Caulobacter Crescentus Chromosome Segregation is an Ordered Multistep Process

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.

Click above image for video of cell division

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.

(A) Overlaid images from a time-lapse of a cell coexpressing mCherry-ParB (red) and GFP-ParA (green). The arrowhead indicates the translocating mCherry-ParB and the arrow indicates the edge of the GFP-ParA comet tail. (B) Kymographs of a cell coexpressing mCherry-ParB and GFP-ParA Composite of the cell’s fluorescence intensities at 2 minute intervals over a 2 hour period. (top) mCherry-ParB. (middle) GFP-ParA. (bottom) mCherry-ParB and GFP-ParA merge. Red and black curves display the positions of the proximal and distal mCherry-ParB labeled oris. The white line indicates a normalized threshold of GFP-ParA intensity.

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.

(A) Kymograph showing GFP-ParB labeled oris translocating within a single cell. The steps of ori translocation are labeled: 1, polar release; 2, polar retraction; 3, early translocation; 4, late translocation. (B) A schematic summarizing the steps of ori translocation. (C) Plot average of GFP-ParB instantaneous velocity as a function of relative cell position.

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.

Conclusion

  • 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

References

Caulobacter Chromosome Segregation is an Ordered Multistep Process. Conrad Shebulet,  Jonathan Guberman, Sven Teeffelen, Anastasiya Yakhnina, and Zemer Gitai.

Leave a Reply

Your email address will not be published. Required fields are marked *