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Updated: Jul 9, 2026
Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy
Published on: December 13, 2012
In vivo visualization of type II plasmid segregation: bacterial actin filaments pushing plasmids
Christopher S Campbell1, R Dyche Mullins
1School of Medicine, University of California, San Francisco, San Francisco, CA 94158, USA.
Abstract:
Type II par operons harness polymerization of the dynamically unstable actin-like protein ParM to segregate low-copy plasmids in rod-shaped bacteria. In this study, we use time-lapse fluorescence microscopy to follow plasmid dynamics and ParM assembly in Escherichia coli. Plasmids lacking a par operon undergo confined diffusion with a diffusion constant of 5 x 10(-5) microm(2)/s and a confinement radius of 0.28 microm. Single par-containing plasmids also move diffusively but with a larger diffusion constant (4 x 10(-4) microm(2)/s) and confinement radius (0.42 microm). ParM filaments are dynamically unstable in vivo and form spindles that link pairs of par-containing plasmids and drive them rapidly (3.1 microm/min) toward opposite poles of the cell. After reaching the poles, ParM filaments rapidly and completely depolymerize. After ParM disassembly, segregated plasmids resume diffusive motion, often encountering each other many times and undergoing multiple rounds of ParM-dependent segregation in a single cell cycle. We propose that in addition to driving segregation, the par operon enables plasmids to search space and find sister plasmids more effectively.
Insights
Bacterial plasmids use dynamic protein filaments to segregate themselves, ensuring accurate cell division. These filaments actively move plasmids to opposite cell poles, then disappear, allowing plasmids to search for their sisters.
Area of Science:
- Microbiology
- Molecular Biology
- Cell Biology
Background:
- Type II par operons utilize the actin-like protein ParM for low-copy plasmid segregation in bacteria.
- Plasmid segregation is crucial for maintaining genetic stability during cell division.
Purpose of the Study:
- To investigate the dynamics of plasmid segregation mediated by ParM in Escherichia coli.
- To characterize the movement and assembly of ParM filaments in vivo.
Main Methods:
- Time-lapse fluorescence microscopy was employed to observe plasmid dynamics and ParM assembly.
- Quantitative analysis of plasmid diffusion and ParM filament behavior was performed.
Main Results:
- Plasmids lacking par operons exhibited confined diffusion.
- ParM filaments formed dynamic spindles that actively transported plasmids to opposite cell poles at 3.1 microm/min.
- Segregated plasmids resumed diffusive motion and could undergo multiple segregation rounds per cell cycle.
Conclusions:
- The par operon actively drives plasmid segregation and facilitates the spatial search for sister plasmids.
- Dynamic instability of ParM filaments is essential for efficient plasmid segregation and bacterial cell division.
