Doctor of Philosophy, Stanford University, BIOC-PHD (2017)
Bachelor of Science, Bucknell University, Chemistry (2011)
Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment.IMPORTANCE Lacking internal membrane-bound compartments, bacteria achieve subcellular organization by establishing self-assembling protein-based microdomains. The asymmetrically dividing bacterium Caulobacter crescentus uses one such microdomain to link cell cycle progression to morphogenesis, but the mechanism for the generation of this microdomain has remained unclear. Here, we demonstrate that the ordered assembly of this microdomain occurs via the polymeric network protein PopZ directly recruiting the polarity factor SpmX, which then recruits the histidine kinase DivJ to the developing cell pole. Further, we find that overexpression of the bridge protein SpmX in Caulobacter disrupts this ordered assembly, generating ectopic cell poles containing both PopZ and DivJ. Together, PopZ and SpmX assemble into a cooligomeric network that forms the basis for a polar microdomain that coordinates bacterial cell polarity.
View details for DOI 10.1128/mBio.02238-16
View details for PubMedID 28246363
View details for PubMedCentralID PMC5347347
Cellular functions in Bacteria, such as chromosome segregation and cytokinesis, result from cascades of molecular events operating largely as self-contained modules. Regulated timing of these cellular modules stems from global genetic circuits that allow precise temporal activation with respect to cell cycle progression and cell differentiation. Critically, many of these functions occur at defined locations within the cell, and therefore regulators of each module must communicate to remain coordinated in space. In this perspective, we highlight recent discoveries in Caulobacter crescentus asymmetric cell division to illuminate diverse mechanisms by which a cellular compass, composed of scaffolding and signaling proteins, directs cell cycle modules to their exact cellular addresses.
View details for DOI 10.1016/j.mib.2016.06.007
View details for PubMedID 27517351
View details for PubMedCentralID PMC5069156
All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation.
View details for DOI 10.1038/ncomms11454
View details for PubMedID 27117914
View details for PubMedCentralID PMC4853435
This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.
View details for DOI 10.1261/rna.049502.114
View details for Web of Science ID 000356316200002
View details for PubMedID 25883046
View details for PubMedCentralID PMC4436661
Chemical mapping experiments offer powerful information about RNA structure but currently involve ad hoc assumptions in data processing. We show that simple dilutions, referencing standards (GAGUA hairpins), and HiTRACE/MAPseeker analysis allow rigorous overmodification correction, background subtraction, and normalization for electrophoretic data and a ligation bias correction needed for accurate deep sequencing data. Comparisons across six noncoding RNAs stringently test the proposed standardization of dimethyl sulfate (DMS), 2'-OH acylation (SHAPE), and carbodiimide measurements. Identification of new signatures for extrahelical bulges and DMS "hot spot" pockets (including tRNA A58, methylated in vivo) illustrates the utility and necessity of standardization for quantitative RNA mapping.
View details for DOI 10.1021/bi5003426
View details for PubMedID 24766159
View details for PubMedCentralID PMC4033625