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

Kwok, Alvin Chun Man; Mak, C.C.M.; Wong, Francis T. W.; Wong, Joseph T.Y.

doi:10.1128/ec.00301-06

Cited by 15 publications

(21 citation statements)

References 27 publications

(22 reference statements)

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“…Several prior attempts have been made to produce protoplasts of armored dinoflagellates (Adamich and Sweeney ; Kwok et al. ; Pozdnyakov et al. ; Trench and Blank ), but success has been limited.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Levin

Suggett

Nitschke

et al. 2017

J Eukaryotic Microbiology

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Dinoflagellates within the genus Symbiodinium are photosymbionts of many tropical reef invertebrates, including corals, making them central to the health of coral reefs. Symbiodinium have therefore gained significant research attention, though studies have been constrained by technical limitations. In particular, the generation of viable cells with their cell walls removed (termed protoplasts) has enabled a wide range of experimental techniques for bacteria, fungi, plants, and algae such as ultrastructure studies, virus infection studies, patch clamping, genetic transformation, and protoplast fusion. However, previous studies have struggled to remove the cell walls from armored dinoflagellates, potentially due to the internal placement of their cell walls. Here, we produce the first Symbiodinium protoplasts from three genetically and physiologically distinct strains via incubation with cellulase and osmotic agents. Digestion of the cell walls was verified by a lack of Calcofluor White fluorescence signal and by cell swelling in hypotonic culture medium. Fused protoplasts were also observed, motivating future investigation into intra- and inter-specific somatic hybridization of Symbiodinium. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Generation of Symbiodinium protoplasts opens exciting, new avenues for researching these crucial symbiotic dinoflagellates, including genetic modification.

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confidence: 99%

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confidence: 99%

mentioning

confidence: 99%

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

Levin

Suggett

Nitschke

et al. 2017

J Eukaryotic Microbiology

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“…Knockdown experiments and cell-cycle studies were conducted with C. cohnii cells. There are established transfection and coccoid stage-swarmer release synchronization protocol [24,25].…”

Section: Dinoflagellate Cell Cultures and Flow Cytometrymentioning

confidence: 99%

Knockdown of Dinoflagellate Condensin CcSMC4 Subunit Leads to S-Phase Impediment and Decompaction of Liquid Crystalline Chromosomes

Yan

Kwok

et al. 2020

Microorganisms

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Dinoflagellates have some of the largest genomes, and their liquid-crystalline chromosomes (LCCs) have high degrees of non-nucleosomal superhelicity with cation-mediated DNA condensation. It is currently unknown if condensins, pentameric protein complexes containing structural maintenance of chromosomes 2/4, commonly involved in eukaryotic chromosomes condensation in preparation for M phase, may be involved in the LCC structure. We find that CcSMC4p (dinoflagellate SMC4 homolog) level peaked at S/G2 phase, even though LCCs do not undergo global-decondensation for replication. Despite the differences in the chromosomal packaging system, heterologous CcSMC4p expression suppressed conditional lethality of the corresponding fission yeast mutant, suggesting conservation of some canonical condensin functions. CcSMC4p-knockdown led to sustained expression of the S-phase marker PCNAp, S-phase impediment, and distorted nuclei in the early stage of CcSMC4p depletion. Prolonged CcSMC4p-knockdown resulted in aneuploidal cells and nuclear swelling with increasing LCC decompaction–decondensation. Cumulatively, our data suggested CcSMC4p function was required for dinoflagellate S-phase progression, and we propose that condensin-mediated higher-order compaction provisioning is involved in the provision of local rigidity for the replisome.

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“…The percentage of FDA-stained viable protoplasts was determined using a FACSCalibur Flow Cytometer (BD Bioscience, Heidelberg, Germany) and calculated using the CellQuest Software (BD Bioscience). Cellulose staining with Calcofluor White (CW, Sigma-Aldrich) was used to detect regenerating primary cell walls (Kwok et al, 2007). A 400-lL aliquot of protoplast suspension was inverted gently for 10 min with 0.04 mg ⁄ mL CW solution.…”

Section: Protoplast Stainingmentioning

confidence: 99%

Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

Kirchhoff

Raven

Boes

et al. 2012

Plant Biotechnology Journal

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SummaryPlant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY-2 cells expressing the human antibody M12 by selecting the co-expressed fluorescent marker protein DsRed located on the same T-DNA. Separation yielded 35% wells containing single protoplasts and 15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 90-96% in the six monoclonal lines resulted in an up to 13-fold increase in M12 production that remained stable for 10-12 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins.

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

Cited by 15 publications

References 27 publications

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

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

Knockdown of Dinoflagellate Condensin CcSMC4 Subunit Leads to S-Phase Impediment and Decompaction of Liquid Crystalline Chromosomes

Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

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