A propagating ATPase gradient drives transport of surface-confined cellular cargo

Abstract

Significance The process of DNA segregation is of central importance for all organisms. Although the basic mechanism of eukaryotic mitosis is relatively well established, the most common mechanism used for bacterial DNA segregation has been unclear. ParA ATPases form dynamic patterns on the bacterial nucleoid to spatially organize plasmids, chromosomes and other large cellular cargo, but the force-generating mechanism has been a source of controversy and debate. A dominant view proposes that ParA-mediated transport and cargo positioning occurs via a filament-based mechanism that resembles eukaryotic mitosis. Here, we present direct evidence against such models. Our cell-free reconstitution supports a non–filament-based mode of transport that may be as widely found in nature as filament-based mechanisms.

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

“…Whereas the spatial ParA gradient was correlated with directed and persistent movement in the in vitro experiments (14), key questions remained: What is the causal relationship between the motion and the gradient? What is the molecular basis for the highly persistent movement?…”

“…However, recent experiments cast doubt on the feasibility of this proposal (14)(15)(16)(17). A diffusion ratchet model was then put forward, which posited that a concentration gradient of ParA dimers on the nucleoid could act as the driving force for DNA segregation (18).…”

“…A diffusion ratchet model was then put forward, which posited that a concentration gradient of ParA dimers on the nucleoid could act as the driving force for DNA segregation (18). This proposed mechanism gained direct support from a recent in vitro experiment that reconstituted the segregation system of the Escherichia coli F plasmid (14). In this experiment, the ParB protein (F SopB) specifically bound to centromere sites (F sopC) that were immobilized on a microbead.…”

“…Interestingly, the microbead movement left a ParA-depleted zone in its wake, consistent with the proposed diffusion ratchet model for cargo motion. Simulations of a continuum model indicate that it is possible to drive persistent ParBbound cargo motion by maintaining a ParA concentration gradient around the cargo if the cargo diffusion constant is reduced to that observed during persistent bead motion, which is much smaller than that of the free microbead (14,19). However, the previous study did not address the origin of the cargo diffusion Significance Cells typically use processive motor proteins or the growth/ shrinkage of cytoskeletal filaments to power directed and persistent movement of cellular structures.…”

Surfing on Protein Waves: Proteophoresis as a Mechanism for Bacterial Genome Partitioning

“…This process entails the oscillation of ParA from pole to pole and the separation of the ParBS partition complex into two complexes with distinct sub-cellular trajectories and long-term localization. Overall, these interactions result in an equidistant, stable positioning of the duplicated DNA molecules along the cell axis.The specific modeling of ParABS systems falls into two categories: either "filament" (pushing/pulling the cargos, similar to eukaryotic spindle apparatus [3]) or reaction-diffusion models [8][9][10][11][12][13][14][15]. Recent superresolution microscopy experiments have been unable to observe filamentous structures of ParA [5,13], disfavoring polymerization-based models [12].…”