High-throughput identification of protein localization dependency networks

Abstract

Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.

Fig. 1.

Cell cycle localization patterns of PleC, DivJ, and CpaE. (A) The composite microscope images and cell schematics show the position of DivJ-mCherry (red), PleC-YFP (green), and CFP-CpaE (blue) foci within Caulobacter cells at 30-min time intervals. (B) Normalized average localized fluorescence signal of DivJ-mCherry (red squares), PleC-YFP (green triangles), and CFP-CpaE (blue circles) are shown as a function of the cell cycle, fit to an exponentially damped sine function.

Fig. 2.

Single cell quantification of subcellular protein distribution. (A) The fluorescence intensity profile of a single cell expressing a chromosomal DivJ-mCherry fusion is shown, with the peak intensity mapped to red and lowest intensity to blue. The localized fluorescence signal (λ) is the difference between the peak level and dispersed fluorescence signal (δ) after background correction. (B) Cell shape parameters estimated from the phase contrast image are shown overlaid on the fluorescence intensity profile (Inset). Changing numbers of discrete image regions found by dynamic thresholding are plotted as a function of the relative threshold level applied. The maximum number of confined image regions found is a measure of the amount of dispersed fluorescence signal (δ) present in the cell (SI Text). (C) Each dot indicates the localized fluorescence signal (λ) from a single cell for the PleC-YFP (green), DivJ-mCherry (red), and CFP-CpaE (blue) reporters. The transposon insertions in tacA and shkA (arrows) led to a significant decrease in localized DivJ-mCherry and increased localized CFP-CpaE fluorescence signal as compared to the unmutagenized strains (control).

Fig. 3.

Identification of gene modules coordinating temporal and spatial protein localization by hierarchical clustering analysis. Heat map representation of the z-scores (SI Text) for localized (L), dispersed (D), bipolar (B), and monopolar (M) metrics for CFP-CpaE, PleC-YFP, and DivJ-mCherry are shown for four statistically significant clusters of 52 localization mutants (SI Text). Dendrograms representing the linkage of mutant alleles within cluster A (blue), cluster B (green), cluster C (purple) and the cluster D (red) are shown.

Fig. 4.

Protein localization patterns of a set of mutants identified by the automated fluorescence microscopy screen. (A) An in-frame deletion of cpaE showed a 47% reduction in polar localized PleC-YFP levels as compared to the control. Providing cpaE (pBX::cpaE) on a plasmid restored PleC-YFP localization. (B) Subcellular fluorescence intensities of DivJ-mCherry (red), PleC-YFP (green), and CFP-CpaE (blue) are merged onto the phase contrast images (gray). Transposon insertions in shkA, shpA, pleC, tacA, rpoN, flbE, spmX, and murI alter the localization pattern of PleC, CpaE, and DivJ as compared to the control. (C) A transposon insertion within cckN caused conditional loss of DivJ-mCherry localization and bipolar localization of PleC-YFP and CFP-CpaE upon addition of xylose. The orientation of the Tn5Pxyl insertion in cckN positioned the Pxyl promoter toward the neighboring divJ gene yielding constitutive DivJ expression.

Fig. 5.

Protein localization dependency network. The localization dependency network for PleC, DivJ, and CpaE inferred from the automated quantitative fluorescence microscopy and epistasis analyses is shown. The fluorescently tagged reporter proteins used are DivJ-mCherry (dark red), PleC-YFP (green), and CFP-CpaE (dark blue). Proteins belonging to cluster A (light blue), cluster C (purple), and the cluster D (light red) are shown. The thick solid line indicates that SpmX mediates the removal of PleC and CpaE from the incipient stalked pole. The thin solid lines indicate that SpmX mediates the localization of DivJ to the stalked pole, as previously reported (25). PodJ has been reported to be involved in the localization of PleC and CpaE (19, 21, 22), and here we show that PodJ partially mediates the polar localization of both PleC (60%) and CpaE (60%) (Fig. 3 and Fig. S6). We also found that CpaE partially facilitates the polar localization of PleC (47%) (Figs. 3 and 4 and Table S1). The dashed lines show a localization pathway derived from the results of the epistasis experiments (Tables S2 and S3) and DivJ, PleC and CpaE mislocalization phenotypes observed in Tn5 insertion mutants in shkA, shpA, tacA, rpoN, and spmX (Figs. 3 and 4).