Assessing the Toxoplasma Tachyzoite Cell Cycle Phases Using Fluorescent Ubiquitination-Based Cell Cycle Indicator
基于荧光泛素化细胞周期指示系统评估弓形虫速殖子细胞周期阶段
发布: 2026年01月20日第16卷第2期 DOI: 10.21769/BioProtoc.5588 浏览次数: 19
评审: Marcelo S. da SilvaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jul 2025
Abstract
Toxoplasma gondii is an apicomplexan parasite that infects a wide variety of eukaryotic hosts and causes toxoplasmosis. The cell cycle of T. gondii exhibits a distinct architecture and regulation that differ significantly from those observed in well-studied eukaryotic models. To better understand the tachyzoite cell cycle, we developed a fluorescent ubiquitination-based cell cycle indicator (FUCCI) system that enables real-time visualization and quantitative assessment of the different cell cycle phases via immunofluorescence microscopy. Quantitative immunofluorescence and live-cell imaging of the ToxoFUCCIS probe with specific cell cycle markers revealed substantial overlap between cell cycle phases S, G2, mitosis, and cytokinesis, further confirming the intricacy of the apicomplexan cell cycle.
Key features
• This protocol describes the development of the transgenic lines capable of detecting individual cell cycle phases and processes of the Toxoplasma tachyzoite cell cycle.
• Quantitative immunofluorescence analysis and real-time microscopy enable the measurement of each cell cycle phase.
• The ToxoFUCCIS probe helps to gain new insights into the highly flexible, overlapping nature of cell cycle organization in apicomplexan parasites.
Keywords: Apicomplexa (顶复门寄生虫)
Toxoplasma gondii (刚地弓形虫)
Cell cycle (细胞周期)
FUCCI (FUCCI)
PCNA1 (PCNA1)
Graphical overview
Background
Fluorescent ubiquitination–based cell cycle indicator (FUCCI) is a powerful technology that enables real-time visualization of the cell cycle [1–6]. FUCCI technology exploits the phase-specific proteolysis of critical cell cycle regulators. Sequential activation of major E3 ligases, APC/C and SCFSkp2, orchestrates the precise regulation of DNA replication factors. This temporal machinery controls the production of Geminin accumulation during S and G2 phases to prevent DNA re-replication. On the contrary, Cdt1 (chromatin licensing and DNA replication factor 1) is expressed in M and G1 phases and assists in licensing the origins of replication [7–9]. The original FUCCI probes invented by Sakauo-Sawana et al. in 2008 used fluorescent protein fusions of Geminin and Cdt1 and accurately detected cell cycle progression in mammalian cells in real time [2]. This technology has revolutionized cell cycle analysis. It provided a new dimension for studying cellular dynamics, enabled the assessment of drug effects on cell replication, visually distinguished proliferating and quiescent cells, and allowed the development of various stem cell populations [3,5]. Due to the lack or altered expression of the conventional FUCCI markers, the FUCCI adaptation to the unicellular apicomplexan parasite Toxoplasma gondii required technical adjustments [10,11]. To satisfy FUCCI marker requirements, a candidate protein should be abundant and localized in distinguishable cellular compartments such as the nucleus and cytoplasm, and its cell cycle expression should be controlled. According to these criteria, a DNA replication factor, TgPCNA1 (proliferating cellular nuclear antigen 1), was identified as a suitable FUCCI marker and successfully used to determine the organization of the T. gondii tachyzoite cell cycle [10]. While the ToxoFUCCIS cell line alone is sufficient to detect major cell cycle phases such as G1, S, and mitosis concurrent with cytokinesis (budding), the combination of ToxoFUCCIS with quantitative immunofluorescence microscopy analysis (qIFA) using a set of cell cycle markers facilitates the identification of the intervals within the composite cell cycle phase [10]. Previous studies validated cell cycle–dependent expression and localization of the suggested markers, including basal complex component TgBCC0 [12], MORN (membrane occupation and recognition nexus) repeat-containing protein TgMORN1 [11,13–15], and inner membrane complex protein TgIMC1 [16,17].
Materials and reagents
Biological materials
1. Human foreskin fibroblasts (HFF) (ATCC SCRC-1041)
2. Toxoplasma gondii RH TIR1 strain (genotype: RHΔhxgprtΔku80; TUB1:TIR1-3xFLAG, SAG1:CAT) [18]
3. Toxoplasma gondii RH Tati Δku80 strain [19]
Reagents
1. Dulbecco’s modified Eagle’s medium (DMEM) (Millipore Sigma, catalog number: D6429)
2. Fetal bovine serum (FBS) (Millipore Sigma, catalog number: F0926)
3. Antibiotic antimycotic solution (PSA) (Millipore Sigma, catalog number: A5955)
4. Hank’s balanced salt solution (Millipore Sigma, catalog number: H9394)
5. Mycoplasma Detection PCR kit (MP biomedicals, catalog number: 93050201)
6. Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231)
7. 0.5% Trypsin EDTA solution (Fisher Scientific, catalog number: 15400054)
8. Adenosine triphosphate (ATP) (New England Biolabs, catalog number: P0756S)
9. Reduced L-glutathione (RPI, catalog number: 70188)
10. 6× gel loading dye (New England Biolabs, catalog number: B7025)
11. dNTPs (New England Biolabs, catalog number: N0447S)
12. Ethanol (Fisher Scientific, catalog number: BP28184)
13. DNA oligomers (Integrated DNA technologies, IDT, 25 nmole)
14. DNA Extraction kit (Monarch Genomic DNA purification Kit, NEB, catalog number: T3010S)
15. Miniprep kit (Qiagen, catalog number: 27106)
16. PCR Cleanup kit (Macherey Nagel, catalog number: 740609.50)
17. Phusion DNA Polymerase (New England Biolabs, catalog number: M0535S)
18. 10× Tris-Borate-EDTA (TBE) buffer (Bio-Rad, catalog number: 1610770)
19. Orange G powder (Fisher Scientific, catalog number: AC416550100)
20. 1 kb+ DNA ladder (New England Biolabs, catalog number: N3232L)
21. Gibson Assembly master mix (New England Biolabs, catalog number: E2611S)
22. E. coli NEB5α chemically competent cells with SOC media (New England Biolabs, catalog number: C2987H)
23. Agarose (Millipore Sigma, catalog number: A9539-500)
24. LB agar (Fisher Scientific, catalog number: BP1425-500)
25. LB broth (Fisher Scientific, catalog number: BP1426-500)
26. Ampicillin sodium salt (Fisher Scientific, catalog number: BP-1760-25)
27. 7.5 M ammonium acetate solution (Millipore Sigma, catalog number: A2706)
28. 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Millipore Sigma, catalog number: MBD0015)
29. Rabbit anti-myc antibodies (clone 71D10) (Cell Signalling, catalog number: 2278)
30. Rabbit anti-NeonGreen antibodies (Cell Signaling Technology, catalog number: 53061S)
31. Mouse anti-Centrin antibodies (clone 20H5) (Millipore Sigma, catalog number: 04-1624)
32. Rabbit anti-TgMORN1 serum (provided by Dr. Marc-Jan Gubbels, Boston College, MA)
33. Rabbit anti-TgIMC1 serum (provided by Dr. Gary Ward, University of Vermont, VT)
34. Sodium azide (NaN3) (Fisher Scientific, catalog number: S227I-500)
35. Proteinase K (Fisher Scientific, catalog number: EO0491)
36. Anti-rabbit, cross-adsorbed secondary antibody (Invitrogen, catalog number: A11012)
37. Anti-mouse, cross-adsorbed secondary antibody (Invitrogen, catalog number: A11032)
38. Potassium chloride (KCl) (Fisher Scientific, catalog number: P217-500)
39. Monopotassium phosphate (KH2PO4) (Fisher Scientific, catalog number: BP362-500)
40. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: S374-500)
41. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358)
42. Calcium chloride (CaCl2) (ThermoFisher Scientific, catalog number: 10035)
43. Magnesium chloride (MgCl2) (Fisher Scientific, catalog number: 7791-18-6)
44. HEPES (RPI, catalog number: 7365)
45. Tween-20 (Millipore Sigma, catalog number: P1379)
46. Pyrimethamine (Millipore Sigma, catalog number: SML3579)
47. Triton X-100 (Fisher Scientific, catalog number: AAA16046AE)
48. PacI endonuclease (New England Biolabs, catalog number: R0547S)
49. NsiI endonuclease (New England Biolabs, catalog number: R0127S)
50. EcoRV endonuclease (New England Biolabs, catalog number: R3195)
51. 4% paraformaldehyde solution (PFA) (Fisher Scientific, catalog number: AAJ19943K2)
52. 10 mg/mL ethidium bromide solution (Millipore Sigma, catalog number: E1510)
Laboratory supplies
1. Microcentrifuge tubes (1.7 mL) (Fisher Scientific, catalog number: 14222-171)
2. PCR tubes (Millipore Sigma, catalog number: AXYPCR02D)
3. T-25 and T-75 culture flasks (Fisher Scientific, catalog numbers: FB012936, FB012938)
4. 96-well, 24-well, and 6-well plates (Fisher Scientific, catalog numbers: FB012931, FB012929, FB 012927)
5. 15 mL conical centrifuge tubes (VWR, catalog number: 21008-212)
6. 50 mL conical centrifuge tubes (Fisher Scientific, catalog number: 12-565-270)
7. Snap cap tubes (Fisher Scientific, catalog number: 07-000-212)
8. 10, 200, 1,000 μL pipette tips (USA Scientific, catalog numbers: 1120-3710, 1120-8710, 1122-1730)
9. 5 mL, 10 mL, 25 mL serological pipettes (Fisher Scientific, catalog number: 13-678-11)
10. 10 mL Luer lock syringes (Fisher Scientific, catalog number: 14-955-459)
11. Blunt end needles 23G (Grainger, catalog number: 5FVK7)
12. Stericup filter units 500 and 1,000 mL (Fisher Scientific, catalog numbers: S2GPU05RE, S2GPU10RE)
13. 3.0 μm pore size filter membrane (Millipore Sigma, catalog number: TSTP02500)
14. Cell scrapers (Fisher Scientific, catalog number: 07-000-553)
15. 0.22 μm sterile syringe filters (Fisher Scientific, catalog number: 50-209-3153)
16. Glass pipettes (Fisher Scientific, catalog number: 13-678-20C)
17. Nitrocellulose/filter sandwich (Bio-Rad, catalog number: 1620213)
18. Petri dishes (VWR, catalog number: 25384-342)
19. 2 mm electroporator cuvette (Fisher Scientific, catalog number: FB102)
20. MyFugeTM Minispin (Alkali Scientific, catalog number: 50-233-6060)
21. 15 mm, 22 mm glass coverslips (Fisher Scientific, catalog numbers: 501215160, 12541016)
Solutions
1. Tachyzoite medium (see Recipes)
2. Pyrimethamine selection media (see Recipes)
3. 10× phosphate-buffered saline (PBS) (see Recipes)
4. Cytomix electroporation buffer pH 7.6 [20] (see Recipes)
5. 3% IFA block buffer (see Recipes)
6. IFA permeabilization buffer (see Recipes)
7. 0.8% agarose gel (see Recipes)
Recipes
1. Tachyzoite medium
DMEM supplemented with 3% FBS and 1% PSA. Store at 4 °C.
2. Pyrimethamine selection media
Tachyzoite medium (Recipe 1) with 1 μM pyrimethamine. Store at 4 °C.
3. 10× PBS
1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 20 mM KH2PO4 in a final volume of 1 L. Adjust pH to 7.4. Store at room temperature.
4. Cytomix electroporation buffer pH 7.6 [20]
10 mM KPO4 (pH 7.6), 120 mM KCl, 0.15 mM CaCl2, 5 mM MgCl2, 25 mM HEPES, 2 mM EDTA. Adjust the solution to the final volume of 1 L. Filter-sterilize and store at 4 °C.
5. 3% IFA block buffer
500 mL of 1× PBS and 15 mL of FBS. Run through a Stericup filter and add 500 μL of 2 mM NaN3. Store at 4 °C.
6. IFA permeabilization buffer
900 mL of nanopure water, 100 mL of 10× PBS, 1 mL of 2 mM NaN3, and 5 mL Triton X-100.
7. 0.8% agarose gel
0.8 g of agarose in 100 mL of 1× TBE buffer. Microwave until clear, cool down, and add 5 μL of 10 mg/mL ethidium bromide solution.
Equipment
1. Autoclave (Steris, model: Amsco Lab 250)
2. Biological safety cabinet (Labconco, model: 302411000)
3. Benchtop centrifuge with 15 and 50 mL conical vial holders (Eppendorf, model: 5910R)
4. Water bath at 37 °C (Shel Lab, model: SWB15)
5. Cell culture incubator at 34 and 37 °C with 5% CO2 (Eppendorf, catalog number: 6731010015)
6. Incubator at 37 °C (Fisher Scientific, model number: FFCO300DABB)
7. Bacterial shaker at 37 °C (New Brunswick Scientific, model: I26R)
8. Sterile Pyrex borosilicate glassware for solutions and reagents (test tubes, beakers, storage vials)
9. Amaxa Nucleofector II electroporator (Lonza, catalog number: 40700053)
10. Hemacytometer (Fisher Scientific, catalog number: 2671154)
11. Inverted-phase contrast microscope (Nikon, model: TMS-F and Olympus, model: CKX53SF)
12. Zeiss Axio Observer inverted microscope equipped with filters for multichannel fluorescence imaging (green, DAPI, and red)
13. Gel imaging system (Foto dyne Incorporated, model: 1-1430)
14. Microcentrifuge (Eppendorf, catalog number: 2231001179)
15. Microwave (Black+Decker, catalog number: 810004819726)
16. Pipettes and carousel (Fisher, catalog numbers: 13684251, 3116000015)
17. Motorized pipette controller (Eppendorf, catalog number: 4430000018)
18. Protein transfer apparatus (Bio-Rad, model: Trans-Blot Turbo System)
19. SDS-PAGE electrophoresis apparatus (Bio-Rad, model: Mini-Protean Tetra Cell)
20. Thermocycler (ThermoFisher Scientific, catalog number: 4484073)
21. Refrigerator (LG, model: LRTLS2403S)
22. Freezers -20 and -80 °C (Fisher Scientific, catalog number: FBH07CFSA and Eppendorf, model: CryoCube F740hi)
23. Liquid nitrogen storage (Thermo Scientific, catalog number: 7400)
24. Nanodrop spectrophotometer (Thermo Scientific, catalog number: 13400519)
25. Vacuum aspirator (Barnant Company, catalog number: 400-3910)
26. Heat block (Fisher Scientific, catalog number: 88-860-023)
Software and datasets
1. Google Chrome (Google), Safari, or any other web browser
2. SnapGene (Version 8.0)
3. Zen 3.2 (Zeiss)
4. NEB ligation calculator (New England Biolabs) (nebiocalculator.neb.com)
Procedure
文章信息
稿件历史记录
提交日期: Nov 7, 2025
接收日期: Dec 15, 2025
在线发布日期: Jan 8, 2026
出版日期: Jan 20, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Batra, M. and Suvorova, E. S. (2026). Assessing the Toxoplasma Tachyzoite Cell Cycle Phases Using Fluorescent Ubiquitination-Based Cell Cycle Indicator. Bio-protocol 16(2): e5588. DOI: 10.21769/BioProtoc.5588.
Download Citation in RIS Format
分类
微生物学 > 微生物细胞生物学
细胞生物学 > 细胞工程
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