Unveiling the Inner Workings of Live Bacteria Using Super-Resolution Microscopy

Tuson, Hannah H.; Biteen, Julie S.

doi:10.1021/ac5041346

Cited by 63 publications

(70 citation statements)

References 274 publications

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“…However, due to the small size of the injectisome, previous approaches have not successfully resolved individual machines within the bacterial cell and therefore have not been able to address critical questions that require this higher level of resolution. The development of superresolution imaging techniques has made possible the visualization of subcellular structures at an unprecedented level of resolution (29,30). Here we have used 2D (31) and 3D (32) single-molecule switching nanoscopy (SMSN) to visualize the different structural elements of the type III secretion system in live bacteria, providing major insight into the assembly and function of this bacterial nanomachine.…”

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confidence: 99%

Visualization and characterization of individual type III protein secretion machines in live bacteria

Zhang

Lara‐Tejero

Bewersdorf

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

103

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Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine's assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.bacterial nanomachines | protein secretion | superresolution microscopy | bacterial pathogenesis | Salmonella virulence T ype III secretion systems are remarkable molecular machines with the capacity to inject multiple bacterial proteins into eukaryotic cells (1, 2). These machines play an essential role in the pathogenic or symbiotic relationships between the bacteria that encode them and their respective hosts. Consequently, type III secretion systems are rapidly emerging as targets for the development of novel therapeutic and prevention strategies with the potential to combat several important infectious diseases (3-5). The components of type III secretion machines are highly conserved across bacterial species although the effector proteins that they deliver are customized for the specific bacteria that harbor them (1, 6, 7). The entire type III secretion machine or injectisome is ∼40 nm in width and ∼150 nm in length and is organized in defined substructures (Fig. 1A). The bestcharacterized substructure is the needle complex, which is a multi-ring cylindrical structure embedded in the bacterial envelope with a needle-like extension that projects several nanometers from the bacterial surface. The needle complex is traversed by a narrow channel that serves as the conduit for the proteins that travel this secretion pathway (8). The needle is capped at its end by the tip complex, which is thought to coordinate the activation of the secretion machine upon contact with target cells (9, 10). Because the needle complex can be isolated in a manner suitable for single-particle cryo-electron microscopy, its organization at the quasi-atomic level is known (11-18). Passage of the secreted proteins through the inner membrane requires the export apparatus, which is composed of a group of poly-topic inner membrane proteins located at the center of the needle complex base (19). Finally, several cytoplasmic proteins form a large complex known as the...

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mentioning

confidence: 99%

Visualization and characterization of individual type III protein secretion machines in live bacteria

Zhang

Lara‐Tejero

Bewersdorf

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

103

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“…SPT and SR provide improved resolution down to ~20-50 nm using fluorescent proteins (FPs). 14,15 Moreover, both SPT and SR are single-molecule (SM) methods, allowing us to investigate the heterogeneity in protein behavior and distribution across many different cells. To avoid artifacts from projecting SM localizations onto two dimensions, we have used the double-helix point spread function (DH-PSF) which encodes axial information in each single-molecule image.…”

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confidence: 99%

Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus

Bayas

Wang

Lee

et al. 2017

Preprint

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We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single particle tracking and super-resolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in E. coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Moreover, RNase E cluster positions correlate with the subcellular location of chromosomal loci of two highly transcribed ribosomal RNA genes, suggesting that RNase E's function in ribosomal RNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/228122 doi: bioRxiv preprint first posted online Dec. 3, 2017; In bacteria, the RNA degradosome mediates the majority of messenger RNA (mRNA) turnover and ribosomal RNA (rRNA) and transfer RNA (tRNA) maturation. 1 The RNA degradosome assembles on the C-terminal scaffold region of the RNase E endoribonuclease.2 RNase E in Escherichia coli (E. coli) and RNase Y, the RNase E homolog in Bacillus subtilis (B. subtilis),both associate with the cell membrane through a membrane-binding helix. [3][4][5][6] One proposed rationale behind the observed membrane association is to physically separate transcription within the nucleoid from RNA degradation, thus providing an inherent time delay between transcript synthesis and the onset of transcript decay, avoiding a futile cycle. Caulobacter crescentus (Caulobacter) is an alpha-proteobacterium widely studied as a model system for asymmetric cell division. Unlike E. coli or B. subtilis, Caulobacter contains a pole-tethered chromosome that fills the cytoplasm. 7-9 Surprisingly, Caulobacter RNase E does not have a membrane targeting sequence and is not membrane-associated. 2, 10 Furthermore, diffraction-limited (DL) images of RNase E exhibited a patchy localization throughout the cell, with RNase E directly or indirectly associating with DNA. 10Fluorescently labeled ribosomal proteins S2 and L1, proxies for ribosomes in E. coli and B. subtilis, respectively, were found enriched at the cell poles, spatially excluded from the nucleoid on the hundreds of nanometer scale. 11,12 Similar fluorescence imaging studies in Caulobacter revealed no apparent separation of ribosome...

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“…In Fig. 1 A, <50% of the one-frame displacements are recoverable when M C is >20, even though 20 is a reasonable value for subcellular diffusion in a typical single-molecule microscope (40). For instance, M C ¼ 20 could correspond to a molecule diffusing with a rate of 2.88 mm 2 /s measured with a microscope that has a point-spread function standard deviation of 98 nm, a camera integration time of 50 ms, and a pixel width of 49 nm in the object plane.…”

Section: Resultsmentioning

confidence: 91%

“…During imaging, the cells were given a 1-to 10-ms dose of 406 nm light every~30 s. Movie acquisitions lasted 2-5 min each. Due to the small (~1 mm) cell thickness, a low background noise is maintained even when single-molecule imaging in bacteria cells is done with a wide-field microscopy configuration (40). Thus, this work describes the analysis of wide-field microscopy, though these methods could be readily applied to total internal fluorescence microscopy.…”

Section: Imaging Conditionsmentioning

confidence: 99%

“…Although single-molecule imaging has successfully answered a host of questions in cell biology (40,(42)(43)(44), one current challenge is a limited ability to characterize the dynamics of the fastest molecules-especially when confinement precludes stroboscopic illumination-for instance, freely diffusing proteins in the bacterial cytoplasm (5). We imaged E. coli cells expressing the photoswitchable fluorescent protein mMaple3, which is switched from a green state to a red state upon activation with 406-nm light (37).…”

Section: Cytosolic Mmaple3 Diffusion In E Colimentioning

confidence: 99%

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Resolving Fast, Confined Diffusion in Bacteria with Image Correlation Spectroscopy

Rowland

Tuson

Biteen

2016

Biophysical Journal

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By following single fluorescent molecules in a microscope, single-particle tracking (SPT) can measure diffusion and binding on the nanometer and millisecond scales. Still, although SPT can at its limits characterize the fastest biomolecules as they interact with subcellular environments, this measurement may require advanced illumination techniques such as stroboscopic illumination. Here, we address the challenge of measuring fast subcellular motion by instead analyzing single-molecule data with spatiotemporal image correlation spectroscopy (STICS) with a focus on measurements of confined motion. Our SPT and STICS analysis of simulations of the fast diffusion of confined molecules shows that image blur affects both STICS and SPT, and we find biased diffusion rate measurements for STICS analysis in the limits of fast diffusion and tight confinement due to fitting STICS correlation functions to a Gaussian approximation. However, we determine that with STICS, it is possible to correctly interpret the motion that blurs single-molecule images without advanced illumination techniques or fast cameras. In particular, we present a method to overcome the bias due to image blur by properly estimating the width of the correlation function by directly calculating the correlation function variance instead of using the typical Gaussian fitting procedure. Our simulation results are validated by applying the STICS method to experimental measurements of fast, confined motion: we measure the diffusion of cytosolic mMaple3 in living Escherichia coli cells at 25 frames/s under continuous illumination to illustrate the utility of STICS in an experimental parameter regime for which in-frame motion prevents SPT and tight confinement of fast diffusion precludes stroboscopic illumination. Overall, our application of STICS to freely diffusing cytosolic protein in small cells extends the utility of singlemolecule experiments to the regime of fast confined diffusion without requiring advanced microscopy techniques.

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Unveiling the Inner Workings of Live Bacteria Using Super-Resolution Microscopy

Cited by 63 publications

References 274 publications

Visualization and characterization of individual type III protein secretion machines in live bacteria

Visualization and characterization of individual type III protein secretion machines in live bacteria

Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus

Resolving Fast, Confined Diffusion in Bacteria with Image Correlation Spectroscopy

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