Differences in the origins of kinetochore-positive and kinetochore-negative micronuclei: A live cell imaging study

Area of Science:

• Cell Biology
• Genetics
• Cytogenetics

Background:

• Micronuclei (MNi) are biomarkers for genotoxicity and chromosomal instability.
• Distinguishing kinetochore-negative (K-MNi) and kinetochore-positive (K+MNi) micronuclei enhances the MN test's accuracy.
• Traditional methods limit understanding the fundamental origins of K-MNi and K+MNi.

Purpose of the Study:

• To investigate the distinct origins of K-MNi and K+MNi using advanced live-cell imaging.
• To elucidate the specific chromosomal events leading to K-MNi and K+MNi formation.
• To improve the mechanistic understanding of micronuclei generation in genotoxicity assessment.

Main Methods:

• Construction of dual-color fluorescent HeLa cells expressing histone 2B (H2B) and centromere protein B (CENP B) as fusion proteins.
• Utilizing H2B-mCherry for micronuclei identification and CENP B-GFP for kinetochore labeling.
• Long-term live cell imaging to observe micronuclei formation and track chromosomal dynamics.

Main Results:

• Kinetochore-negative micronuclei (K-MNi) originate from kinetochore-negative displaced chromosomes (K-DCs), lagging chromosomes (K-LCs), and broken chromosome bridges (CBs).
• Kinetochore-positive micronuclei (K+MNi) originate from kinetochore-positive displaced chromosomes (K+DCs), lagging chromosomes (K+LCs), and broken CBs.
• Lagging chromosomes (K+LCs and K-LCs) contribute more significantly to K+MNi and K-MNi formation, respectively, than displaced chromosomes.

Conclusions:

• Kinetochore-negative and kinetochore-positive micronuclei have demonstrably different origins in HeLa cells.
• Specific chromosomal aberrations contribute differentially to the overall counts of K-MNi and K+MNi.
• This research provides a deeper mechanistic insight into micronuclei formation, crucial for genotoxicity evaluation.