Cellular division requires partitioning of chromosomes into two daughter cells. In most cells, chromosome segregation and cytokinesis are symmetric events. Some cells, however, have asymmetric division.
The polarized cell cycle of C. crescentus makes it well-suited for studying asymmetric division.
- Origin of replication (ori)
- Par System
Results and Discussion
Caulobacter ParA Forms a Polarized Gradient Whose Retraction Mediates the Latter Part of ParB Translocation.
To examine the potential functions of ParA and ParB: their relative spatial and temporal localization patterns were determined (Figure 1).
- GFP-ParA was primarily localized to the distal pole, showed a graded “comet tail-like” localization
- mCherry-ParB initially localized to the polar focus at the pole
- ParB focus duplicated into two foci
- One foci advances into the vicinity of the ParA “comet tail”
To help visualize ParA and ParB, the localization dynamics of each fusion was reduced to a kymograph and the peak of the mCherry-ParB fluorescence and a normalized threshold of GFP-ParA intensity were labeled.
Much of ParB’s motion early in the process of chromosome segregation occurred in a region of the cell that is largely devoid of ParA. ParA is necessary for Caulobacter chromosome segregation, but only mediates in the latter stage of this process.
Quantitative Analysis of ParB Trajectories Identifies Distinct Phases of Chromosome Segregation.
More than one type of process might regulate ParB dynamics. ParB dynamics in living cells were imaged, producing hundreds of images per cell cycle (temporal resolution of 15 s). To circumvent complications introduced by exogenous chromosome labeling methods, a strain in which the endogenous parB gene was replaced with a fully functional gfp-parB fusion that binds and labels native parS sites. The ori translocation does not proceed via a single continuous motion, but rather a complex process with four definable steps.
ParA Mediates an Irreversible Commitment Step.
ParA could mediate the late translocation step. This was suggested by the localization of ParA at the distal pole and the incomplete segregation phenotype of ParAK20R-mCherry. The position in the cell where ParB arrested in the presence of the dominant negative ParAK20R-mCherry corresponded well to the position where the translocation phase began in unperturbed cells. Perturbing ParA specifically disrupts the late translocation step of ori motion. The ParA machinery gets disassembled during ParB movement, ensuring that once ParA moves the distal ori it cannot later move the second ori. Par-mediated translocation represents an irreversible commitment step after which no futher oris can be segregated.
Early Phases of Motion May Initiate Segregation and Distinguish the Proximal and Distal Chromosomes.
ParA retraction appears to translocate ParB. Immediately after ori replication , the duplicated oris are equivalent and in close proximity. If ParA encountered them in this state, it would be unable to distinguish the two oris and could inadvertently move both oris to the distal pole. DNA replication is blocked with novobiocin and the single ori is occasionally translocated across the cell, resulting in a cell with an inverted ParB polarity. This aberrant translocation depends upon ParA, as it is never observed when cells expressing the ParAK20R-mCherry dominant-negative mutant were treated with novobiocin.
- Complex cellular processes mediated by an ordered series of steps
- Chromosome segregation is a complex process with distinct events
- ori is released from polar tethering
- ori is duplicated, two daughter oris are spatially differentiated
- Distal ori committed to moving across cell by irreversible ParA-mediated mechanism
Caulobacter Chromosome Segregation is an Ordered Multistep Process. Conrad Shebulet, Jonathan Guberman, Sven Teeffelen, Anastasiya Yakhnina, and Zemer Gitai.