Jerod L. Ptacin

A spindle-like apparatus guides bacterial chromosome segregation

Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe’s diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20–40 nm in 3D) over a large field of view (12 × 12 μm).

Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions.

We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.

Bacterial scaffold directs pole-specific centromere segregation

Significance Bacteria use molecular partitioning systems based on the ATPase ParA to segregate chromosome centromeres before cell division, but how these machines target centromeres to specific locations is unclear. This study shows that, in Caulobacter crescentus, a multimeric complex composed of the PopZ protein directs the ParA machine to transfer centromeres to the cell pole. Spent ParA subunits released from the mitotic apparatus during segregation are recruited throughout a 3D PopZ matrix at the pole. ParA recruitment and sequestration by PopZ stimulates the cell-pole proximal recycling of ParA into a nucleoid-bound complex to ensure pole-specific centromere transfer. PopZ therefore utilizes a 3D scaffolding strategy to create a subcellular microdomain that directly regulates the function of the bacterial centromere segregation machine. Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.

Oligomerization and higher‐order assembly contribute to sub‐cellular localization of a bacterial scaffold

In Caulobacter crescentus, the PopZ polar scaffold protein supports asymmetric cell division by recruiting distinct sets of binding partners to opposite cell poles. To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub‐cellular localization and recruitment activity. We identified a domain within the C‐terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N‐terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. Mutations that inhibited dynamic PopZ localization inhibited the recruitment of other factors to cell poles. Mutations in the C‐terminal domain also blocked discrete steps in the assembly of higher‐order structures. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self‐associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into filaments, and these structures are important for sub‐cellular localization. Thus, PopZ undergoes multiple orders of self‐assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter.

Chromosome architecture is a key element of bacterial cellular organization

The bacterial chromosome encodes information at multiple levels. Beyond direct protein coding, genomes encode regulatory information required to orchestrate the proper timing and levels of gene expression and protein synthesis, and contain binding sites and regulatory sequences to co‐ordinate the activities of proteins involved in chromosome repair and maintenance. In addition, it is becoming increasingly clear that yet another level of information is encoded by the bacterial chromosome – the three‐dimensional packaging of the chromosomal DNA molecule itself and its positioning relative to the cell. This vast structural blueprint of specific positional information is manifested in various ways, directing chromosome compaction, accessibility, attachment to the cell envelope, supercoiling, and general architecture and arrangement of the chromosome relative to the cell body. Recent studies have begun to identify and characterize novel systems that utilize the three‐dimensional spatial information encoded by chromosomal architecture to co‐ordinate and direct fundamental cellular processes within the cytoplasm, providing large‐scale order within the complex clutter of the cytoplasmic compartment.

Initiating bacterial mitosis: understanding the mechanism of ParA-mediated chromosome segregation.

Comment on: Ptacin JL, et al. Nat Cell Biol 2010; 12:791–8.