Organisation of axial regions of isolated mitotic chromosomes visualised by cryo correlative light and electron tomography

The formation of mitotic chromosomes is essential for the accurate segregation of genetic material during cell division. Increasing evidence suggests that chromosome formation involves the reorganization of DNA into loops anchored within chromosomal axial regions, whose structural organization remains insufficiently characterized. Taking advantage of DT40 cells, an avian cell model characterized by the presence of a range of chromosome sizes from 3.2-197 Mb, we have established a preparation of entire close-to-native native mitotic chromosomes for cryogenic correlative light and electron microscopy (cryo-CLEM). The size of the smallest chromosomes allows imaging of their axial regions without further thinning. Cryo-electron tomography of the chromosome axial regions reveals the presence of heterogeneous non-histone macromolecular densities (NHMDs), approximately 30-45 nm in size, interspersed within chromatin/DNA regions. We propose that NHMDs may contain condensins and contribute to chromosome architecture. In addition to NHMDs, we identified dense clusters of particles, similar in size, near the chromosome surface, likely associated with ribosomal components. To quantitatively differentiate NHMDs from these surface clusters, we developed an analytical approach based on particle interspacing and spatial distribution within the chromosome volume. By establishing a cryo-CLEM workflow for whole, near-native mitotic chromosomes, our study provides a foundation for investigating their ultrastructural architecture.