Electrorotational spectra of protoplasts generated from the giant marine algaValonia utricularis

Abstract

Smnmary. Protoplasts of Valonia utricularis lacking the large central vacuole can be generated by cutting multi-nucleated, giant "'mother" cells into small pieces after short exposure to air. When the protoplasmic content was squeezed out into sea water, irregularly shaped, green coloured aggregates were formed which changed into spherical protoplasts (radius of 20-60 gm) after about 2 h. In these protoplasts the dense internal material (consisting mainly of organelles) was separated from the plasmalemma by a thin transparent layer containing a large number of small lipid vesicles. Cell wall regeneration occurred rapidly after protoplast formation. A central vacuole developed alter about 10 h. The regenerated cells continued to grow and were viable for several months. Electrorotation studies on 2-3 h old protoplasts at pH 7 in low-and fairly high-conductivity solutions showed one or two anti-field rotation peaks (depending on medium conductivity) between 10 kHz to 1 MHz as well as one cofield rotation peak between 10 MHz to 100 MHz. The rotation spectra could not be fitted on the basis of the single-(or multi-) shell model (i.e., by modelling the cells as a homogeneous sphere surrounded by one or more layers). However, fairly good agreement between the experimental data and theory could be obtained by assmning that the rotational behaviour of the protoplasts depends not only on passive eleclrical properties of the plasmalemma but is influenced by "'mobile charges" of carrier transport systems and/or the dielectric behaviour of the aggregated chloroplasts and vesicles.

A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis

“…The preparation of protoplasts from giant "mother cells" was described in detail by Wang et al (1997b). Briefly, cylindrical cells with a length of 10-30 mm and a diameter of 3-5 mm were taken.…”

“…They were formed within 2 hr and regenerated very rapidly to walled cells with a central vacuole which grew to large-sized cells over the following months. Besides this, there was evidence (Wang et al, 1997b) that the membrane of the protoplasts contained exclusively plasmalemma material from the "mother cells." Problems arising from large central vacuoles and cell walls do not exist provided that newly formed protoplasts are used immediately for patch-clamp studies (after short treatment of the protoplasts with enzymes for removal of cell wall fibrils).…”

“…In this communication, we applied the patch-clamp technique to protoplasts produced from the "mother cells" of V. utricularis (Wang et al, 1997b) in order to identify the transport elements of the plasmalemma in more detail and to reject or to support previous conclusions. These protoplasts could easily be obtained when the protoplasmic content of turgorless "mother cells" was squeezed out in sea water or in solutions suitable for patch-clamp studies.…”

Multiple-Cylindrical Electrode System for Rotational Electric Field Generation in Particle Rotation Applications

“…The simulations further show that particles polarize in both cases, but for the eight-electrode system rotational fields consistently polarize the particle, resulting in a uniform rotation rate, as seen in the supplementary videos. Buffer medium (DI water) [44] 78 5.5e-3 Table 3. Parameter constants of particles and medium electric-field frequency and amplitude.…”

Electroporation and lysis of marine microalga Karenia brevis for RNA extraction and amplification

“…At an applied voltage of 60 V, the cell sees a field of the order 3 Â 10 6 V m 21 . For a cell radius of 10 mm , a frequency of 600 kHz and an assumed membrane capacitance of 8 mF m 22 (reported for similar marine alga [49,50]), the trans-membrane potential is calculated as 3 V, more than sufficient to cause dielectric breakdown of the membrane and complete cell lysis [44,46]. The experimental conditions used for cell lysis were selected to maximize the applied voltage dropped across the cell membrane.…”