Probing Asymmetric Behavior of a Cell Cycle Regupatory Protein in Live Caulobacter using Single-Molecule Imaging

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Asymmetric cell division is a fundamental event in developmental programs where two daughter cells are produced and exhibit distinct characteristics and fates. Remarkably, asymmetric cell division is also observed in the bacterium Caulobacter crescentus (∼3μm x 0.5μm). Caulobacter regulates cell cycle events through its master regulator CtrA, which is specifically degraded at one cell pole (stalked) during the swarmer-to-stalk (G1-to-S) transition and in late predivisional cell to enable replication onset and the transcription of over 90 genes. Surprisingly, the CtrA degradation adaptor protein, PopA, localizes symmetrically to both cell. PopA binds a secondary messenger molecule, c-di-GMP (cdG). cdG accumulates asymmetrically at only one of the two daughter cells (stalked). (Christen et al. 2010, Science) We hypothesized that the differential distribution of cdG could imply that PopA proteins at the two cell poles are intrinsically different. We report the first single-molecule tracking studies in live Caulobacter of wild type (WT) and mutant PopA that lacks cdG binding capability. By quantifying our single-molecule trajectories, we found that PopA that cannot bind cdG diffuses 1.28x faster than the WT. The difference in the diffusion coefficients is consistent with the possibility that WT, when cdG is bound, forms a complex twice the size of mutant that does not bind cdG. We also obtained evidence for preferential dimerization of PopA at the stalked pole, the site of cdG synthesis, using bimolecular fluorescence complementation. Furthermore, the different diffusion coefficients of PopA at the two poles, as well as the distinct impact on the diffusion coefficient when cdG cannot bind, suggest different localization mechanisms at the two poles. We propose that by forming different oligomeric states, PopA can perform multiple functions at the two poles and contribute to asymmetric cell division.