Cell Cycle Synchrony in Giardia intestinalis Cultures Achieved by Using Nocodazole and Aphidicolin

Giardia intestinalis is a ubiquitous intestinal protozoan parasite and has been proposed to represent the earliest diverging lineage of extant eukaryotes. Despite the importance of Giardia as a model organism, research on Giardia has been hampered by an inability to achieve cell cycle synchrony for in vitro cultures. This report details successful methods for attaining cell cycle synchrony in Giardia cultures. The research presented here demonstrates reversible cell cycle arrest in G₁/S and G₂/M with aphidicolin and nocodazole, respectively. Following synchronization, cells were able to recover completely from drug treatment and remained viable and maintained synchronous growth for 6 h. These techniques were used to synchronize Giardia cultures to increase the percentages of mitotic spindles in the cultures. This method of synchronization will enhance our ability to study cell cycle-dependent processes in G. intestinalis.Giardia intestinalis is a ubiquitous intestinal protozoan parasite causing disease in humans and animals worldwide (1, 11). In developing countries, diarrheal disease is responsible for 80% of the deaths of children under 2 years of age (21), and Giardia is one of the major causes of this condition. As a diplomonad, Giardia has been proposed to represent the earliest diverging lineage of extant eukaryotes, based on single rRNA and single and/or concatenated protein phylogenies developed by considering an archaeal out-group (2, 3, 5, 15, 23), making it a valuable organism for studying the evolution of biological processes in all eukaryotes. Characteristic of the order Diplomonadida, Giardia trophozoites contain two nuclei that remain separate during mitosis, with each daughter cell inheriting one copy of each parental nucleus (19). The trophozoite form, which attaches to the small intestine of the host, has a tetraploid (4N) DNA content in G₁ since each nucleus is 2N (4). Following a round of DNA synthesis, each G₂ nucleus is 4N, making the cell 8N. According to previous flow cytometry results, actively growing Giardia cultures spend the majority of the cell cycle in the G₂/M phase and significantly less time in the G₁ and S phases (4); in contrast, many tissue culture cells display a lengthy G₁ phase. Until recently, an inability to synchronize in vitro Giardia cultures to any degree has severely hampered the ability of researchers to study cell cycle-dependent processes (16, 20).This work demonstrates successful cell cycle arrest by using nocodazole, a microtubule-destabilizing drug that leads to the depolymerization of spindle microtubules in Giardia (6, 20). A brief nocodazole treatment resulted in cells arrested early in mitosis or at the end of G₂, presumably by the activation of a mitotic spindle checkpoint (22). G₂ arrest using nocodazole was combined with G₁ arrest using aphidicolin, a drug that presumably acts through the inhibition of polymerase-dependent DNA synthesis (8, 12, 14, 25). By combining these two treatments, we were able to effectively synchronize Giardia cultures while maintaining cell viability. These synchronization methods were used to enrich cultures with mitotic spindles at the M phase. Moreover, these methods will be a valuable tool for studying other aspects of Giardia biology such as encystation, the time in the life cycle when the trophozoite transforms into an infectious cyst.