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Double minute chromosomes in mouse methotrexate-resistant cells studied by atomic force microscopy
Xinyu Deng1, Liangyu Zhang, Yu Zhang
1Laboratory of Medical Genetics, Harbin Medical University, Harbin 150086, China.
Abstract:
Double minute chromosomes (DMs) are acentric, autonomously replicating extra-chromosomes and frequently mediate gene amplification in tumor and drug resistant cells. Atomic force microscopy (AFM) is a powerful tool in microbiology. We used AFM to explore the ultrastructure of DMs in mouse fibroblasts 3T3R500. DMs in various phases of cell cycle were also studied in order to elucidate the mechanisms of their duplication and separation. Metaphase spread and induced premature condensed chromosomes (PCCs) were observed under the AFM. DMs were detected to be composed of two compact spheres linked by fibers. The fibers of DMs directly connected with metaphase chromosomes were observed. Many single-minutes and few DMs were detected in G1 PCCs, while more DMs were detected in S PCCs than in G1 PCCs. Besides, all of the DMs in G2 PCCs were coupled. Our present results suggested that DMs might divide into single-minutes during or before G1-phase, followed by duplication of the single-minutes in S-phase. Moreover, we introduced a new powerful tool to study DMs and got some ideal results.
Insights
Atomic force microscopy visualized double minute chromosomes (DMs), which are extra-chromosomes driving gene amplification. Researchers observed DM duplication and separation mechanisms across the cell cycle in mouse fibroblasts.
Area of Science:
- Cell Biology
- Genetics
- Microscopy Techniques
Background:
- Double minute chromosomes (DMs) are extrachromosomal elements crucial for gene amplification in cancer and drug-resistant cells.
- Understanding DM replication and segregation is vital for cancer research and therapeutic development.
- Traditional microscopy methods have limitations in visualizing the fine ultrastructure of DMs.
Purpose of the Study:
- To investigate the ultrastructure and cell cycle-dependent behavior of double minute chromosomes (DMs).
- To elucidate the mechanisms of DM duplication and separation using advanced imaging.
- To evaluate Atomic Force Microscopy (AFM) as a novel tool for studying DMs.
Main Methods:
- Utilized Atomic Force Microscopy (AFM) to examine the ultrastructure of DMs in mouse fibroblasts (3T3R500).
- Analyzed DMs in various cell cycle phases, including metaphase spreads and premature condensed chromosomes (PCCs).
- Observed DMs in G1, S, and G2 phases of the cell cycle.
Main Results:
- AFM revealed DMs composed of two compact spheres connected by fibers, with direct connections to metaphase chromosomes.
- DM distribution varied across cell cycle phases: few DMs in G1 PCCs, more in S PCCs, and all coupled in G2 PCCs.
- Evidence suggests DMs divide into single-minutes before or during G1, followed by duplication in S-phase.
Conclusions:
- Atomic Force Microscopy (AFM) provides high-resolution insights into the ultrastructure and dynamics of double minute chromosomes.
- The study proposes a model for DM replication and segregation, involving division in early G1 and duplication in S-phase.
- AFM is introduced as a powerful new tool for advancing the study of DMs and their role in cellular processes.
