequences are diacidic motifs that function as endoplasmic reticulum (ER) export signals. So far it is unclear if similar also exist in plant channels. In this study we analyzed four DXE/DXD K(+) channel KAT1. Mutation first DXE motif resulted strong reduction KAT1 conductance both guard cell protoplasts and HEK293 cells (human embryonic kidney cells)....

The correct functioning of ion channels depends not only on the control their activity but also regulation number in membrane. For example, it has been proposed that density plant K(+)-channel KAT1 may be adjusted by controlling its export from site synthesis, endoplasmic reticulum (ER). Efficient transport channel to plasma membrane was found depend a di-acidic ER signal C-terminus protein. Studies yeast and mammals indicate motifs are essential for enrichment proteins into ER-derived coat...

The dynamics of macroscopic currents underlying the electrically triggered action potential (AP) in giant alga Chara corallina were directly recorded with an clamp method. In this technique AP is and repetitively replayed as command voltage to same cell under control. Upon adding channel blockers niflumic acid and/or Ba2+ bath, excitation current, i.e. current crossing membrane during AP, can be dissected into a transient, fast-appearing Cl− inward transient delayed K+ outward current. onset...

Osmotically driven swelling and shrinking of guard‐cell protoplasts (GCPs) requires adjustment surface area which is achieved by addition removal plasma membrane material. To investigate the mechanism for adaptation we have used patch‐clamp capacitance measurements. The recorded ( C m ) trace GCPs occasionally revealed discrete upward downward deflecting steps, respectively, with a median value about 2 fF. observed steps resulted from fusion fission single vesicles diameter around 300 nm. We...

Exocytosis allows the release of secretory products and delivery new membrane material to plasma membrane. So far, little is known about underlying molecular mechanism its control in plant cells. We have used whole-cell patch-clamp technique monitor changes capacitance study exocytosis barley aleurone protoplasts. To investigate involvement Ca 2+ GTP-binding proteins exocytosis, protoplasts were dialyzed with very low (<2 nM) high (1 μM) free nonhydrolyzable guanine nucleotides guanosine...

The activity of the two dominant K(+) channels in plasma membrane Vicia faba guard cell protoplasts was examined during pressure-driven swelling. For this purpose, currents and capacitance (C(m)) were recorded parallel. A rise C(m), reflecting an increase surface area, coupled to a proportional conductance both inward outward rectifier. activation kinetics not affected process. quantitative temporal coupling C(m) can hence be interpreted as result addition active rectifier area.

The halophyte Mesembryanthemum crystallinum adapts to salt stress by uptake and switching from C3 photosynthesis CAM (crassulacean acid metabolism). An important role in this process is played transport proteins the tonoplast of central vacuole. In present study we examine dynamic changes protein composition during salt-stress adaptation microsomes M. leaves. Plants challenged with 400 mM NaCl accumulate day 4 treatment malic only at 12; a hence follows any initial steps delay. Using...

Changes in membrane capacitance (C m) after photolysis of the caged Ca2+ compound dimethoxynitrophenamine were studied protoplasts from maize coleoptiles. C m values resulting increased concentrations free cytoplasm ([Ca2+]cyt) interpreted as representing changes [Ca2+]cyt–sensitive exocytosis and endocytosis. A continuous increase [Ca2+]cyt resulted a sigmoidal with half-maximal concentration at ∼1 μM. The steep was followed by variable slow phase changing values. When rate 0.6 μmol L–1...

For a number of ion channels, including the potassium (K(+)) inward rectifying channel from Arabidopsis thaliana (KAT1), diacidic endoplasmic reticulum (ER) export motifs have been identified. These consist two acidic amino acids (aspartate (D) and/or glutamate (E)) separated by any acid. To specify role single for efficiency ER export, we analysed sequence KAT1 that included originally identified motif (DxE) plus an additional D just upstream motif. Analysis single, double and triple...

With patch‐clamp recording, we detect in yeast protoplasts individual exo‐ and endocytotic events as discrete steps membrane capacitance. The high‐resolution data show that vesicles undergo, similar to other eukaryotes, permanent transient fusion/fission with a bias for events. electrical are good representation of the dynamics growing cell.

Much of our knowledge ion-transport mechanisms in plant cell membranes comes from experiments using voltage-clamp. This technique allows the measurement ionic currents across membrane, whilst voltage is held under experimental control. The patch-clamp was developed to study flowing through single channels, but its whole-cell configuration it probably most widely used voltage-clamp technique. article provides a basic introduction several advanced techniques, and evaluates methods for...