Alvin C.M. Kwok

Cellulose Synthesis Is Coupled to Cell Cycle Progression at G1 in the Dinoflagellate Crypthecodinium cohnii

Cellulosic deposition in alveolar vesicles forms the “internal cell wall” in thecated dinoflagellates. The availability of synchronized single cells, the lack of secondary deposition, and the absence of cellulosic cell plates at division facilitate investigation of the possible roles of cellulose synthesis (CS) in the entire cell cycle. Flow cytograms of cellulosic contents revealed a stepwise process of CS in the dinoflagellate cell cycle, with the highest rate occurring at G1. A cell cycle delay in G1, but not G2/M, was observed after inhibition of CS. A cell cycle inhibitor of G1/S, but not G2/M, was able to delay cell cycle progression with a corresponding reduction of CS. The increase of cellulose content in the cell cycle corresponded well to the expected increase of surface area. No differences were observed in the cellulose to surface area ratio between normal and fast-growing G1 cells, implicating the significance of surface area in linking CS to the coupling of cell growth with cell cycle progression. The coupling of CS to G1 implicates a novel link between CS and cell cycle control, and we postulate that the coupling mechanism might integrate cell wall integrity to the cell size checkpoint.

Alveolata histone-like proteins have different evolutionary origins.

Prokaryotic histone‐like proteins (Hlps) are abundant proteins found in bacterial and plastid nucleoids. Hlps are also found in the eukaryotic dinoflagellates and the apicomplexans, two major lineages of the Alveolata. It may be expected that Hlps of both groups were derived from the same ancestral Alveolates. However, our phylogenetic analyses suggest different origins for the dinoflagellate and the apicomplexan Hlps. The apicomplexan Hlps are affiliated with the cyanobacteria and probably originated from Hlps of the plastid genome. The dinoflagellate Hlps and the proteobacterial long Hlps form a clade that branch off from the node with the proteobacterial short Hlps.

The Activity of a Wall-Bound Cellulase Is Required for and Is Coupled to Cell Cycle Progression in the Dinoflagellate Crypthecodinium cohnii

Cellulases are ubiquitous enzymes that play an essential role during plant cell growth and development. This study demonstrates that the activity of a dinoflagellate cell wall–bound cellulase is required for cell cycle progression and that its expression is downregulated in response to specific inhibitors in the G2/M phase. Cellulose synthesis, but not its degradation, is generally thought to be required for plant cell growth. In this work, we cloned a dinoflagellate cellulase gene, dCel1, whose activities increased significantly in G2/M phase, in agreement with the significant drop of cellulose content reported previously. Cellulase inhibitors not only caused a delay in cell cycle progression at both the G1 and G2/M phases in the dinoflagellate Crypthecodinium cohnii, but also induced a higher level of dCel1p expression. Immunostaining results revealed that dCel1p was mainly localized at the cell wall. Accordingly, the possible role of cellulase activity in cell cycle progression was tested by treating synchronized cells with exogenous dCelp and purified antibody, in experiments analogous to overexpression and knockdown analyses, respectively. Cell cycle advancement was observed in cells treated with exogenous dCel1p, whereas the addition of purified antibody resulted in a cell cycle delay. Furthermore, delaying the G2/M phase independently with antimicrotubule inhibitors caused an abrupt and reversible drop in cellulase protein level. Our results provide a conceptual framework for the coordination of cell wall degradation and reconstruction with cell cycle progression in organisms with cell walls. Since cellulase activity has a direct bearing on the cell size, the coupling between cellulase expression and cell cycle progression can also be considered as a feedback mechanism that regulates cell size.

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

ABSTRACT 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.

Multiplex fluorimetric assays for monitoring algal toxins

Most known algal toxins act on ion channels either directly or indirectly, resulting in a change in intracellular ion concentrations when administered to targeted cells. The present project developed the working conditions for the use of fluorescent dyes in monitoring changes in membrane potential, intracellular calcium, and intracellular sodium levels in mammalian cell lines. Using these conditions, we were able to demonstrate specific changes in fluorescent signals in response to several purified toxins. We were also able to generate algal extracts which, when administered to the developed fluorimetric assays, were able to elicit different pattern of changes in membrane potential, intracellular calcium, and intracellular sodium levels. The differential pattern of responses induced by the different algal toxins in the three fluorimetric assays serve as a proof of concept for the use of multiplex fluorimetric assays in the laboratory monitoring of algal toxins.