^★Track: Inferred counting and tracking of replicating DNA loci

Abstract

Fluorescent microscopy is the primary method to study DNA organization within cells. However the variability and low signal-to-noise commonly associated with live-cell time lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present Star Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate ★Track's abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, Star Track can help to uncover the mechanistic principles of chromosome organisation and dynamics across a range of systems.

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes

“…At the exposure used, we did not observe statistically significant incidences of cell cycle arrest or cell death (in any case such cells would be excluded by our segmentation pipeline). However, the mean cell cycle duration was 13% longer than what we previously observed while studying plasmid dynamics of F plasmid pJY243 (miniF, parB-mVenus) without CFP excitation (31, 64). Cells were also 26% longer at birth (Fig.…”

“…For the majority of our study, we use the miniF plasmid pJYB249 (miniF parA-mVenus parB-mTurquoise2 ) that carries functional fluorescent fusion to ParA and ParB under the native promoter (40). Figure S1G-I also presents data of the strain DLT1215 and plasmid pJYB234 (miniF parB-mVenus ) from our earlier publications (64). These and similar fusions have previously been shown not to affect plasmid stability or expression levels (43, 44, 52, 65).…”

“…Cell segmentation was performed using a previously described image analysis pipeline (31). In addition, detected fluorescent foci were tracked using our recently developed ★ Track algorithm (https://gitlab.gwdg.de/murray-group/StarTrack, commit 38141270) (64). See SI Appendix for details.…”