3.1.

Wild-Type Cells

We began by analyzing the dry mass surface density (DMSD) of wild-type cells (strain 972 $h^{-}$ ). Changes in the DM through the cell cycle were obtained by recording the mean phase shift of the cell and its projected surface according to Eq. 1.

The results obtained from analysis of five cells are presented in Fig. 1, which shows the change in the DM (pg), projected cell surface, and DM surface density (DMSD in $\mathrm{pg}∕\mu {\mathrm{m}}^2$ ), defined as the ratio of the last two quantities, through the cell cycle. Four DMSD images of a cell at various stages of the division cycle are shown. The region where the septum forms Fig. 1d(3) has a high DM density $(>1\mathrm{pg}∕\mu {\mathrm{m}}^2)$ , which probably reflects the recruitment of proteins and carbohydrates involved in the synthesis and subsequent cleavage of the division septum.

Fig. 1

Time evolution of: (a) dry mass (fit: linear regression), (b) projected cell surface, and (c) DM concentration. Vertical lines: red, end of surface growth; green, cytokinesis. (d) Representative DM surface density images recorded at the beginning (1), at the end of cell growth (2), just before cytokinesis (arrow: septum) (3), and at the end of the recording period (4). Scale bar $5\mu \mathrm{m}$ . The two sister cells after cell division have been considered as a single cell in order to coherently appreciate the evolution of the measured parameters. The results are representative of five wild-type cells. (Color online only.)

Figure 1a shows a constant production rate of DM at around $10.7\mathrm{pg}∕\mathrm{h}$ (as indicated by linear regression) during the whole cell cycle; a mean value of $11.1\pm 1.5\mathrm{pg}∕\mathrm{h}$ [ $\text{mean}\pm \text{standard}$ error of the mean, (SEM)] was found for five wild-type cells. This production rate predicts that the cell should doubles its protein content in about $2.3\mathrm{h}$ , which is consistent with the doubling time of fission yeast at $36°\mathrm{C}$ reported by a study of S. pombe growth by Mitchison⁴ using a Baker interference microscope.

The cell surface exhibits linear growth during interphase and is followed by a nongrowing period of $35\min$ (indicated by the green vertical line) before cytokinesis when cells enter mitosis (see Introduction).

Specific variations of the DMSD were observed through the cell cycle. At first, the DMSD slowly declines, indicating a larger surface growth than dry mass production. In contrast, during the $35\min$ preceding cytokinesis, as the DM production remains constant and the surface growth pauses, the DMSD showed a significant increase with a maximum value occurring a few minutes before the cytokinesis. This peak value is likely related to the recruitment and/or synthesis of components involved in the mitosis, cytokinesis, and the subsequent S-phase, which occurs prior to cell separation.⁴ As cells divide, the rapid surface increase and the septum degradation induce an abrupt decrease in DMSD [Fig. 1c, green line].

3.2.

Cdc16-116 Mutant

We have also characterized the DM, cell surface, and DMSD parameters in a cdc16-116 mutant.¹⁶ Cdc16p is required to limit the cell to a single septum per cell cycle; if it is inactivated, the cell synthesizes multiple additional septa, producing anucleate cell compartments,¹⁶ cf. Fig. 2d (4).

Fig. 2

Time evolution of: (a) dry mass (fit: two linear regressions), (b) cell surface, and (c) DM concentration. Vertical red line: end of surface growth. (d) representative dry mass density images recorded at the beginning (1), at the first end of cell growth (2), at the second end of cell growth (3), and at the end of the recording period (4). Arrows indicate septum position. Scale bar $5\mu \mathrm{m}$ . (Color online only.)

In contrast to the wild-type cells, the production of DM shows a bilinear pattern¹⁷ with linear segments representing constant growth rates, separated by a transitional period around a rate-change point (RCP, here at minute 120) during which the rate of growth increases. However, there is considerable controversy concerning the exact time profile of size-related parameters, including length, volume, and surface during the cell cycle, and several models (linear, exponential, bilinear, etc.) have been proposed.¹⁷

The data for a typical cell presented here show a twofold increase of the DM production rate, from 9.9 to $19.7\mathrm{pg}∕\mathrm{h}$ after the RCP, which occurs at the end of the mitotic “plateau” [Fig. 2b]. At this time, the two daughter cells are separated by a septum but remain attached to each other.

The mean hour DM production of the Cdc16-116 mutant cell, $13.8\pm 3.5\mathrm{pg}∕\mathrm{h}$ ( $\text{mean}\pm \mathrm{SEM}$ , $n=5$ cells), was found to be slightly higher than the DM production of the wild-type cells. It is possible that this represents the continued accumulation of dense septal material in these cells.

Although the cell surface area of the cdc16-116 and wild-type yeasts both show alternating plateau and expansion phases, the abrupt change in DSMD that accompanies cell separation in wild-type [Fig. 1b] is absent from the cdc16-116 mutant, which shows a more symmetrical sawtooth pattern. It is likely that the increase occurs while the septa are being synthesized, while cell elongation has stopped, and that the decrease represents a resumption of cell elongation. A more detailed explanation of these data will require a greater understanding of the growth patterns of cdc16-116 under restrictive conditions.

A comparison of typical wild-type and mutant DSMD time courses (Fig. 3 ) shows that the DMSD values in cdc16-116 are approximately 25% larger than wild-type. Although the absence of the rapid postcytokinesis surface enlargement and the persistence of the septum in cdc16-116 contribute to this difference, DMSD calculations performed before septum formation (or excluding the contribution of additional septa, $\sim 5$ pg each) still show a significantly larger DMSD (15%) than for the wild type. The reason for this is unclear at present. It is also important to note that at the end of the cell cycle of the wild-type yeast the dry mass concentration returns to the basal level [cf. Figs. 1c and 2c], indicating a homeostasis of the dry mass along generation.

Fig. 3

Comparison of the DM concentration for wild-type (triangle) and cdc16 mutant (circle) fission yeast. Data expressed as $\text{mean}\pm \mathrm{SEM}$ for five cells. The horizontal line denotes time of cytokinesis for wild-type and the stage of maximal DM concentration for mutant.