Cellulose synthesis is coupled to cell cycle progression at G1 in the dinoflagellate Crypthecodinium cohnii

Abstract

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 G(1). A cell cycle delay in G(1), but not G(2)/M, was observed after inhibition of CS. A cell cycle inhibitor of G(1)/S, but not G(2)/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 G(1) 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 G(1) 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.

Figure 1

Schematic diagram of the amphiesma of a typical thecate dinoflagellate based on Morrill and Loeblich (1984). The amphiesma consists of a continuous outermost membrane (the plasma membrane), the outer plate membrane, and a single-membrane-bounded thecal vesicle. Inside this vesicle, there are a number of cellulosic thecal plates subtended by a pellicular layer. The structural component, cellulose, is mainly deposited in the thecal plates.

Figure 2

Cellulose biosynthesis in normal C. cohnii cell cycle. A, Flow cytograms of propidium iodide (PI)-stained synchronous C. cohnii. T, Time (hour) that the samples were harvested after cell cycle synchronization. Cellulose deposition of C. cohnii is a stepwise process within a cell cycle. The CFW:forward scatter (FSC²) ratio increased mainly in G₁ phase and peaked at T = 6. The ratio decreased when the cells entered G₂/M phase (T = 7 to T = 10). B, G₁ and G₂/M cells are marked on the density plot with CFW plotted against PI. Percentage of G₁ and G₂/M cells were determined by using WinMDI. Corresponding CFW intensities are written in brackets. C, Photomicrographs of CFW-stained cells (same exposure time, 4 s) corresponding to the specific time points after cell cycle synchronization. Scale bar = 10 μm. D, Fluorescence photomicrographs of CFW-stained cells (focusing on the cell surface) corresponding to T = 6 and T = 8. The white arrows indicate the cellulosic thecal plates. At T = 6, individual thecal plates were clearly demarcated. At T = 8, the margins between individual thecal plates became blur. Scale bar = 10 μm. E, Cell growth is coupled to CS. Cells growing at 32°C are larger than control at T = 4 (G₁ phase) with more cellulosic contents. No significant difference in CFW:FSC² ratio was observed for the cells growing at 32°C compared with control at T = 4. F, Line regression analyses of the CFW fluorescence intensities (determined by flow cytometry) and cellulose amount (determined by acetic-nitric cellulose assay) yield a significant positive correlation. Dotted line represents the regression line including the data obtained from T = 12 (R² = 0.6248, P = 0.0344) whereas the solid line represents the regression line excluding the data obtained from T = 12 (R² = 0.9797, P = 0.0002). Data were obtained from a time course experiment.

Figure 3

DCB-arrested C. cohnii cells at G₁ phase. A, Flow cytograms of PI-stained synchronous C. cohnii cells treated with different concentrations of DCB. T, Time (hour) that the samples were harvested after cell cycle synchronization. Note that there is a G₁ arrest for cells treated with 100 μm DCB. B, DCB-treated cells are smaller in cell size than the control. At T = 4, FSC data from DCB treatment (100 μm) were extracted and overlain on the control. Photographs (same exposure time) of CFW-stained cells corresponding to the time point T = 4 under different treatments: I, control; ii, 25 μm DCB; and iii, 100 μm DCB. Scale bar = 10 μm. C, At both T = 4 (G₁ phase) and T = 10 (late G₂/M phase), significant (P < 0.05) differences were observed for DCB concentrations above 25 μm. D, DCB dose dependently reduced the CFW:FSC² ratio.

Figure 4

DCB failed to arrest C. cohnii cells at G₂/M phase. A, Flow cytograms of PI-stained synchronous C. cohnii cells treated with different concentrations of DCB. DCB was added to synchronous cell culture at T = 7 h with final concentration at 25 and 100 μm. B, Relative CFW intensities obtained from different treatments were plotted: upper, T = 10 (late G₂/M phase); lower, T = 12. Asterisks are used to indicate significant (P < 0.05) differences as compared with control.

Figure 5

HU-arrested cells reveal that CS was coupled to cell cycle progression in C. cohnii. A, Long S phase delay occurred for the HU-treated cells. About 18 h were spent to finish the cell cycle. B, During the S phase delay (T = 8 to T = 12), no significant (P < 0.05) differences were observed for the CFW intensities. When the cells entered G₂/M phase, CFW intensity increased substantially and peaked at T = 18, where the cells finished the cell cycle. C, HU-treated cells grew to a larger cell size caused by the G₁/S arrest after a cell cycle time. Note that the cell size of HU-treated cells (mainly G₁ cells) is even larger than that of control at T = 8 (mainly G₂/M cells). D, CS in G₂/M became deregulated after HU-induced G₁/S arrest. The CFW:FSC² ratio of HU-treated cells was much lower than that of control.

Figure 6

The effects of nocodazole on C. cohnii. A, Nocodazole arrested cells in G₂/M phase, but not in G₂/M phase. Flow cytograms of PI-stained synchronized cells treated with nocodazole are plotted, with the x axis representing the relative DNA content, the y axis representing the cell number, and the z axis representing the time (hour). The arrow indicates the G₂/M delay upon nocodazole treatment. B, CFW intensity of nocodazole-treated cells increased at T = 8. No significant (P < 0.05) differences were observed for the following time points.

Figure 7

Schematic diagram illustrating the variation of amphiesma in the C. cohnii cell cycle. A, Ultrastructure of the variation of C. cohnii amphiesma was drawn based on Morrill and Loeblich (1984). i, Cellulosic thecal plates are absent in newly emerged daughter cells. As the cell grows in G₁ phase, cellulose accumulated and thecal plates thickened (ii). iii, Small prethecal vesicles formed beneath the cytoplasmic membrane after the cells shed the flagella. iv, Prethecal vesicles then enlarged and fused, with a new pellicle formed within. Only the cytoplasmic membrane remains and becomes the outer most membrane of the emerged daughter cell. New thin thecal plates are formed shortly after ecdysis. OM, Outermost membrane; OP, outer plate membrane; TV, thecal vesicle; TP, thecal plate; P, pellicle; CM, cytoplasmic membrane; PTV, prethecal vesicle; MCW, mother cell wall; DCW, daughter cell wall. B, Hypothetical model describing the bidirectional flow of information between cell wall integrity and cell cycle control in G₁ phase of C. cohnii cell cycle. Our results demonstrated a novel link between CS and cell cycle progression.