STED microscopy reveals mitotic stage-dependent CENP-A organization

Chromosome segregation is vital. Its disruption can cause aneuploidy, a hallmark of cancer. Centromere protein A (CENP-A) is an important protein during the segregation as it marks the location of the centromere, where the kinetochore assembles for microtubule attachment. Each mammalian centromere contains hundreds of CENP-A nucleosomes, whose spatial arrangement is expected to be critical for kinetochore function and error-free chromosome segregation. However, previous studies— mostly in fixed cells and focused on metaphase—have yielded conflicting results on CENP-A organization. Given the centromere’s sub-diffraction size, we used stimulated emission depletion (STED) super-resolution microscopy to visualize the CENP-A organization in both living (about 45 nm resolution) and fixed (about 30 nm resolution) immortalized human retinal pigment epithelial cells (hTERT RPE-1). We found that CENP-A organization does not adopt a single architecture but spans a spectrum from dense clusters to fragmented subclusters, with mitotic stage-dependent abundance and morphology. CENP-A chromatin is most dispersed in prophase, and progressively compacts during prometaphase and metaphase as microtubules attach. In meta- and anaphase predominantly dense organizations are formed, often with a plate-like morphology. Yet, non-dense and non-plate-like organizations persist through metaphase and anaphase, suggesting CENP-A spatial reorganization is heterogeneous during cell division.