Spatial Organization and Dynamics of Rnase E and Ribosomes in Caulobacter Crescentus
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Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus Camille A. Bayasa, Jiarui Wanga,b, Marissa K. Leea, Jared M. Schraderb,1, Lucy Shapirob,c, and W. E. Moernera,2 aDepartment of Chemistry, Stanford University, Stanford, CA 94305; bDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305; and cChan Zuckerberg Biohub, San Francisco, CA 94158 Contributed by W. E. Moerner, March 5, 2018 (sent for review December 15, 2017; reviewed by Zemer Gitai and James C. Weisshaar) We report the dynamic spatial organization of Caulobacter cres- components of the degradosome or the ribosome that are on centus RNase E (RNA degradosome) and ribosomal protein L1 (ri- length scales of less than ∼200 nm. bosome) using 3D single-particle tracking and superresolution We used a combination of live-cell single-particle tracking microscopy. RNase E formed clusters along the central axis of the (SPT) and fixed-cell superresolution (SR) microscopy to study the cell, while weak clusters of ribosomal protein L1 were deployed dynamics and spatial distribution of eYFP-labeled (13) RNase E throughout the cytoplasm. These results contrast with RNase E and ribosomal protein L1 in Caulobacter on subdiffraction limit and ribosome distribution in Escherichia coli, where RNase E coloc- length scales. SPT and SR provide improved resolution down to alizes with the cytoplasmic membrane and ribosomes accumulate in ∼20–50 nm using fluorescent proteins (FPs) (14, 15). Moreover, Cau- polar nucleoid-free zones. For both RNase E and ribosomes in both SPT and SR are single-molecule (SM) methods, allowing us lobacter , we observed a decrease in confinement and clustering to investigate the heterogeneity in protein behavior and distribu- upon transcription inhibition and subsequent depletion of nascent tion across many different cells. To avoid artifacts from projecting RNA, suggesting that RNA substrate availability for processing, deg- SM localizations onto two dimensions, we have used the double- radation, and translation facilitates confinement and clustering. Im- helix point spread function (DH-PSF), which encodes 3D infor- portantly, RNase E cluster positions correlated with the subcellular mation in each SM image (16). location of chromosomal loci of two highly transcribed rRNA genes, Using these methods, we observed that the number of RNase suggesting that RNase E’s function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome E clusters changed as a function of the cell cycle, while the size of and ribosome assembly are spatiotemporally organized in Caulo- the clusters remained constant. However, ribosomes formed only bacter, with chromosomal readout serving as the template for weak clusters, which were deployed throughout the cell, as we α this organization. found to be the case in another -bacterium, Sinorhizobium meliloti. For both RNase E and ribosomes, we observed a decrease ribosomes | RNA degradation | RNA processing | superresolution in confinement and clustering when transcription was inhibited, microscopy | single-molecule tracking likely due to depletion of RNA substrates for degradation, pro- cessing, and translation. We found that the localization of RNase E n bacteria, the RNA degradosome mediates the majority of clusters correlates with the subcellular chromosomal position of the ImRNA turnover and rRNA and tRNA maturation (1). The RNA degradosome assembles on the C-terminal scaffold region Significance of the RNase E endoribonuclease (2). RNase E in Escherichia coli and RNase Y, the RNase E homolog in Bacillus subtilis, both Regulated gene expression involves accurate subcellular posi- associate with the cell membrane through a membrane-binding tioning and timing of RNA synthesis, degradation, processing, helix (3–6). One proposed rationale behind the observed mem- and translation. RNase E is an endoribonuclease that forms the brane association is to physically separate transcription within scaffold of the RNA degradosome in bacteria, responsible for the nucleoid from RNA degradation, thus providing an inherent the majority of mRNA turnover and RNA processing. We used time delay between transcript synthesis and the onset of tran- 3D superresolution microscopy and single-particle tracking to script decay, avoiding a futile cycle. Caulobacter crescentus (Cau- quantify the spatial distribution and dynamics of RNase E and lobacter) is a Gram-negative α-proteobacterium widely studied as ribosomes in the asymmetrically dividing bacterium Caulo- a model system for asymmetric cell division. Unlike E. coli or B. bacter crescentus. Our results show that active transcription subtilis, Caulobacter contains a pole-tethered chromosome that and RNA substrate availability facilitate confinement and fills the cytoplasm (7–9). Surprisingly, Caulobacter RNase E does clustering of both RNase E and ribosomes. RNase E clusters not have a membrane-targeting sequence and is not membrane colocalized with the subcellular position of the two Caulo- associated (2, 10). Furthermore, diffraction-limited (DL) images bacter rRNA gene chromosomal loci, indicating that RNA pro- of RNase E exhibited a patchy localization throughout the cell, cessing can be spatially organized in a bacterium according to with RNase E directly or indirectly associating with DNA (10). its transcriptional profile. Moreover, the copy number of RNase E is regulated as a function of Author contributions: C.A.B., J.W., M.K.L., J.M.S., L.S., and W.E.M. designed research; the Caulobacter cell cycle. The copy number reaches maxima during C.A.B., J.W., and M.K.L. performed research; C.A.B., J.W., M.K.L., and J.M.S. contributed the swarmer to stalked cell transition and before cell division (2). new reagents/analytic tools; C.A.B., J.W., M.K.L., and W.E.M. analyzed data; and C.A.B., Fluorescently labeled ribosomal proteins S2 and L1, proxies J.W., L.S., and W.E.M. wrote the paper. for ribosomes in E. coli and B. subtilis, respectively, were found Reviewers: Z.G., Princeton University; and J.C.W., University of Wisconsin–Madison. enriched at the cell poles, spatially excluded from the nucleoid The authors declare no conflict of interest. on the hundreds of nanometer scale (11, 12). Similar fluores- Published under the PNAS license. cence imaging studies in Caulobacter revealed no apparent sep- 1Present address: Department of Biological Sciences, Wayne State University, Detroit, aration of ribosomes and the nucleoid, but did suggest that MI 48202. ribosome diffusion throughout the cell was spatially confined 2To whom correspondence should be addressed. Email: [email protected]. (10). However, because the Caulobacter cell is only ∼500 nm by This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ∼3.5 μm in size, the DL resolution of conventional fluorescence 1073/pnas.1721648115/-/DCSupplemental. microscopy would obscure any organization or motion of the Published online April 2, 2018. E3712–E3721 | PNAS | vol. 115 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1721648115 Downloaded by guest on September 24, 2021 highly expressed rRNA genes that are positioned at two distinct and 0.1944 ± 0.0010 μm2/s (mobile). For rif-treated cells, the PNAS PLUS sites on the chromosome, consistent with processing occurring at diffusion coefficient for confined subtrajectories is 0.0060 ± the site of rRNA synthesis. Thus, cotranscriptional activities exhibit 0.0001 μm2/s and for mobile subtrajectories is 0.1270 ± dynamic subcellular organization that is dependent on the site of 0.0072 μm2/s. To determine whether the confined subtrajectories chromosomal readout. are stationary or not, the apparent diffusion coefficient of sta- tionary RNase E-eYFP molecules was calculated using the Results ensemble-averaged MSD of trajectories from fixed cells. Stationary, Diffusion of RNase E and Ribosomal L1 Proteins Is Influenced by fixed particles were found to have an apparent diffusion coefficient Transcriptional Activity. of 0.0022 ± 0.0015 μm2/s (error is SEM from 143 trajectories). RNase E. The endoribonuclease RNase E forms the core and Using this comparison, we determined that the observed diffusion scaffold of the Caulobacter RNA degradosome. Other compo- coefficient for WT confined subtrajectories is consistent with a nents include the exoribonuclease polynucleotide phosphorylase, stationary particle, whereas the diffusion coefficientforrifconfined a DEAD-box RNA helicase RhlB, the Krebs cycle enzyme aco- subtrajectories is larger than would be expected for a stationary nitase, and the exoribonuclease RNase D (2, 17). To study the particle given our localization precision (32/33/49 nm in x/y/z). motional dynamics of RNase E, we performed 3D SPT in a mixed The difference in diffusion between WT and rif-treated cells is population of live Caulobacter cells using a strain expressing not an artifact due to a potential rif-induced chromosome struc- RNase E-eYFP as the only copy of RNase E. We performed an ture disruption (22). We performed the same 3D SPT of RNase E initial photobleaching and waited for stochastic recovery and molecules in a ΔHU2 background with significantly reduced nu- blinking of eYFP SMs (18) (Fig. 1A, Left). We then obtained 3D cleoid compaction (23). Results show similar confinement as that trajectories of individual molecules by joining DH-PSF localiza- of WT cells (SI Appendix,Fig.S6), arguing against the possibility tions over consecutive frames (Materials and Methods) (Fig. 1A, of a difference in chromosome packaging between WT and rif- Right). In contrast to E. coli in which RNase E diffuses on the treated cells contributing to the difference in RNase E diffusion. membrane (3), RNase E molecules in Caulobacter (average copy Ribosomes. To obtain information about ribosome dynamics and number is 3,100 molecules per cell as determined for a mixed cell translation, we performed 3D SPT of ribosomes through imaging population; SI Appendix, Supplementary Methods)werefound eYFP-labeled ribosomal protein L1 in a mixed population of throughout the cytoplasm of the cell, diffusing limited distances cells. We found that L1 (average copy number is 16,300 mole- (Fig.