Revealing and manipulating the structure of mitotic chromosomes using atomic force microscopy

Abstract

Although the mitotic cell cycle has been extensively characterized, very little is known about the highly condensed structure of metaphase chromosomes. The hierarchical organization of the chromatin fibers and their extrusion into complex loop structures by the Structural Maintenance of Chromosome (SMC) proteins are widely acknowledged features of the chromosomal architecture; however, the small size and complex topology of these structures are hindering their unequivocal detection and characterization. Even though Atomic Force Microscopy (AFM) has the potential to access these features, chromosome deformability and susceptibility to alterations to their external environment have so far limited the application of this technique. In the present work we overcome these issues and employ AFM to study the structure of human metaphase chromosomes in a liquid environment that closely resembles their physiological conditions, without the use of fixation agents and enzymatic treatments. Our analysis reveals the finer details of the unperturbed chromosome structure, shedding light on the organization of the chromatin fiber network and testing its stability against changes in the environment ionic conditions. Moreover, by complementing the image analysis with AFM-based Force Spectroscopy we probe the response of mitotic chromosomes to nanoscale deformations in the presence and absence of specific SMC proteins, namely Condensin I and II, thereby providing new insights into their contribution to the chromosome structural stability. The presented results enrich our current understanding of chromosome organization and provide new valuable tools to study the structure of mitotic chromosomes in both healthy and pathological conditions.