Characterizing kinetochore size in the spatial organization of chromosomes during metaphase alignment

Abstract

To maintain genomic integrity, cells rely on accurate chromosome segregation during mitosis, which in turn depends on faithful progression through the mitotic phases. Proper attachment of microtubules to chromosomes is essential for correct alignment in metaphase. However, how individual chromosomes are spatially organized within the metaphase plate is not yet fully understood. Recent studies have shown that kinetochores vary in size between chromosomes, revealing chromosome-specific differences not seen before. Whether this heterogeneity influences the spatial organization of chromosomes during metaphase alignment is unknown. We used fixed-cell imaging of HAP1 cells endogenously tagged for the kinetochore component Spc25 with mNeonGreen to quantify kinetochore intensity from the center to the periphery of the metaphase plate. This revealed no consistent pattern in the distribution of kinetochore size across the metaphase plate, which suggests that kinetochore size heterogeneity is not a driving factor in chromosomal positioning at this stage in mitosis. To confirm the previously observed organization of chromosomes, we assessed chromosomal positioning in metaphase by employing dCas9-Scarlet with chromosome-specific gRNAs to label chromosomes. This allowed us to assess that chromosome 18 — which is a chromosome with a relatively large centromere — exhibits non-random spatial positioning during metaphase. These findings suggest that while spatial positioning of chromosomes in mitosis is not random, kinetochore size does not contribute to this organization. This offers new insights into the principles guiding metaphase alignment and their potential implications for chromosome segregation fidelity.