Super-resolution microscopy reveals the number and distribution of topoisomerase IIα and CENH3 molecules within barley metaphase chromosomes.

Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes. We detected a dispersed Topo IIα distribution along chromosome arms but an accumulation at centromeres, telomeres, and nucleolus-organizing regions. With a precision of 10-50 nm, we counted ~ 20,000-40,000 Topo IIα molecules per chromosome, 28% of them within the (peri)centromere. With similar precision, we identified ~13,500 CENH3 molecules per centromere where Topo IIα proteins and CENH3-containing chromatin intermingle. In short, we demonstrate PALM as a useful method to count and localize single molecules with high precision within chromosomes. The ultrastructural distribution and the detected amount of Topo IIα and CENH3 are instrumental for a better understanding of their functions during chromatin condensation and centromere determination.