In this project, we set out to understand how the chromosome moves, folds, and interacts with the cell’s replication machinery over time. To this end, we developed a suite of new experimental and computational tools that allowed us to watch the chromosome in action inside living cells.
One major advance was the ability to read out the identity of individual cells directly on a microfluidic chip using chromosome‑encoded barcodes. This “in situ genotyping” approach allowed us to perform optical pooled screens in bacteria, where we can measure spatial phenotypes in thousands of cells and still know exactly which genetic variant each one carries. Using this platform, we analyzed libraries of strains with fluorescently labeled chromosome positions and mapped how different loci behave inside the cell.
To track how pairs of chromosome sites move relative to each other, we developed a robust two‑color labeling system. After extensive optimization, we identified a combination of fluorescent tags that provides stable, high‑precision tracking of two loci at once. Using deep‑learning–based 3D localization, we can now pinpoint chromosome positions with better than 60‑nanometer accuracy throughout the cell cycle.
These tools revealed new principles of chromosome behavior. We found that gene loci move in a restricted, “subdiffusive” manner but still explore the full width of the cell within about a minute. We also discovered that the replication machinery, the replisomes, remain confined to a small region of the cell, and that chromosome sites move toward them rather than the replisomes traveling along the DNA. This supports a model in which the DNA is actively brought to the replication machinery, not the other way around.
To complement these dynamic measurements, we developed a time‑resolved version of Hi‑C, a method that captures physical contacts between different parts of the chromosome. By profiling around 100,000 cells per experiment and sorting them into 25 cell‑cycle stages, we obtained a detailed view of how short‑range chromosome interactions change as the cell grows and divides.
Finally, we explored how chromosome dynamics are coordinated with the cell cycle. By tracking replication machinery across thousands of generations in different mutant strains, we showed that the timing of DNA replication initiation depends more on the balance between ATP‑ and ADP‑bound forms of the initiator protein DnaA than on its absolute concentration. We also discovered that two regulatory systems, DARS and datA, act redundantly to stabilize this process. Using a new method we call Tweez‑seq, which isolates individual cells directly from the microfluidic chip, we identified additional genes, both known and previously uncharacterized, that influence the coupling between DNA replication and cell division.
