Quantification of mRNA translation in live cells using single-molecule imaging

Khuperkar, Deepak; Hoek, Tim A.; Sonneveld, Stijn; Verhagen, Bram M.P.; Boersma, Sanne; Tanenbaum, Marvin E.

doi:10.1038/s41596-019-0284-x

Cited by 14 publications

(15 citation statements)

References 39 publications

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“…Although a mathematical estimation of a HILO illumination PSF is not available, it is well understood for a confocal microscope . Live-cell dual-color SPT is commonly performed on both HILO ,,,,, and spinning-disk confocal microscopes, ,− whereas confocal microscopes are more commonly used for bulk fluorescence detection requiring more homogeneous illumination across the FOV . We decided to adopt the Gaussian approximation of a 3D PSF on the confocal microscope: G σ x y , σ z ( x , y , z ) = e − x 2 + y 2 / 2 σ x y 2 − z 2 / 2 σ z 2 σ x y = 2 k em NA σ z = 2 6 n k em NA 2 …”

Section: Methodsmentioning

confidence: 99%

“…44 Live-cell dual-color SPT is commonly performed on both HILO 8,9,11,13,45,46 and spinning-disk confocal microscopes, 10,47−51 whereas confocal microscopes are more commonly used for bulk fluorescence detection requiring more homogeneous illumination across the FOV. 50 We decided to adopt the Gaussian approximation of a 3D PSF on the confocal microscope: 44…”

Section: Live-cell Dual-color Sptmentioning

confidence: 99%

See 1 more Smart Citation

Critical Assessment of Condensate Boundaries in Dual-Color Single Particle Tracking

Gao,

Walter

2023

J. Phys. Chem. B

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Biomolecular condensates are membraneless cellular compartments generated by phase separation that regulate a broad variety of cellular functions by enriching some biomolecules while excluding others. Live-cell single particle tracking of individual fluorophore-labeled condensate components has provided insights into a condensate's mesoscopic organization and biological functions, such as revealing the recruitment, translation, and decay of RNAs within ribonucleoprotein (RNP) granules. Specifically, during dual-color tracking, one imaging channel provides a time series of individual biomolecule locations, while the other channel monitors the location of the condensate relative to these molecules. Therefore, an accurate assessment of a condensate's boundary is critical for combined live-cell single particle-condensate tracking. Despite its importance, a quantitative benchmarking and objective comparison of the various available boundary detection methods is missing due to the lack of an absolute ground truth for condensate images. Here, we use synthetic data of defined ground truth to generate noise-overlaid images of condensates with realistic phase separation parameters to benchmark the most commonly used methods for condensate boundary detection, including an emerging machine-learning method. We find that it is critical to carefully choose an optimal boundary detection method for a given dataset to obtain accurate measurements of single particle-condensate interactions. The criteria proposed in this study to guide the selection of an optimal boundary detection method can be broadly applied to imaging-based studies of condensates.

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Section: Methodsmentioning

confidence: 99%

Section: Live-cell Dual-color Sptmentioning

confidence: 99%

Critical Assessment of Condensate Boundaries in Dual-Color Single Particle Tracking

Gao,

Walter

2023

J. Phys. Chem. B

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“…In the future, these approaches promise to aid structure-based drug design not only against SARS-CoV2 but also other emerging RNA viruses [ 201 ]. It is finally important to note that other novel and innovative techniques developed to study translation such as single-molecule approaches [ 203 ] or ribosome profiling by deep sequencing [ 204 ] are revealing the complexity of vRNA interaction with host ribosomes. For instance, single-molecule studies of IRES-mediated translation have revealed insights into the dynamics of HCV and CrPV IRES interaction with ribosomes and clarified decades of biochemical research, providing an outline of the conformational and compositional trajectory of the ribosome during initiation [ 151 ].…”

Section: Technology Guiding Discovery; the Case Of Rbp-vrna Interactimentioning

confidence: 99%

Roadblocks and fast tracks: How RNA binding proteins affect the viral RNA journey in the cell

Girardi¹,

Pfeffer²,

Baumert³

et al. 2021

Seminars in Cell & Developmental Biology

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As obligate intracellular parasites with limited coding capacity, RNA viruses rely on host cells to complete their multiplication cycle. Viral RNAs (vRNAs) are central to infection. They carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. Regardless of its origin or tropism, vRNA has by definition evolved in the presence of host RNA Binding Proteins (RBPs), which resulted in intricate and complicated interactions with these factors. While on one hand some host RBPs recognize vRNA as non-self and mobilize host antiviral defenses, vRNA must also co-opt other host RBPs to promote viral infection. Focusing on pathogenic RNA viruses, we will review important scenarios of RBP-vRNA interactions during which host RBPs recognize, modify or degrade vRNAs. We will then focus on how vRNA hijacks the largest ribonucleoprotein complex (RNP) in the cell, the ribosome, to selectively promote the synthesis of its proteins. We will finally reflect on how novel technologies are helping in deepening our understanding of vRNA-host RBPs interactions, which can be ultimately leveraged to combat everlasting viral threats.

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“…Messenger RNA (mRNA) molecules interact with RNA-binding proteins throughout their lifespan to carry genetic information and provide precise spatiotemporal regulation within cells. Live-cell single-molecule imaging techniques have enabled in-depth characterization of dynamics for mRNA-processing steps, including transcription, translation, splicing, export, degradation, and interactions with ribonucleoprotein (RNP) granules ( Bertrand et al., 1998 ; Buxbaum et al., 2014 ; Czaplinski, 2017 ; Horvathova et al., 2017 ; Katz et al., 2016 ; Khuperkar et al., 2020 ; Larson et al., 2011 ; Mateju et al., 2020 ; Moon et al., 2019 ; Mor et al., 2010 ; Sato et al., 2020 ; Vargas et al., 2011 ; Vítor et al., 2019 ; Wan et al., 2021 ; Wang et al., 2016 ; Wilbertz et al., 2019 ; Wu et al., 2016 ; Yan et al., 2016 ). However, continuous imaging of single mRNAs has numerous challenges coupled to low imaging sensitivity that is exacerbated by rapid photobleaching ( Pichon et al., 2018 ; Tutucci et al., 2018a ).…”

Section: Introductionmentioning

confidence: 99%