Design of daughter arrester
Our goal was to design a gene that would selectively prevent daughter cells from dividing. Thus the protein product of this gene needed to meet design specifications: it would (i) arrest cell growth if and only if the drug were present (otherwise the strain could never propagate), (ii) be expressed only in daughter cells after cytokinesis, but be degraded before the daughter cell completed its first cell division, (iii) be in the appropriate cellular compartments to carry out the above functions, and (iv) be readily detectable. To meet these criteria, we combined genetic modules for a conditionally lethal enzyme with cell-cycle dependent synthesis, cell-cycle dependent degradation, visualization and subcellular localization (C) as described in detail below.
Our first major consideration was to use a gene encoding an enzyme capable of interacting with certain substrates, generating a toxic chemical and inducing cell death—we term this the ‘conditional killer gene’. We selected URA3
as the conditional killer gene; it codes for the protein orotidine-5′-phosphate (OMP) decarboxylase which converts 5-FOA into the cytotoxic pyramidine analog 5-fluorouracil (5-FU) (21
). 5-FU misincorporates into RNA and DNA in place of uracil or thymine, hampering normal RNA and DNA processing, and leads to cell arrest (22
). In strains lacking the URA3
gene, 5-FOA is not converted into 5-FU, and cell growth is not affected (23
The second key design requirement was to restrict this conditional killer protein to daughter cells. Therefore, we paired a cell-cycle dependent promoter with a cell-cycle dependent protein degradation sequence to limit this protein to daughter cells between G1
- and S-phase. To prevent the conditional kill protein from being present past G1
), we fused the N-terminal region of Sic1p in frame to the conditional kill gene coding sequence. This protein region, a so-called G1
/S degradation tag, causes protein fusions (26
) to be specifically degraded at the G1
/S transition with the same timing as the native Sic1p (27
). Since our engineered protein must be present long enough to convert 5-FOA into 5-FU, yet should not be present before G1
, we chose two different promoters with slight different temporal patterns of expression to create two versions of our device. While both the CTS1
) specifically drive gene expression in newly budded cells, CTS1
lags slightly behind DSE4
in driving the expression of any gene coupled to it (29
). Hence we term the DSE4
promoter-variant of our device the ‘early daughter arrester’ and the CTS1
-variant the ‘late daughter arrester’.
Lastly, we included genetic modules for subcellular localization and visualization in our device. Since OMP decarboxylase is usually a cytosolic protein, yet RNA and DNA processing occur in the nucleus, we reasoned that we could increase the probability of arrest by targeting our fusion protein to both compartments. A partial nuclear localization is also necessitated by our G1
/S degradation tag: the degradation of endogenous Sic1 is triggered in the nucleus, where it is phosphorylated by B-type cyclins (27
). Thus, the coding sequence for both a nuclear localization signal (NLS) and a nuclear export signal (NES) were fused to the URA3
module. A protein that contains both signals continuously shuttles (30
) between the cytoplasm and the nucleus. As a final component, a yeast codon optimized YFP DNA sequence was fused to the Sic1 module in order to quantify the presence of the engineered protein with spatial and temporal resolution.
Assuming these components maintain their modularity, we can build a timeline predicting how our device should selectively arrest daughter cells (D). Starting at birth (panel 1), the daughter-specific promoters are up-regulated and result in expression of the fluorescent conditional kill protein (yellow ovals). Throughout G1 (panel 2), this protein will continue to be expressed, will shuttle continuously between the cytoplasm and the nucleus, and will convert 5-FOA (if present) into 5-FU. At the G1/S transition (panel 3), the daughter-specific promoters will no longer be active and the conditional kill protein will be phosphorylated in the nucleus, ubiquitinated, and rapidly degraded by the proteasome. Subsequently (panel 4), the daughter cell will arrest due to incorporation of 5-FU in RNA and DNA. In contrast, mother cells should not express the conditional kill protein and should divide normally.
The daughter arrester conditionally inhibits growth of bulk cultures
To test whether this genetic device could conditionally arrest cell growth on the population level, the wild-type, early daughter arrester and late daughter arrester strains were grown in liquid culture, serially diluted starting from the same initial cell density, and plated on rich media and synthetic complete plates with and without the added drug 5-FOA (A–C). All three strains cells grew to a similar extent when grown on rich YEPD (A) or synthetic complete media (B), suggesting that the device does not affect growth in the absence of a drug. Likewise, cell growth of the wild-type strain in the presence of drug was not affected (C), indicating that the drug does not impact its growth. In comparison, both the early and late daughter arrester strains displayed reduced growth, as demonstrated by the growth of fewer colonies in the serial dilution (C). Additionally, the early daughter arrester strain displayed a slower growth rate compared to late daughter arrester strain. Based on these results, we used only the early daughter arrester strain in the subsequent experiments.
Figure 2. The daughter arrester strain shows reduced growth on the population level only in the presence of drug. Serial dilutions of the parent strain (PSY580), the early daughter arrester (pCTS1, PSY3652) and the late daughter arrester strain (pDSE4, PSY3651) (more ...)
Under normal growth conditions, the biomass and cell density of a yeast cell culture grow exponentially over time after the lag-phase and before stationary-phase. In a population where the newly budded cells arrest their transit through the cell cycle, biomass will be generated in a linear fashion since current mother cells will bud new cells, but these new cells will fail to bud (B). The plot of cell growth versus time can be qualitatively described by one of the following behaviors: (i) concave up, typical of a wild-type population during exponential growth; (ii) concave down, expected for a growing population where mother and daughter cells arrest or die and cease to bud; (iii) linear, expected for a growing population where mother cells continuously bud new daughter cells that get arrested. In order to assay growth rate over time we indirectly quantified biomass by measuring light scattering at 600 nm using a spectrophotometer. Measurements were obtained while growing yeast cell cultures in a temperature controlled well-stirred mini-reactor. D shows a representative time course of the early daughter arrester strain growing in the mini-reactor over time in the presence and absence of the stimulus. Cells grown in the absence of drug (circles) display exponential growth similar to wild-type cells (data not shown), whereas cells grown in the presence of drug (squares) display reduced growth that fits perfectly to a linear regression [OD600 = 0.215 + 0.123 × time (h), R2 = 1.00]. These observations indicate that engineered genetic device reduces the rate of cell division in a manner that is qualitatively consistent with our design.
The daughter arrester conditionally inhibits growth of daughter cells
In order to determine whether the reduced growth of the populations expressing our genetic device was due to the arrest of the newly budded daughter cells, we followed individual cells in a microfluidic device by differential interference contrast and fluorescence microscopy. The microfluidic format conferred two important advantages: the media composition could be changed within less than 1 min without altering the field of view and growing cells remained in the same focal plane because they are trapped in a 4 µm high region. In a typical experiment, the microfluidic chamber was seeded with cells, the cells were grown for 1 h in synthetic complete media, and then the media was switched to either drug-containing media (t = 0 in ), and individual cells were tracked over many generations.
Figure 3. Time-lapse microscopy and single-cell analysis of daughter arrester cells growing in a microfluidic device shows specific arrest of daughter cells only in the presence of drug. Cells were seeded into a microfluidic device and grown in the absence of drug (more ...)
We analyzed representative cell trajectories for the early daughter arrester strain grown in the presence and absence of drug (). A shows a mother cell (outlined in yellow), its first, second and third daughter cells (outlined in red, green and blue, respectively) and their progeny (outlined in red, green and blue) when grown in the absence of drug. While the mother cell divides several times over the course of the experiment, it never shows significant fluorescence. In contrast, newly budded cells (e.g. first daughter cell outlined in red) rapidly accumulate bright fluorescence centered in the nucleus (A, panel 1), which persists until the cell enters into S-phase and then it rapidly disappears and does not reappear (A, panels 2–6). This pattern of expression and degradation indicates that the CTS1
daughter-specific promoter and G1
/S degradation tag can be combined in a modular fashion to tightly restrict an arbitrary protein to a particular phase of the cell cycle. The predominately nuclear localization of the conditional killer protein is in accordance with previous observations in human fibroblasts that a fusion protein carrying both the PKI NES and the SV40 NLS leads to predominately nuclear localization (31
), indicating that in this configuration the NLS prevails. Additionally, both mother and daughter cells of the daughter arrester strain divided many times over the course of the experiment and displayed a growth rate inside the microfluidic device comparable to their wild-type counterparts (~90 min).
When grown in the presence of the drug, both mother cells and post-G1 daughter cells of the daughter arrester strain are not significantly fluorescent (B) and divide at slightly slower rate compared to wild-type cells in the absence of drug (140 versus 90 min, respectively). We speculate that this slower growth could be due to very low expression of conditional killer protein or a bystander effect originating from nearby arrested daughter cells. As in the absence of drug, fluorescence appeared only in newly budded cells, e.g. red cell in panel 2, and then precipitously disappeared as the cells entered into S-phase. However, these cells subsequently arrested as they started to bud new cells, e.g. red cell in panels 3–6, and never completed a cell division over the next ~14 h, i.e. until the end of the experiment.
In order to quantitatively assess the function of our daughter arrester strain on the population level and to identify possible modes of failure, we analyzed the trajectories of many cells grown and imaged under identical conditions as B. Of the daughter cells born in the presence of drug (n = 185), 86% arrested after the G1/S transition and did not complete a cell division during the remainder of the experiment (>30 min). Additionally, a very small subset of the daughter cells died before the G1/S transition. Interestingly, the daughter cells that completed a division displayed noticeably slower growth and in all cases that we observed, their daughters do arrest. Thus we suggest that these cells have divided in spite of significant accumulated damage rather than mutating in such a way that 5-FOA is no longer converted into toxic 5-FU.
Quantifying the average fluorescence intensity of the YFP reporter, which is indicative of the protein concentration, allows a fine-grained examination of device function over multiple cell divisions (C). The fluorescence intensity as a function of time for a mother cell and three of its daughter cells is plotted in B and C, in where mother cell is circled in yellow and daughter cells are indicated by red, green, and blue outlines, respectively. The mother cell only displays a very small fluorescent signal above its own time averaged background level some minutes before the newly budded cell shows a fluorescent signal. Newly budded cells display a fluorescent signal with a remarkable features that are reproduced over several generations (C), such as the peak protein level (~85 A.U. at hours 2, 5 and 8), the period that the fluorescent protein is present (~2 h), the rate of protein production and the precipitous degradation as cells go through the G1/S transition (C). Additionally, the peaks centered around hours 2, 5 and 8 lack a plateau, indicating that the system never reaches a steady-state before the protein is degraded.
The daughter arrester enriches the population for oldest cells
During exponential phase, a wild-type culture of budding yeast will roughly contain 50% new daughter cells, 25% cells that have divided once, 12.5% that have budded twice, etc., so that the oldest n-generational cells should be 100/2n% of the population. While the population distribution of the daughter arrester strain should be unchanged relative to the wild-type strain in absence of drug, it should be significantly altered in the presence of the drug since newly budded daughters do not divide. To explore to what degree our device can enrich for this oldest population of cells, we identified mother–daughter lineages present at the beginning of the microfluidic imaging experiment in either the presence or absence of drug, mapped the number of divisions undergone by these cells and the descendents, and calculated the percentage of the population corresponding to the oldest mother cell. Using the mother–daughter lineage shown in A and B, we calculated and plotted in the percentage of the population corresponding to the oldest mother cell as a function of time for representative trajectories. In the absence of drug (A, red line), the percentage of oldest cells drops sharply as would be expected. However, in the presence of drug (green line), the fraction of oldest cells is much higher, reflecting the much greater number of oldest cells in the total population. This trend is reproduced over many manually tracked mother–daughter lineages (n = 15 mother cells), confirming that the daughter arrester strain can be used to create populations enriched in old cells.
Figure 4. The daughter arrester strain can enrich a yeast cell population in older cells. (A) Starting populations of two cells—one mother and daughter—were tracked over time and the percentage of original mother cells (highest replicative age) (more ...)
Mapping cellular divisions also reveals that the replicative age distribution of the daughter arrester strain (B, analysis of A and B) is radically altered in the presence of drug (red) relative to without drug (green). In the absence of drug, the population distribution of the daughter arrester strain closely resembles the expected wild-type distribution. In the presence of drug (B, red), the age distribution is bimodal. While most of the cells in the population are arrested daughter cells, the mother cells that budded four times are highly enriched (20%) compared with the population in the absence of drug (4%). Again, these results were qualitatively reproduced over several mother–daughter lineages (n = 15 mother cells), and we never observed a mother cell arrest or die. Thus growing the daughter arrester strain in the presence of drug radically changes the population replicative age distribution, creating one peak population of newly born daughter cells and a second peak corresponding to cells with n number of cell divisions. The number of cell divisions—n—can be manipulated by growing the cells for longer times, e.g. different stages of the time course in B.