Abstract
Eukaryote cells completely reorganize their long chromosomal DNAs to folded mitotic chromosomes to facilitate their physical segregation during mitosis. The internal organization of mitotic chromosomes remains unclear. We report biophysical experiments on single mitotic chromosomes from human cells, where isolated single human chromosomes were studied by micromanipulation and nanonewton-scale force measurement to understand chromosome connectivity and topology. We demonstrated that 4 base blunt-cutting restriction enzymes completely dissolved single metaphase human chromosomes, while proteases did not cut through human chromosomes, and led to reduction of their elasticity. Our results rule out the possibility that the mitotic chromosome is structured on a mechanically contiguous internal protein scaffold. Instead, mitotic chromosomes have a “chromatin network” organization, where chromatin fiber inside each chromosome is tethered to itself, by crosslinking proteins. We further looked into the effect of RNAi knockdowns of a major chromosome-organizing protein– condensin on mototic chromosome organization. We found the stiffness of human chromosomes goes down by almost 10 fold in condensin depleted cells, compared to wildtype cells. Moreover, the distinct effect of condensin I and condensin II is also discussed. Our studies provide a quantitative analysis of the effect of condensin on mitotic chromosome condensation.
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