Localized Patch Clamping of Plasma Membrane of a Polarized Plant Cell

Abstract

We used an ultraviolet laser to rupture a small region of cell wall of a polarized Fucus spiralis rhizoid cell and gained localized access to the plasma membrane at the growing apex. Careful control of cell turgor enabled a small portion of plasma membranebound cytoplasm to be exposed. (1,11,17,19,21), and more recently, the technique has been used to investigate their regulation (7,10,15,16,18,20).Removal of the cell wall to expose plasma membrane suitable for patch recording was predicted to be a major problem in the advancement of single-channel studies in plant cells by Takeda and coworkers (22). In all but a few cases, removal of the cell wall is achieved by enzymic treatment to produce a spherical protoplast. the low success rates have often proved unsuccessful (6), although recent improvements to the procedure of enzymically isolating protoplasts may increase the number of successful GU seals (4).Plant cell protoplasts do not retain the structural order or polarity of the parent cell. This problem is of particular importance in the study of ion channel activity, distribution, and regulation in polarizing and polarized plant cells. In Fucus zygotes, the plant cell wall is required for the axis of polarity to develop (13); protoplasts from young Fucus zygotes are, therefore, of limited use in a single-channel study of polarity because localized cell wall removal and exposure of the underlying plasmalemma is required. Furthermore, the extraction and purification of the enzymes required for cell wall removal in Fucus are complex and time consuming (12). Although mechanical or surgical techniques have been used to gain access to the plasmalemma of Chara (3, 14), these are not suitable for smaller and less robust cells.In this report, we describe a novel technique for localized removal of the cell wall of Fucus rhizoid cells using a UV laser. Under appropriate conditions of turgor, the tip of the rhizoid cell is exposed, enabling high resistance GO seals to be obtained on the plasmalemma. MATERIALS AND METHODS Growth of ZygotesThallus tips bearing mature conceptacles of the dioecious marine alga Fucus spiralis were collected, washed, and stored at 40C in the dark for 1 to 3 d before they were placed in FSW2 at 180C to release zygotes. Zygotes were washed and transferred into small plastic chambers with a glass coverslip base. Dishes were placed in unilateral white fluorescent light (50 ,Em-2 s-1) at 180C to promote polarization, and zygotes were used 2 to 3 d after release. Preparation of ZygotesDishes were placed on the stage of an inverted microscope (Nikon Diaphot, Nikon, Tokyo, Japan) and treated with

Elemental propagation of calcium signals in response-specific patterns determined by environmental stimulus strength

“…Fluorescence of injected cells was monitored at least 3 h after injection and recovery in SW. Photometric measurements of Ca 2ϩ were made on fura-2-dextran-injected rhizoid cells (19,20). For confocal imaging, zygotes were imaged after Ͼ3 h with a Bio-Rad (Hemel Hempstead, Hertfordshire, U.K.) 1024 confocal laser scanning microscope equipped with a Nikon ϫ40 (numerical aperture ϭ 1.3) oil immersion lens and an argon͞krypton laser.…”

“…Fucus serratus embryos were cultured to the two-celled stage in seawater (SW) for 24 h in unidirectional white light at 17°C as described (19,20). Rhizoid cells were pressure microinjected with 10-50 M concentrations of dye prepared in artificial cytoplasm solution (19,20). Dyes used included fura-2-dextran (10 kDa), Calcium Green-dextran (10 kDa), or a mixture of Calcium Green-dextran and Texas Red-dextrans (10 kDa) (Molecular Probes).…”

Laser ablation of the cell wall and localized patch clamping of the plasma membrane in the filamentous fungus Aspergillus: characterization of an anion‐selective efflux channel

“…Laser ablation of the cell wall to expose the plasma membrane has been used as a tool for investigations of algal cells (Taylor & Brownlee, 1992;DeBoer et al, 1994;Taylor et al, 1996) and higher plant cells (Kurkdjian et al, 1993;Henricksen et al, 1995); though only two studies have then successfully applied the patch-clamp technique to characterize ion channels in the exposed plasma membrane (Henricksen et al, 1995;Taylor et al, 1996). In the present study, we have shown that it is feasible to use the laser ablation technique to produce plasma membrane-bound blebs at defined locations along the hyphae of Aspergillus.…”

“…A pulsed nitrogen laser (VSL-337, Laser Science Inc., Cambridge, MA) was used to produce a high intensity u.v. beam into the microscope objective (Nikon UV-Fluor x 40, numerical aperture 0-8-1-3) and focused to a spot of c. 1 fim (see Taylor & Brownlee, 1992). The laser was triggered remotely using 12-V pulses from a stimulator.…”

“…laser is used to ablate the cell wall and expose the plasma membrane without the use of cell walldegrading enzymes circumvents these problems (Taylor & Brownlee, 1992). To date, the laser ablation approach has been successfully used in conjunction with the patch clamp technique to study plasma membrane ion channels in Fucus rhizoid cells (Taylor et al., 1996) and guard cells (Henricksen et al, 1995).…”