Live Imaging Characterization of Centromere Movements During Male Meiotic Prophase in Arabidopsis thaliana

Our research explores plant meiosis, aiming to uncover molecular mechanisms guiding homologous chromosome recognition, alignment, and pairing during meiotic progression. The challenge is to visualize chromosome movement in meiotic profiles, requiring live tissue imaging with high resolution and speed to track trajectories. To begin, remove the protective film of the spacer on one side and place it at the center of a glass slide.

Then remove the second protective film. Deposit eight microliters of tap water into the center of the spacer. Using forceps, pick an inflorescence from one of the main stems.

Place the inflorescence on a new slide positioned under a stereo microscope fitted with a ruler. Select flower buds that measure approximately 0.5 millimeters in length. Using sharp needles, gently open the buds by pulling apart the sepals and petals to retain only the anthers.

Transfer the anther bunches delicately into the water in the spacer cavity immediately after removing the sepals and petals to prevent dehydration. Deposit several anther bunches inside the spacer cavity. To cover the slide with a cover slip while avoiding air bubbles, add eight microliters of water onto a cover slip.

Then carefully invert the cover slip with the drop of water facing the spacer cavity. Ensure the anther bunches are immersed in a total of 16 microliters of water, and that the cover slip adheres well to the spacer. Set up the microscope by exciting GFP at 488 nanometers and detecting it with a hybrid detector between 494 nanometers and 547 nanometers.

Then localize the anther using brightfield illumination. Using the brightfield channel, determine the meiotic stage based on the shape of the cells. Visualize the anther on the software and use the RFP signal as an additional indicator of the meiotic stage of the cells.

To perform continuous acquisitions, set the time interval to zero in the time section. Then use the spot module to automatically track spots. Choose the green source channel and set the XY diameter to one micrometer for the GFPCENH3-marked centromere.

Then modify the quality of detection based on signal intensity using the graph at the bottom of the control window. Use the auto regressive motion tracking algorithm to trace centromere trajectories and visualize all trajectories to easily distinguish meiocytes from somatic tissues. Next, use the spot module to track spots over time on the selected nucleus.

Define a region of interest by selecting the option segment only a region of interest and perform spot tracking within it. Select a region of interest that properly includes the nucleus in all three dimensions and over time. In a lineage plot, use the software's 3D viewer to visualize all trajectories.

Remove the trajectories outside the analyzed meiocyte by selecting them and pressing the delete key on the keyboard. In edit mode, delete or add an object, ensuring that the object is added in the correct plane. In edit tracks mode, manually connect the new objects on the lineage plot after thoroughly analyzing the cell in 3D.

Compute the instantaneous speed at each time step to monitor variations in movement velocity over time. Calculate the turning angle as the change in direction of movement between consecutive time steps to reveal trajectory patterns. Store the result together with the instantaneous speed.

Compute the outreach ratio using cell tracker to measure how far a tracked object moves relative to its starting position, indicating exploratory behavior. Compute the average speed for each track to summarize movement characteristics across the observation period. Calculate the average turning angle per track to analyze movement, directionality and behavioral patterns and store the result.

Next, compute the centroid size at each time step to measure the spatial dispersion of centromeres around their average position. Use variations in centroid size over time to evaluate global consistency across centromere tracks and store the result. Compute the speed cross correlation to analyze relationships between the movement dynamics of different tracked objects revealing potential coordinated behaviors.

Then compute the mean square displacement for each track to quantify the average square distance traveled by centromeres over a given time interval. Meiotic centromeres exhibited a significantly higher average speed of 109.35 nanometers per second compared to somatic centromeres. Meiotic centromeres showed a broader range and higher normalized outreach ratio compared to somatic centromeres.

The turning angle distribution differed between meiotic and somatic centromeres. The mean square displacement of meiotic centromeres increased over time while somatic centromeres remained largely static. This protocol enabled first time quantification of meiotic chromosome movement in Arabidopsis, revealing dramatic centromeres dynamics during zygotene and pachytene stage.

This protocol enables studying genomic region movements during meiotic prophase and can be adapted for application in other plant species. This works enables exploration of chromosome movement and recombination interplay, deepening understanding of mechanism driving genetic diversity during plant gamete formation.