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High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 143 - Stretched Out DNA rods Observed in Anaphase Consist of Chromatin

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

    Presenting Author(s)

  • Tina Li

    Student
    San Dieguito High School Academy
    Carlsbad, California, United States

    • Co-Author(s)

  • Katelyn Lee

    Undergraduate
    UCSD, California, United States

    • Co-Author(s)

  • Sarah Chen (she/her/hers)

    High School Student
    Pacific Ridge School, United States

    • Co-Author(s)

  • NL

    Naomi E. Lee (she/her/hers)

    High School Student
    Canyon Crest Academy, United States

    • Co-Author(s)

  • Selina Carandang

    High School Student
    Pacific Ridge School
    Carlsbad, California, United States

    • Co-Author(s)

  • AF

    Arthur Forer

    Professor
    York University, United States

    • Primary Investigator(s)

  • Linda Shi, PhD (she/her/hers)

    OPALS Program Director, Project Scientist, Co-director IEM
    University of California, San Diego: Institute of Engineering in Medicine
    La Jolla, California, United States

    • Last Author(s)

  • VG

    Veronica Gomez-Godinez

    Scientist
    University of California, San Diego
    La Jolla, California, United States


Introduction: During anaphase of mitosis, we studied DNA rods that signify the presence of inter-telomeric tethers. According to our past findings, we concluded that the presence of tethers occurred most frequently in early anaphase. To further our study of inter-telomeric tethers, we researched DNA-wrapped proteins called histones. Among the four variations of histones, we specifically focused on histone H3, an unmodified histone, as well as a modified and phosphorylated histone called Phospho-H3S10. We observed an overlap between the DNA rods and histone H3. Similar to DNA rods, the appearance of Phospho-H3S10 rods is correlated with the progression of anaphase, as anaphase progresses we see fewer histone rods. We hypothesized that the presence of tethers may relate to the phosphorylation of H3S10. Through our findings, we will be able to learn more about the relationship between histones and inter-telomeric tethers.

Materials and

Methods: To study the presence of stretched-out DNA during mitosis, the mammalian cells osteosarcoma U2OS cells and rat-kangaroo Ptk2 cells are utilized. To get a visual on these cells the dyes, DAPI, a fluorescence that binds to A-T DNA bases, and Alexa 488 and Alexa 594 are used to provide a visual on chromosomes and tethers through the images being processed by an Olympus Fluoview FV1000 IX81 Spectral Type Laser Scanning Confocal microscope system. To differentiate the two different types of histones, we labeled phospho-H3S10, a phosphorylated histone, with the green dye Alexa 488, and labeled H3, the basic histone protein with a red dye, Alexa 594. After the images are processed through the microscope system they get downloaded and looked at in closer depth with ImageJ. In ImageJ the cells are analyzed through the histone and DAPI channels whilst scrolling through the z-scale to see all aspects of the cells.

Results, Conclusions, and Discussions:

Results: Our analysis showed that stretched-out histones predominantly overlapped with stretched-out DNA fibers, suggesting that inter-telomeric tethers not only stretch out DNA but also stretch out the chromatin. Similar to stretched-out DNA fibers, stretched-out histone was also most present when kinetochore distances were shorter, which for the majority of cells was early anaphase. Unfortunately, we found that the analysis of U2OS cells was much more difficult than PtK2 cells. Due to the U2OS cells' cancerous state and increased number of chromosomes, visualizing stretched-out DNA and histone was more tedious. We also observed an elevated number of abnormalities including DNA fragments and DNA bridges in the U2OS cells.

Conclusion: Our data concludes that the presence of stretched-out histones in the H3S10 (phosphorylated) cells decreases as anaphase progresses since they were most present in early anaphase. These results are consistent with previous research and published findings on stretched-out DNA fibers. Knowing that phosphorylation decreases over time during mitosis leads us to hypothesize that there may be a correlation between the stage of phosphorylation and the frequency of inter-telomeric tethers. Additionally, because we saw most tethers in histone and DAPI, our observations suggest that the tethers also pull on chromatin, not just DNA, since chromatin is made of DNA and histone. Future studies will include the continued quantification of histone data/images to draw more solid conclusions. We will also investigate the co-localization of stretched-out DNA fibers and telomere cap proteins to study where inter-telomeric tethers are specifically attached to.

Acknowledgements (Optional): This work was supported by a generous gift from Beckman Laser Institute Inc. to LS and VGG. The high school students participated in the UCSD IEM OPALS internship. Special thanks to Dr. Shu Chien from UCSD Bioengineering and Dr. Lizhu Chen from CorDx Inc. for their donations.

References (Optional):