Nadja Hümpfer
Freie Universität Berlin, Germany
“Nanoscale spatiotemporal organization of cytokinesis revealed by multidimensional microscopy”
Abstract: After the genetic material is duplicated, mitosis is concluded by cytokinesis. This highly conserved process starts with the ingression of a cleavage furrow between the two daughter cells by actomyosin and septins. A thick and extremely dense microtubule bundle builds a connection between the dividing cells known as the midbody, which later is cut when the cells abscise by an ESCRT-dependent process. The cytoskeletal family of septin GTPases was discovered in budding yeast as essential for the completion of cytokinesis. This role is conserved in mammalian cells where septins act in multimeric complexes as scaffolding proteins, linking actomyosin with cytokinetic proteins such as anillin, the ESCRT machinery as well as the plasma membrane.
While the main players that execute the ingression of the cleavage furrow, the formation of the midbody and finally the abscission of the dividing cells have long been identified, the temporal progression of the nanoscale organization of septins and interacting molecules during cytokinesis remains only rudimentarily understood. This is due to the relatively fast process of furrow ingression and its three-dimensional nature, which is difficult to study at high spatiotemporal resolution, especially below the diffraction limit of light.
Here we set out to combine fast volumetric imaging using lattice lightsheet microscopy with expansion microscopic (ExM) investigation of the nanoscale organization of the midbody to develop a dynamic description of cytokinesis. The analysis of dozens of dividing cells allowed us to extract a trajectory of microtubule bundle thinning during cytokinesis, which we correlate to the progression of cytokinesis and the recruitment and localization of key molecules in ExM.
In this talk I will focus on ExM, highlight its advantages and discuss possible pitfalls. A comprehensive overview about different ExM techniques should allow the audience to get set with their own ExM experiments.
I will also show how I model the nanoscale progression of events at the midbody based on thousands of super-resolved images of 18 cytokinetic molecules. ExM-inherent decrowding allowed the staining of midbody components that otherwise remain inaccessible for staining while providing three-dimensional and contextual information with a resolution of up to 70 nm. Our data offer unprecedented spatiotemporal understanding of mammalian cytokinesis and an assay to further explore its perturbation.