Following Chromosome Movement in 3D

Lisa L. Hua and Takashi Mikawa from the University of California, San Francisco are using Imaris to better understand how cells control recombination and homologous chromosome pairing. These processes must be precisely regulated to prevent genomic instability during mitosis while still allowing for genetic diversity during meiosis. Characterizing the precise mechanisms involved in maintaining mitotic fidelity could lead to new ways to prevent genetic instability.

Tracking chromosomes in 3D space

The researchers used Imaris for 3D reconstruction and analysis of homologous chromosome positions in 3D space. “The software was user friendly and the final 3D reconstructions are very aesthetically pleasing,” said Hua.

Using the Imaris measurement tool, the researchers performed 3D angular measurements between the homologous chromosomes. They did this by first selecting three points for angular measurements with the center of mass function (in volumes) and then determining the closest 3D point to another object.

The researchers also calculated the distance between homologous chromosomes using edge-to-edge measurements with the MATLAB plugin and performed sample drift correction to correct for translational drift when analyzing time-lapse images of mitotic cells. They applied the Imaris Spot tracking function to follow individual centromere trajectories over time. The MATLAB plugin also allowed them to calculate signal intensities between the homologous chromosomes.

Overlaying images reveal antipairing

“By using Imaris to overlay multiple mitotic cells, we were able to discover an antipairing pattern between the homologous chromosomes,” said Hua. “Imaris also allowed us to analyze the 3D confocal stacks very carefully with many user-friendly tools such as zoom, crop, and moving the objects in space.”

The Imaris analysis revealed that human and mouse cells keep two haploid chromosome sets apart along a subnuclear division axis during mitosis. “Abnormal homologous pairing has been previously shown to have detrimental consequences such as allelic mis-regulation and abnormal gene transcription,” explained Hua. “We found that this haploid set-based antipairing motif is shared by multiple cell types and is lost in cancer cells.”

The new discovery provides insight into the biological mechanism that a cell employs to prevent mitotic recombination and maintain genetic stability across daughter cells. The new findings could be used to ensure successful use of stem cell-based therapies to treat retinal disease. In the US, there are no approved stem cell-based therapies for retinal disease, although several candidates are undergoing clinical trials. The safety of these stem cell-based therapies has been a concern because of the possibility of genetic mutations, chromosomal abnormalities, and tumorigenicity when these cells are grown in culture prior to transplantation or injection.

The researchers are continuing their work by using Imaris to investigate the underlying mechanisms of haploid set-based antipairing.

• Video 1: A time-lapse movie of a RPE1 CENP-A/centrin1-GFP human cell undergoing mitosis from metaphase to anaphase. Four centromeres are labeled/tracked (colored spots, lines corresponding to colored spots, respectively) at an initial time point at metaphase and tracked to anaphase. Courtesy of Lisa L. Hua, University of California, San Francisco.
• Video 2: A 3-D reconstructed anaphase cell stained with TO-PRO3 (blue) painted with the X (red) and Y (green) chromosome of male-derived HUVECs. Courtesy of Lisa L. Hua, University of California, San Francisco.

Research Paper: Hua LL, Mikawa T. 2018. Mitotic antipairing of homologous and sex chromosomes via spatial restriction of two haploid sets. Proc Natl Acad Sci U S A. 115(52):E12235-E12244. doi: 10.1073/pnas.1809583115