Quantifying Nuclear Shape Fluctuations During Early Mitosis.

This chapter presents a detailed methodology for monitoring nuclear shape fluctuations and their correlation with chromatin condensation, developed for our analyses of early prophase, but applicable to other contexts. Nuclear shape dynamics play a key role in mitotic progression, and understanding the mechanical and biophysical properties associated with the fast-paced fluctuations may offer insights into key cellular processes like chromatin condensation and nuclear envelope breakdown. By employing live-cell imaging and computational analysis such as segmentation and flickering spectroscopy techniques, this approach leverages high-resolution temporal tracking of nuclear shape changes during cell cycle progression to derive insights into the mechanical forces driving chromatin condensation and nuclear envelope instabilities leading to the nuclear envelope breakdown. The experimental protocol provides a step-by-step guide for synchronizing HeLa cells at a specific cell-cycle transition and manipulating chromatin condensation and cytoskeletal structures through pharmacological perturbations. The data analysis section includes methods for extracting relevant biophysical parameters, such as nuclear effective tension and nuclear invaginations, as well as image-processing analysis, to correlate nuclear deformations with chromatin dynamics. We hope this robust and accessible workflow will serve as a powerful tool for exploring the mechanical coupling between chromatin condensation and nuclear structure across different conditions, which is crucial for our fundamental understanding of the nuclear function, as well as relevant for diseases resulting in nuclear abnormalities and disrupted cellular functions.