Rebuilding interphase nuclei into mitotic chromosomes

"The remarkable morphological transformation that cells undergo during mitosis has been studied for >140 years, but how mitotic chromosomes are built from interphase nuclei remains unknown and challenging to study. We propose to uncover the mechanisms for this process at high-resolution in cells and in vitro, a task that requires coordinated implementation of innovative experimental designs in several disciplines. We will engineer mutant and hybrid cell lines that enter mitosis with minute-by-minute synchrony, allowing us to develop new “kinetic sectioning” methods to map the intracellular distribution of chromatin near the nuclear envelope, from nucleosomes to mitotic chromosomes at unprecedented resolution, trace the action of individual condensin complexes as they produce loops during mitotic chromosome formation and efficiently isolate mitotic chromosomes for biochemical (4D proteomics) and structural analysis. We will combine genome-wide chromatin mapping, super-resolution microscopy and cryo-ET with innovative image analysis and modelling. In addition to studies of conventional chromosomes, we will study tiny natural ""dot chromosomes"" from chicken cells. Only a few megabases in size, these natural models for their larger counterparts will be isolated for detailed 4D proteomic and structural analysis and used to map scaffold and loop organisation at high resolution. Our team will address mitotic chromosome formation from multiple complementary angles in a synergistic approach where insights from collaborative computational analysis, cross-linking and structural data will inspire and define questions, both predicted and unforeseen. We will discover new insights as condensed mitotic chromosomes are built from active interphase chromatin across a wide range of physical and temporal dimensions. The information and technology that we develop will be generally applicable to other cellular systems, contributing to a paradigm change in structural and cellular biology."