Focus on the 3D architecture of mitotic spindles in human cells

Abstract text (incl. figure legends and references)

Mitosis in eukaryotes follows organism- as well as cell-specific patterns. One common feature of mitosis is the assembly of a bipolar spindle apparatus that is involved in the segregation of the genetic material to the newly forming daughter cells. The spindle is a complex microtubule-based cellular machinery. Within the spindle, microtubules are interacting with molecular motors and passive crosslinkers to align chromosomes in metaphase and move chromosome halves in anaphase. It is crucial to study the 3D complexity of mitotic spindles in order to better understand chromosome missegregation. Almost any chromosome missegregation results in aneuploidy, which is a hallmark of most types of cancer. Studying the 3D ultrastructure of the mitotic spindle apparatus at the level of microtubule organization requires electron microscopy. For 3D analysis, we apply high-pressure freezing, freeze substitution and resin embedding. Subsequently, we pre-screen embedded cells using confocal light microscopy to identify cells in desired stages of mitotic cell division. Once a cell has been chosen, we apply serial sectioning followed by electron tomography to reconstruct spindles in silico with single-microtubule resolution. For 3D reconstruction and quantitative analysis of spindle organization, we are applying a routine pipeline, which involves various software tools such as IMOD and Amira. With this approach, we recently analyzed bipolar spindles in HeLa cells (Kiewisz et al., Elife, 2022). During the meeting we will also report on our newest results on the ultrastructure of kinetochore microtubules (KMTs) in monopolar spindles in retinal pigment epithelial 1 (RPE1) cells. In both bipolar and monopolar mammalian spindles, KMTs are organized in so-called kinetochore (k)-fibers. Comparing spindle organization in various cell types, it is our goal to refine our recently developed biophysical model of k-fiber self-organization in mammalian cells (Conway et al., Elife, 2022).