The control was cells incubated with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen; A11008) only. the G2/M content (2N). In the G1 phase, the first peak (1N DNA content) appears as the major peak in a histogram. As the cells progress to the S phase, the first (G1, 1N) peak drops, and the second (G2/M, 2N) increases. During the G2/M phase, the DNA content doubles. Eventually, the cells complete mitosis and reenter the next G1 phase (the second peak drops and the first peak increases again). (B) Flow cytograms of PI-stained synchronous and and green with SYTOX Xanthotoxol Green for and with the assistance of fluorescent DNA stains (Kwok and Wong, 2003). Fluorescent photomicrographs suggest that the addition of cellobiose (100 M) resulted in the appearance of more multinucleated cells in both species (Figure 1C). Control experiments using other small oligosaccharides that do not inhibit cellulase activity (e.g., cellotriose and maltose) were performed to verify that the observed cell cycle effects were due to the specific inhibition of cellulase activity. Cellotriose and maltose were individually added to the synchronized cells (at T = 7) at concentrations essentially the same as for the cellobiose. Unlike cellobiose, neither cellotriose nor maltose delayed the cell cycle when compared with the control (see Supplemental Figure 1 online). This confirms that the observable cell cycle delay following the addition of cellobiose was a consequence of cellulase activity inhibition. Cellulase Activity Peaks at G2/M To determine if Xanthotoxol there is any cell wallCassociated cellulase activity in the cells, cell walls were isolated and purified, and cell wallCbound proteins were extracted. Cellulosic cell walls in the cell wall fraction were stained blue with Calcofluor White (Figure 2A). To detect the presence of cellulase activity in the cell wallCbound protein sample, a Congo red-carboxymethylcellulose (CMC) staining method was employed that locates bands of cellulase activity in a polyacrylamide gel following electrophoresis (Schwarz et al., 1987). By staining the CMC-containing gel replica with Congo red, light-yellow bands (of 60 kD, corresponding to the molecular mass of dCel1p) against a red background demonstrated the breakdown of CMC substrate (Figure 2B) and suggested the presence of cellulase activity in the samples. Open in a separate window Figure 2. Cellulase Activity in the Cell Cycle. (A) Fluorescence photomicrograph of cellulosic cell walls prepared for the isolation of the cellulase-containing cell wallCbound protein fraction. Cellulose in the cell wall was stained blue with Calcofluor White. Bar = 10 m. (B) Cellulase activity in CMC-containing agarose replica was stained with Congo red following gel electrophoresis. Yellow bands (60 kD) against an orange background were observed in both the cell wallCbound protein fraction Rabbit Polyclonal to PGD and immunoprecipitation (IP) sample. (C) Flow cytograms of PI-stained synchronous cells were plotted, with the axis representing the relative DNA amount, the axis representing the cell number, and the axis representing the time (hours) after synchronization. (D) Cellulase activity of the cell wall protein fraction on Xanthotoxol the CMC substrate was determined by measuring the increase in Xanthotoxol reducing power of the reaction solution. Cell wallCbound protein fractions were harvested at different time points after cell cycle synchronization. Activity was expressed as pmole glucose released per 104 cells or per cellulose content (g). (E) Substrate specificities of cell wallCbound protein sample (harvested at the G2 phase, T = 7) toward different polysaccharides. Data represent means se of three replicate experiments. (F) Effects of different small oligosaccharides and anti-dCel1p antibody on cell wallCbound CMCase activity. Cellobiose (100 M), cellotriose (100 M), maltose (100 M), and anti-dCel1p antibody (3 g mL?1) were tested for their ability to affect cell wallCbound CMCase activity. Cellobiose and anti-dCel1p antibody significantly (P 0.01) reduced the CMCase activity, indicated by the asterisks, when compared with the control. Data represent means se of three replicate experiments. (G) Effects of different small oligosaccharides and anti-dCel1p antibody on cell wallCbound glucomannanase activity. Cellobiose (100 M), cellotriose (100 M), maltose (100 M), and anti-dCel1p antibody (3 g mL?1) were tested for their ability to affect cell wallCbound glucomannanase activity. No significant (P 0.05) difference was found in any of the treatments compared with the control. Data represent means se of three replicate experiments. [See online article for color version of this figure.] During the cell cycle (Figure 2C), cellulase activity per cell (glucose released per 104 cells) increased in the G1 phase (T = 2 to T = 6) (Figure 2D). Cellulase activity per cell peaked at G2/M phase (T = 10) and dropped when the cells entered the.