Conducting Multiple Imaging Modes with One Fluorescence Microscope

Park, Seong-Jin; Zhang, Jiacheng; Reyer, Matthew A.; Zareba, Joanna; Troy, Andrew A; Fei, Jingyi

doi:10.3791/58320

Cited by 7 publications

(10 citation statements)

References 20 publications

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“…Imaging was performed on a custom built microscopy setup as previously described ( Park et al, 2018b ). Briefly, an inverted optical microscope (Nikon Ti-E with 100× NA 1.49 CFI HP TIRF oil immersion objective) was fiber-coupled with a 647 nm laser (Cobolt 06–01), a 561 nm laser (Coherent Obis LS) and a 405 nm laser (Crystalaser).…”

Section: Methodsmentioning

confidence: 99%

Dynamic interactions between the RNA chaperone Hfq, small regulatory RNAs, and mRNAs in live bacterial cells

Park

Prévost

Heideman

et al. 2021

eLife

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RNA-binding proteins play myriad roles in regulating RNAs and RNA-mediated functions. In bacteria, the RNA chaperone Hfq is an important post-transcriptional gene regulator. Using live-cell super-resolution imaging, we can distinguish Hfq binding to different sizes of cellular RNAs. We demonstrate that under normal growth conditions, Hfq exhibits widespread mRNA-binding activity, with the distal face of Hfq contributing mostly to the mRNA binding in vivo. In addition, sRNAs can either co-occupy Hfq with the mRNA as a ternary complex, or displace the mRNA from Hfq in a binding face-dependent manner, suggesting mechanisms through which sRNAs rapidly access Hfq to induce sRNA-mediated gene regulation. Finally, our data suggest that binding of Hfq to certain mRNAs through its distal face can recruit RNase E to promote turnover of these mRNAs in an sRNA-independent manner, and such regulatory function of Hfq can be decoyed by sRNA competitors that bind strongly at the distal face.

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Section: Methodsmentioning

confidence: 99%

Dynamic interactions between the RNA chaperone Hfq, small regulatory RNAs, and mRNAs in live bacterial cells

Park

Prévost

Heideman

et al. 2021

eLife

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“…Imaging buffer was composed of 10 mM NaCl, 50 mM Tris (pH 8), 10% glucose, 50 U/mL glucose oxidase (Sigma Aldrich G2133-10KU), 404 U/mL catalase (EMD Millipore 219001) and 20mM cysteamine (Sigma Aldrich 30070-10G). 3D Image reconstruction and visualization was conducted as previously described (Park et al, 2018).…”

Section: Super-resolution Microscopymentioning

confidence: 99%

K29-Linked Ubiquitin Signaling Regulates Proteotoxic Stress Response and Cell Cycle

Yu¹,

Zheng

Erramilli³

et al. 2020

Preprint

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Protein ubiquitination, one of the most common posttranslational modifications, is involved in numerous cellular processes. It shows remarkable topological and functional diversity, a phenomenon known as the ubiquitin code, through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understanding the physiology of the cell. Among the eight possible linkages, K29-linked polyubiquitin is a relatively abundant type of polyubiquitin in both human and yeast cells (Tsuchiya et al., 2018). Despite previous studies, the lack of specific binders as tools to detect K29-linked polyubiquitin has prevented further understanding of its function. In this study, we screened and characterized a synthetic antigen-binding fragment (named sAB-K29) that can specifically recognize K29-linked polyubiquitin. We determined the crystal structure of sAB-K29 bound to K29-linked diubiquitin (diUb), which revealed the molecular basis of sAB-K29 specificity. Using sAB-K29 as a tool, we identified the involvement of K29-linked ubiquitination in RNA processing, the proteotoxic stress response and cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody at the telophase of mitosis and that downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

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“…1.2-1.4 μL of the sample were placed on the glass slide bottom of the chamber, with a 1% agarose gel pad placed on top to lay the cells flat. Imaging was performed on a custom inverted microscope (Nikon Ti-E with 100x NA 1.49 CFI HP TIRF oil immersion objective) ( 39 ). Multicolor Z-stack images were taken with 0.130 μm step size and 11 slices for each color.…”

Section: Methodsmentioning

confidence: 99%

“…SMLM imaging was conducted using the same microscope as described above with super-resolution modality ( 39 ). Fixed cells were immobilized on the 8-well chambered glass coverslip (Cellvis C8-11.5H-N) using poly-L-lysine (Sigma-Aldrich P8920), and imaged in imaging buffer (50 mM Tris-HCl, 10% glucose,1% 2-Mercapgtoethanol (Sigma-Aldrich M6250), 50 U/mL glucose oxidase (Sigma Aldrich G2133-10KU), 404 U/mL catalase (EMD Millipore 219001) in 2X SSC, pH = 8.0).…”

Section: Methodsmentioning

confidence: 99%

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Kinetic model of small RNA-mediated regulation suggests that a small RNA can regulate co-transcriptionally

Chennakesavalu²,

Em³

et al. 2020

Preprint

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Small RNAs (sRNAs) are important regulators of gene expression in bacteria, particularly during stress responses. Many genetically and biochemically well characterized sRNAs regulate gene expression post-transcriptionally, by affecting translation and degradation of the target mRNA after they bind to their targets through base pairing. However, how regulation at each of these levels quantitatively contributes to the overall efficacy of sRNA-mediated regulation is not well understood. Here we present a general approach combining imaging and mathematical modeling to determine kinetic parameters at different levels of sRNA-mediated gene regulation. Unexpectedly, our data reveal that certain previously characterized sRNAs are able to regulate some targets co-transcriptionally, rather strictly post-transcriptionally, and suggest that sRNA-mediated regulation can occur early in the mRNA's lifetime, perhaps as soon as the sRNA binding site is transcribed. In addition, our data suggest several important kinetic steps that may determine the efficiency and differential regulation of multiple mRNA targets by an sRNA. Particularly, binding of sRNA to the target mRNA is likely the rate-limiting step and may dictate the regulation hierarchy observed within an sRNA regulon.

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Conducting Multiple Imaging Modes with One Fluorescence Microscope

Cited by 7 publications

References 20 publications

Dynamic interactions between the RNA chaperone Hfq, small regulatory RNAs, and mRNAs in live bacterial cells

Dynamic interactions between the RNA chaperone Hfq, small regulatory RNAs, and mRNAs in live bacterial cells

K29-Linked Ubiquitin Signaling Regulates Proteotoxic Stress Response and Cell Cycle

Kinetic model of small RNA-mediated regulation suggests that a small RNA can regulate co-transcriptionally

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