Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping

Abstract

Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100–250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1–5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1–10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented.

Expanding the Symbiodinium (Dinophyceae, Suessiales) Toolkit Through Protoplast Technology

“…; Pozdnyakov et al. ; Trench and Blank ), but success has been limited. Adamich and Sweeney () reported the first protoplast generation of Gonyaulax polyedra using the detergent Liquinox, but trials of the Liquinox method failed to produce Symbiodinium protoplasts (Jit Ern Chen, pers.…”

“…), and finally, treatment of Prorocentrum minimum with a cellulose synthesis inhibitor had low efficacy in preventing cellulose production (Pozdnyakov et al. ).…”

“…The membranous outer layer and thecal vesicle membrane serve as potential obstacles in dinoflagellate protoplast generation by reducing access to the internal cell walls. Several prior attempts have been made to produce protoplasts of armored dinoflagellates (Adamich and Sweeney 1976;Kwok et al 2007;Pozdnyakov et al 2014;Trench and Blank 1987), but success has been limited. Adamich and Sweeney (1976) reported the first protoplast generation of Gonyaulax polyedra using the detergent Liquinox, but trials of the Liquinox method failed to produce Symbiodinium protoplasts (Jit Ern Chen, pers.…”

“…However, cellulase has been indicated to digest the cell walls of the dinoflagellate Lingulodinium (Wang et al 2005). Cell walls of Crypthecodinium cohnii have been reduced, but not removed, by growing cells on agar plates with polyethylene glycol (PEG) (Kwok et al 2007), and finally, treatment of Prorocentrum minimum with a cellulose synthesis inhibitor had low efficacy in preventing cellulose production (Pozdnyakov et al 2014).…”

Trophic strategies in dinoflagellates: how nutrients pass through the amphiesma

“…Ecdysis occurs during the life cycle of many dinoflagellates and can be induced by stress, e.g. mechanical perturbation (centrifugation) or application of chemical agents, such as a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (Morrill, 1984;Morrill and Loeblich, 1984;Sekida et al, 2001;Pozdnyakov et al, 2014). The new cell covering emerges in a relatively short time.…”