We previously showed that holdfasts have the properties of a polysaccharide gel, with wet holdfasts approximately 4 times as thick as when they were dried . With this correction factor, the thickness of wet holdfasts would be between 40 and 50 nm, which is still only about one tenth of their diameter. We conclude that the
holdfast of C. crescentus has the structure of a thin plate. this website Figure 4 AFM images of dried holdfasts of cells at various ages, (a) 17.5 ± 2.5 min, (b) 27.5 ± 2.5 min, (c) 37.5 ± 2.5 min, (d) 47.5 ± 2.5 min, NSC 683864 supplier and (e) 57.5 ± 2.5 min. Scale bars represent 400 nm. A black line is drawn through the center of the holdfast. (f) is the height profile along the black line in (e), showing both the height and width of the holdfast. The holdfast undergoes a two-stage process of spreading and thickening Further AFM measurements were conducted to probe the dynamics of holdfast morphogenesis. Figure 5 shows holdfast diameter and thickness as measured by AFM. The holdfast diameter was quite stable and averaged ~ 360 nm for cells older than 37.5 min, indicating that the holdfast had already attained its maximum diameter at 37.5 min (Figure 5a). Roscovitine cell line We could not reliably measure the holdfast of cells younger than 37.5 min old by AFM because they tended
to be blocked by the cell body. This result is consistent with the fluorescence data, showing no further increase
in intensity beyond the cell age of 35 min (Figure 3). In contrast, holdfast thickness continued to thicken over the next 30 min to about 12 nm in 57.5 min old cells (Figure 5b). The lack of corresponding increase in fluorescence labeling suggests that fluorescein-WGA predominantly labels the surface of the holdfast, which would remain essentially constant as the thin holdfast gradually thickened. Growth in holdfast thickness stopped approximately by the time the attached cells entered their pre-divisional stage. Our experiment did not extend beyond the first cell cycle, thus IMP dehydrogenase it is unclear whether holdfast secretion resumes during subsequent cycles of division. Figure 5 Growth of holdfast attached to a surface measured with AFM. (a) and (b) are the diameter and thickness of dried holdfast measured from AFM images as functions of cell age, averaged over 20 holdfasts for each data point. The error bars are standard errors. The dashed lines are drawn as guide to the eye. Discussion The above results suggest how an attached C. crescentus cell develops its holdfast over time, depicted illustratively in Figure 6. Shortly after attachment, the cell starts to secrete holdfast polysaccharide. This material spreads rapidly on the submerged surface to form a thin plate.