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Eukaryotic Cell, March 2007, p. 563-567, Vol. 6, No. 3
1535-9778/07/$08.00+0     doi:10.1128/EC.00301-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Novel Method for Preparing Spheroplasts from Cells with an Internal Cellulosic Cell Wall

Alvin C. M. Kwok, Carmen C. M. Mak, Francis T. W. Wong, and Joseph T. Y. Wong^*

Department of Biology, Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong SAR, People's Republic of China

Received 18 September 2006/ Accepted 12 January 2007

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Protoplast and spheroplast preparations allow the transfer of macromolecules into cells and provide the basis for the generation of engineered organisms. Crypthecodinium cohnii cells harvested from polyethylene glycol-containing agar plates possessed significantly lower levels of cellulose in their cortical layers, which facilitated the delivery of fluorescence-labeled oligonucleotides into these cells.

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The complete or partial removal of the cell wall, resulting in the generation of protoplasts or spheroplasts, respectively, provides the basic permeabilized system for ultrastructural, genetic, and physiological studies (8, 21). The advent of digestive enzymes, including chitinases, cellulases, and pectinases, as well as mechanical rupture has been instrumental in the production of these permeabilized cells for both plants (4) and fungal cells (8). These permeabilized cells have, in turn, been pivotal for the molecular understanding and exploitation of these cell-walled organisms. The cell walls of dinoflagellates, unlike most other walled cells, are internal to the plasma membrane (14). Membranous cellulosic thecal plates are sandwiched between the outermost plasma membrane and an inner cell membrane. The inner cell membrane is very often strengthened by the deposition of cellulose to form the pellicle layer. The "internal placement" of dinoflagellate cell walls renders the cellulosic components inaccessible to digestive enzymes. The efficiency of dinoflagellate cell wall degradation by cellulase was low, and the cells were also killed by the high optimal temperature (40°C) during digestion (20).

Dinoflagellates are the major causative agents of harmful algal blooms and are the symbiotic alga of corals. Members of the group are infamous for their production of toxins responsible for common seafood poisoning syndromes (e.g., ciguatera poisoning). The nucleosomeless liquid crystalline chromosomes of dinoflagellates are the only known alternative to the histone-based packaging of eukaryotic DNA, but very little is known about their molecular mechanism of gene regulation. Despite their ecological importance, biochemical richness, and biotechnological significance, dinoflagellates are still largely intractable to molecular genetic investigation.

Electron microscopy studies of many dinoflagellate species suggest that the deposition of the cellulosic thecal plates is mediated by vesicular fusion (22). In the course of investigating the cellulose deposition process in Crypthecodinium cohnii cells, we developed a protocol for producing spheroplasts with a significantly lower level of cellulose content. The method is based essentially on the separation of motile daughter cells from the mother cell wall. C. cohnii is one of the two heterotrophic dinoflagellates that have been successfully cultured in a synthetic culture medium (6), and more importantly, it is able to form colonies on agar plates. After cytokinesis, the daughter cells do not immediately separate but instead they stay inside the mother cell for some time before they "break off" and discard the mother cell wall (2). As only the early G₁ daughter cells are motile, highly synchronized cell cultures can be produced by colony release and filtration (24). We made use of this ability to investigate whether agents that mediate membrane fusion (e.g., polyethylene glycol [PEG]) can enhance the deposition of the thecal vesicles through the daughter cell membranes.

Crypthecodinium cohnii Biecheler strain 1649 was obtained from the Culture Collection of Algae at the University of Texas at Austin, maintained in MLH liquid medium (18), and incubated at 28°C in the dark. Two days before the spheroplasts were required, an exponentially growing culture of C. cohnii cells was harvested by centrifugation (2,500 x g for 10 min). After the cell pellet was resuspended in 20% (wt/vol) PEG and vortexed on a Vortex Genie mixer (Scientific Industries) for 10 min, cells were spread on MLH agar plates containing 0.4% (wt/vol) PEG 8000 (Sigma-Aldrich). After incubation at 28°C for 2 days in the dark, we carefully poured fresh MLH medium over the C. cohnii colonies on the agar plates. This first elutant was poured off, thus discarding the loosely attached colonies. Additional MLH medium was then poured carefully over the colonies, and this second elutant was retained. To further enhance the collection of highly synchronized control cells, a filtration step through a 10-µm filter excluded contamination by the larger mother cells (24).

Calcofluor white M2R (CFW; Sigma-Aldrich), a fluorescent brightening agent (UV excitation), has been used extensively to visualize the cellulosic thecal plates of armored dinoflagellates (5, 7). The reliability of CFW in staining cellulose in C. cohnii cells was confirmed with an acetic-nitric cellulose assay (7). In the present study, CFW-stained motile cells eluted from colonies after incubation on 0.4% (wt/vol) PEG-containing MLH agar plates had significantly lower levels of fluorescence (hence lower cellulosic content) than control cells eluted from normal MLH agar plates (Fig. 1A). Flow cytometry comparison suggested that the mean value of relative cellulosic content of the spheroplasts was only 20% of that of the control cells (Fig. 1B).

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FIG. 1. Spheroplast of Crypthecodinium cohnii. Fluorescence photomicrographs (same exposure time, 4 s) (A) and flow cytograms (B) of CFW-stained C. cohnii control cells and spheroplasts. (C) Spheroplasts possessed significantly lower DCVJ fluorescence intensity (P < 0.001) and thereby higher fluidity than control cells. (D) Many cells moved like amoebae after spheroplast preparation. Bar, 10 µm.

To measure membrane fluidity, C. cohnii cells were stained with a viscosity probe, 9-(dicyanovinyl)julolidine (DCVJ; Molecular Probes), as described by Mallipattu et al. (13). The spheroplasts possessed significantly lower levels of DCVJ fluorescence, indicating a higher degree of membrane fluidity (Fig. 1C). Accordingly, many spheroplasts had lost the original (slightly ovoid) dinoflagellate shape. When placed between a glass slide with a coverslip and observed under a light microscope, many spheroplasts moved in an extraordinarily "amoeba-like" fashion (Fig. 1D), which is in contrast to the typical flagellum-mediated "whirling" movement of the control cells. C. cohnii cells were prepared for transmission electron microscopy studies by the standard glutaraldehyde-osmium tetroxide method (12). Electron micrographs demonstrated that two of the cortical layers were missing in the spheroplasts, compared to what was observed with the control cells (Fig. 2). Our method is different from stress-induced ecdysis (1, 3, 17), which would result in pellicle cysts that possess dense and thickened pellicles (3, 16).

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FIG. 2. Transmission electron microscopy of spheroplasts. (A) Schematic diagram showing the swarmer cells and cells in colonies during spheroplast preparation. (B) Swarmer cells and cells in colonies were harvested and observed under transmission electron microscopy at x5,800 (whole cell) and x14,000 (focused at the cortical layers) magnification. For swarmer cells, the layers in red (as shown in the right-hand panel) became thinner or even absent in PEG-treated cells (spheroplasts). For cells in a colony, the green layer (as shown in the right-hand panel) of PEG-treated cells was much thicker than that of the control. Specimens were examined in a JEOL 100CX transmission electron microscope.

The preparation of protoplasts from plant cells per se is a stress-inducing process, particularly during enzymatic digestion, with an accumulation of peroxides which induces cell lysis (15). In our study, no observable differences in cell cycle progression were seen between the spheroplasts and the control cells (Fig. 3A and B). This suggests that the spheroplast preparation method has little or no deleterious effects on the cells. A viable protoplast or spheroplast is capable of cell wall regeneration, cell division, and growth (8, 19). The spheroplasts produced by our method were able to pass through cell division (Fig. 3A) and resynthesize cellulose (Fig. 3C).

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FIG. 3. Cell cycle progression of spheroplasts was not affected by the preparation method. (A) Flow cytograms of propidium iodide-stained synchronous C. cohnii cells. "T" refers to the time (hour) that the sample was harvested after cell cycle synchronization. (B) The percentages of G1 and G2/M cells during the cell cycle were determined by the software WinMDI (version 2.8; The Scripps Research Institute [http://facs.scripps.edu/software.html]). Specific regions corresponding to the G1 and G2/M peaks were gated on flow cytograms by using the "Marker" function. (C) Calcofluor white staining revealed that spheroplasts generally possessed significantly less cellulose than the control throughout the cell cycle. Results are representative of triplicate experiments.

Protoplast or spheroplast preparation allows the transfer of macromolecules into cells. This includes nucleic acids (e.g., RNA and plasmid DNA). We tested the efficacy of liposomal-mediated transfer of macromolecules into spheroplasts with fluorescein isothiocyanate (FITC)-conjugated oligonucleotides and antibodies. Flow cytometry analysis of the transfected cells suggested that the FITC-conjugated oligonucleotides and FITC-conjugated antibodies were 1.9 times (Fig. 4A and B) and 1.7 times (Fig. 5A and B) more efficient at entering the spheroplasts than the control cells. A detailed examination of transfected spheroplasts (transfected with FITC-conjugated oligonucleotides) shows that the fluorescence was localized throughout the cell (Fig. 4A and B). To check whether the spheroplasts were still viable after transfection, transfected cells were spread on MLH agar plates and the number of colonies that formed was counted. No significant difference in the number of colonies formed was observed between transfected control cells and spheroplasts (data not shown). The uptake of DNA into protoplasts or spheroplasts provides the basis for transient or stable nuclear transformation. No fluorescence was observed for either the transfected control cells or the spheroplasts at 24 h (about two cell cycles) posttransfection. It is possible that the fluorescent oligonucleotides, which did not carry any replication origin, were lost after 24 h.

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FIG. 4. Permeability of spheroplasts to an FITC-conjugated oligonucleotide. Control cells (A) and spheroplasts (B) were transfected with an FITC-conjugated oligonucleotide (FP) by using Lipofectamine (Lipo). An FITC-conjugated oligonucleotide (1 µg) was first mixed with 15 µl Lipofectamine and allowed to stand at room temperature for 15 min as described in the manufacturer's manual (Lipofectamine reagent; Invitrogen). Around 1 x 106 C. cohnii cells (in 200 µl MLH medium) per reaction were added, and the mixture was shaken gently on a 24-well microplate at room temperature for 15 min. Cells were washed with phosphate-buffered saline three times prior to flow cytometry analysis. Means of fluorescence intensity (log scale) are indicated within parentheses. Photomicrographs of transfected control cells and spheroplasts were taken with the same exposure time. Fluorescence signals corresponding to the FITC-conjugated oligonucleotides are shown. Bar, 10 µm.

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FIG. 5. Permeability of spheroplasts to FITC-conjugated antibody. FITC-conjugated antibody was delivered into control cells (A) and spheroplasts (B) by Lipofectamine. FITC-conjugated anti-rabbit antibody (Ab) (3 mg) was first mixed with 15 µl Lipofectamine and allowed to stand at room temperature for 15 min as described in the manufacturer's manual (Lipofectamine reagent; Invitrogen). Around 1 x 106 C. cohnii cells (in 200 µl MLH medium) per reaction were added, and the mixture was shaken gently on a 24-well microplate at room temperature for 15 min. Cells were washed with phosphate-buffered saline three times prior to flow cytometry analysis. Means of fluorescence intensity (log scale) are indicated within parentheses.

PEG is a widely used agent for promoting vesicle and membrane fusion (10, 11). Cells grown on PEG-containing agar plates developed thickened mother cell walls (Fig. 2). It is proposed that secretory vesicles, containing cellulose or the precursor materials for cellulose synthesis, moved to the periphery of the cell, flattened, and fused together (22). Our results are in agreement with previous reports that the formation of cellulosic thecal plates on the cell cortex might involve a secretory process (22). We propose that PEG in the agar plates might induce the fusion of cellulose-containing secretory vesicles (or precursors for cellulose synthesis) onto the outer cortical layers of the existing mother cell walls. By promoting vesicle fusion to the mother cell walls, which were subsequently abandoned by the daughter cells in the new cell cycle, PEG treatment led to the formation of daughter cells containing less cellulose. Our method is probably applicable to cells in which the deposition of wall material by membrane vesicles occurs during wall construction, e.g., ciliates (23), and also cells that possess an internal cell wall, e.g., euglenoids and cryptomonads (14). Moreover, our previous study demonstrated that the mechanical properties of the dinoflagellate cellulosic thecal plates are very uniform, which is different from the high variability observed in cellulose microfibrils of higher plants (9). This novel property and the corresponding biofabrication mechanism of the dinoflagellate cellulosic cell wall would be of interest to nanobiotechnology.

 
The present study was partly supported by grant HIA05/06.SC01 from the UGC to J.T.Y.W.

 
* Corresponding author. Mailing address: Department of Biology, Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong SAR, People's Republic of China. Phone: 852-23587343. Fax: 852-23581559. E-mail: botin{at}ust.hk.

Published ahead of print on 26 January 2007.

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Eukaryotic Cell, March 2007, p. 563-567, Vol. 6, No. 3
1535-9778/07/$08.00+0     doi:10.1128/EC.00301-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kwok, A. C. M. Articles by Wong, J. T. Y. Search for Related Content PubMed PubMed Citation Articles by Kwok, A. C. M. Articles by Wong, J. T. Y.