DNA Segregation and Partitioning in Caulobacter Crescentus: Super-Resolving Protein Colocalization at the Cell Pole

Abstract

Biological function relies on biochemical reactions that require spatial proximity between biomolecules. We are investigating how DNA segregation and partitioning is driven by the spatial recruitment of proteins to the cell pole in the bacterial model system Caulobacter crescentus. The polar organizing protein PopZ assembles as a polymeric network at one and then the opposite cell pole where it respectively anchors the original and the newly replicated DNA. Previous super-resolution imaging experiments in our lab have determined the cytosolic locations of the partitioning system proteins, ParAB, during directional DNA segregation (Ptacin et al., 2010), but the role of the PopZ network in the biochemical reaction cycle of these proteins has not yet been explored. To determine precise positioning of the partitioning system proteins in relation to the PopZ polymeric network, we utilized quantitative two-color 3D super-resolution microscopy in live Caulobacter cells. We measured the shapes and volumes of the PopZ networks with tens of nanometer resolution and, by counting the localized PopZ proteins within each network, we determined that PopZ localizes with a globally conserved volume density. These results indicate that PopZ may serve as a homogeneous scaffold that spatially compartmentalizes the biochemical interactions of the partitioning system proteins. This model is supported by two-color super-resolution images that clearly reveal where and how the Par system proteins overlap with the PopZ networks. These data thus provide key mechanistic insights into the spatial regulation of ParAB protein activity during DNA segregation and partitioning.