Cell division is a process fundamental to life. While the genetic material is duplicated in mitosis, the division and separation of cellular organelles, cytoplasm, and surrounding membrane during cytokinesis is just as important. Cytokinesis is initiated during mitotic anaphase, by factors translocating to the midzone of the mitotic microtubule central spindle. It begins with the assembly of a contractile furrow at the division plane, which ingresses inwards to separate the two daughter cells. The central spindle transforms into the intercellular bridge, an extremely dense microtubule bundle connecting the dividing cells. In its center lies the midbody, an organelle discovered by W. Flemming in 1891, which stabilizes the intercellular bridge. In the last step of cytokinesis, the intercellular bridge is severed during abscission, marking the physical separation of the daughter cells. The main players that execute the ingression of the cleavage furrow, the formation of the midbody in the intercellular bridge, and finally the abscission of the dividing cells, have long been identified. However, the temporal progression of their nanoscale organization and redistribution during cytokinesis remains unclear, as cytokinesis is a complex three-dimensional process exerted at various speeds. While ingression of the cleavage furrow is relatively fast, the final remodeling of the intercellular bridge before abscission can take hours depending on the cell type. Additionally, remodeling has to be studied in three spatial dimensions, which is difficult to achieve at high spatiotemporal resolution, especially below the diffraction limit of light. In this thesis, I describe the development of a pipeline for the assembly of a multidimensional atlas of mammalian cytokinesis. I combined fast volumetric imaging using lattice light-sheet microscopy and super-resolution expansion microscopy (ExM) to assemble a multiplexed dynamic representation of several cytokinetic stakeholders. I prove that ExM is an ideal tool to study the organization of the protein-dense midbody region in cytokinesis, while providing increased resolution and allowing multiplexing of targets. Furthermore, I use computational modeling of cytokinesis based on the septin and microtubule cytoskeleton as markers to register and correlate multiple cytokinetic factors along a pseudotime axis. Pseudotemporal modeling of the static ExM data allows the generation of a dynamic and sequential representation of cytokinesis. With the increased resolution of ExM, I discovered a new stage of late cytokinesis, termed the septin manchette stage. I show the importance of this stage for remodeling of the intercellular bridge before abscission, based on key cytokinetic factors, such as the centralspindlin complex, actomyosin, and the endosomal sorting complex required for transport (ESCRT)-machinery. Finally, by correlating all information, I assemble an atlas of cytokinesis, providing unprecedented three-dimensional, dynamic and contextual insights into mammalian cytokinesis at high-resolution and in pseudotime.
