Background

Cell cycle is a multievent cellular process that plays an essential role in maintaining genome stability by coordinating processes like DNA replication, DNA damage repair, and chromosome segregation during cell division. The whole cycle is completed in four phases, where DNA replication is confined to the S phase, G1 is the gap between the M phase and S phase, G2 is the gap between the S phase and M phase, and the division of the nucleus followed by the cytoplasm is restricted to the M phase (Figure 1). G1 and G2 phases are known for cell growth, metabolic activity, and for preparing for the next phase of the cell cycle (Hartwell et al., 1974). When the cells are proliferating, they can be at any phase of the cell cycle; therefore, to properly explore the mechanism of cell cycle progression, synchronization of a heterogeneous population of cells becomes critical. In human cells, serum starvation and double thymidine block are commonly used approaches for synchronizing a cell population (Ma and Poon, 2017). Saccharomyces cerevisiae cells can be synchronized in the G1 phase of the cell cycle with the mating pheromone, α-factor. Similarly, hydroxyurea and nocodazole synchronize budding yeast cells in the early S phase and G2/M phase, respectively (Rosebrock, 2017).

Figure 1. Overview of the cell cycle phases and some routinely used reagents for cell synchronization at the mentioned phases of the cell cycle. For example: thymidine, methotrexate, hydroxyurea, aphidicolin, dedoxy uridine, and mimosine are used to synchronize the cells at G1 or early S phase.

Upon releasing these synchronized cells into a fresh medium, cells undergo similar cell cycle progression that can be monitored after collecting cell populations at different time points. DNA content measurement of a cell population by using DNA-binding fluorescent dyes and a flow cytometer is one of the reliable techniques for getting insights into the cell cycle dynamics of an organism (Haase and Reed, 2002). The existing methodologies of cell synchronization do not work effectively in the case of fungi like Candida albicans. C. albicans is a gut pathogen that survives in at least three morphological forms: yeast, pseudohyphae, and hyphae. Interestingly, these cell types differ even in the rate and order of cell cycle events (Berman, 2006). In addition, most of these reagents used to synchronize cells also induce filamentation in C. albicans (Manohar et al., 2018; Kumari et al., 2023; Patel et al., 2023). Therefore, cell synchronization is one of the major challenges for exploring the cell cycle in C. albicans. Additionally, the use of external chemical reagents for cell synchronization may affect the native cellular physiology by inducing stress in the cells, which will never be a true estimation of the cell cycle and end up in erroneous cell cycle analyses. Therefore, here we describe and demonstrate a chemical method/approach where a time-point-based incubation results in > 90% cell synchronization at the G1 phase so that the cell cycle analysis can be executed without hampering the native physiology of the cells (Figure 2 and Supplementary Figure 1).

Figure 2. Schematic stepwise demonstration of the cell cycle analysis of C. albicans. Synchronized cells will be allowed to grow for 150 min and will be harvested at every 30 min to monitor cell cycle progression. Cells will be fixed with ethanol, stained with SYTOX green, and analyzed by flow cytometry to complete the cell cycle analysis.

One of the key advantages of using flow cytometry for cell cycle analysis in yeast is its ability to analyze a large number of cells at a single-cell level rapidly. This technique employs a laser-based system to measure the fluorescence emitted upon a fluorochrome (in this case, a nucleic acid staining dye) binding to cellular DNA while cells pass through a flow cell. Different nucleic acid staining dyes including 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), and 7-amino actinomycin D (7-AAD) have been used by researchers to access the cell cycle based on the cell types to be analyzed, although PI is the preferred one. In S. cerevisiae, PI staining has been proven effective, and the same was initially adopted by us for cell cycle analysis of C. albicans (Manohar et al., 2018). However, a major concern associated with PI is the relatively high coefficient of variation (CV) and low signal amplification rate of PI-stained cells, which limit the accuracy and reproducibility of the cell cycle (Haase and Reed, 2002). To overcome this problem, we now use SYTOX green as it exhibits low CV, high signal amplification with low signal-to-noise ratio, and low photobleaching rate, thus being an ideal fluorochrome for accurate and reproducible DNA content measurement (Thakur et al., 2015; Patel et al., 2023). In summary, this protocol outlines detailed steps involved in the cell cycle analysis of a wild-type strain of C. albicans, SC5314, using SYTOX green with a simple cell synchronization strategy providing the precise measurement of DNA content in each phase of the cell cycle by flow cytometry (Figure 3). By employing this protocol, researchers can gain valuable insights into the cell cycle dynamics, which will be helpful in understanding the diverse physiological processes of C. albicans. The same protocol can be adopted for other fungal species as well.

Figure 3. Demonstration of cell cycle progression of wild-type C. albicans cells. Using the described protocol, a synchronized population of C. albicans cells (SC5314 strain) was allowed to progress through the cell cycle and, at mentioned time points (0–1 h), cells were harvested, fixed, stained with SYTOX green, and analyzed by flow cytometry.