Characterization of Micronuclei Formations In Human Retinal Pigment Epithelial Cells

Students: Amada Castro Vega, Claudia Tellez

Faculty Mentor: Lisa Hua

Biology

College of Science, Technology, and Business

It was discovered that homologous chromosomes spatially segregate along the centrosome axis throughout mitosis. The spatial restriction of chromosomes was present prior to mitotic spindle assembly and after its disassembly. The specific mechanism for how chromosomes in each haploid set associate together with little chromosome mixing remains elusive. Further research found a high concentration of microtubules present prior to the formation of the mitotic spindle in prometaphase, suggesting microtubules may play a role in spatially segregating homologous chromosomes. Microtubules may play a role in the spatial segregation of chromosomes, seeing as they hold various roles such as maintaining cell shape, attaching to the kinetochores of sister chromatids, and pulling them apart toward opposite nuclear hemispheres. To test the role of microtubules in chromosome organization, we performed drug perturbation experiments targeting microtubules. Paclitaxel is a microtubule-stabilizing agent that disrupts microtubule dynamics by binding to β-tubulin and inhibiting the depolymerization process. Reversine is a checkpoint inhibitor that targets the monopolar spindle (MPS1) checkpoint, which targets the aurora B kinases and prevents apoptosis. Sequential application of the two drugs, Paclitaxel then Reversine, causes the microtubules to be disrupted and allows for cell cycle progression, respectively. We performed immunofluorescence staining for Lamin B1 to visualize the nuclear envelope and a DNA DAPI counterstain. My preliminary data shows the formation of various small nuclear envelopes or micronuclei containing DNA. The preliminary data suggest that microtubules do not play a major role in chromosomal organization of haploid set segregation. The significance of my work is that it aids in further understanding how chromosomes organize when undergoing mitosis in reference to their localization.