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Updated: Jul 5, 2026
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Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi
Published on: May 12, 2023
Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging
T Nicolai Siegel1, Doeke R Hekstra, George A M Cross
1Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Molecular and Biochemical Parasitology
|May 27, 2008
Summary
Kinetoplast DNA replication in Trypanosoma brucei occurs during nuclear S phase, not G2 as previously thought. This study uses quantitative imaging to refine understanding of the cell cycle in this parasite.
Area of Science:
- Cell Biology
- Parasitology
- Molecular Biology
Background:
- Trypanosoma brucei possesses two distinct DNA compartments: nuclear DNA (nDNA) and kinetoplast DNA (kDNA).
- The precise timing of nDNA and kDNA replication within the cell cycle has been a subject of study, with previous assumptions regarding kinetoplast DNA replication timing.
- Morphological indicators have been used to infer cell-cycle stages in T. brucei, but their consistency across strains and conditions has been questioned.
Purpose of the Study:
- To precisely determine the cell-cycle stage of individual procyclic Trypanosoma brucei cells using quantitative DAPI imaging.
- To re-evaluate the timing of kinetoplast elongation relative to nuclear DNA replication.
- To demonstrate the utility of simultaneous multi-wavelength imaging for analyzing cell-cycle-regulated proteins.
Main Methods:
- Quantitative DAPI imaging was employed to assess the cell-cycle stage of individual procyclic cells.
- Cells were sorted by DNA content and analyzed using fluorescence microscopy to confirm findings.
- Simultaneous quantitative imaging at two wavelengths was utilized for co-staining and protein abundance analysis.
Main Results:
- Kinetoplast elongation was found to occur predominantly during the nuclear S phase, challenging the previous assumption of it occurring during G2.
- Confirmation of kinetoplast elongation timing was achieved through DNA content-based cell sorting and fluorescence microscopy.
- The study demonstrated the successful co-staining for histone H4 lysine 4 (H4K4) enrichment during nuclear S phase, validating the multi-wavelength imaging technique.
Conclusions:
- The cell cycle of Trypanosoma brucei has distinct phases for nuclear and kinetoplast DNA replication, with kinetoplast elongation primarily occurring during nuclear S phase.
- Quantitative imaging provides a robust method for accurately determining cell-cycle progression in T. brucei.
- This technique allows for the simultaneous assessment of DNA replication and the abundance of cell-cycle-regulated proteins, offering new insights into parasite cell biology.
