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Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes.

Katz ZB, English BP, Lionnet T, Yoon YJ, Monnier N, Ovryn B, Bathe M, Singer RH - Elife (2016)

Bottom Line: A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors.These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower.This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, United States.

ABSTRACT
Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

No MeSH data available.


Related in: MedlinePlus

HMM-Bayesian analysis of non-co-moving mRNAs.(A) Non-co-moving mRNA trajectories are color-coded according to their diffusive state, as determined by HMM-Bayesian analysis (7931 visits to the fast state D1 are displayed in shades of blue, and 2623 visits to the slow state D2 are displayed in shades of green). Focal adhesions are depicted in white. Scale bar: 5 μm. (B) State-duration histogram for all fast states D1 as determined from the trajectories displayed in (A) Insert: Histogram of only internal state durations of D1 (excluding states at start and end of each track). (C) Corresponding state-duration histogram of all slow state (D2) durations as determined from the trajectories displayed in (A). The insert depicts the internal state durations (excluding states at start and end of each track) for D2.DOI:http://dx.doi.org/10.7554/eLife.10415.023
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fig4s3: HMM-Bayesian analysis of non-co-moving mRNAs.(A) Non-co-moving mRNA trajectories are color-coded according to their diffusive state, as determined by HMM-Bayesian analysis (7931 visits to the fast state D1 are displayed in shades of blue, and 2623 visits to the slow state D2 are displayed in shades of green). Focal adhesions are depicted in white. Scale bar: 5 μm. (B) State-duration histogram for all fast states D1 as determined from the trajectories displayed in (A) Insert: Histogram of only internal state durations of D1 (excluding states at start and end of each track). (C) Corresponding state-duration histogram of all slow state (D2) durations as determined from the trajectories displayed in (A). The insert depicts the internal state durations (excluding states at start and end of each track) for D2.DOI:http://dx.doi.org/10.7554/eLife.10415.023

Mentions: Simultaneous TIRF excitation of GFP labeled β-actin mRNA and PATagRFP labeled ribosomes (fluorescently activated by a 405 nm laser) enabled dual-color single molecule tracking using two synchronized EMCCD cameras (Figure 3A–B). mRNA trajectories were spatially and temporally correlated with ribosome trajectories to identify a co-moving population for diffusion analysis (see Methods and Figure 3—figure supplement 3). Whereas all endogenous β-actin mRNA molecules in the cell are detected using our tdMCP-GFP labeled reporter system, only a few ribosomes (on average 10 molecules/frame) can be tracked at each given time using the sptPALM technique. Consistent with this experimental limit, only a subset of β-actin mRNA were identified as co-moving with ribosomes (Figure 3C–D; Figure 4—figure supplement 3A). We hypothesized those mRNAs that co-moved with ribosomes were likely loaded with multiple ribosomes and may exhibit slower movement. Consistent with this, diffusion analysis showed a shift towards slower movement in the co-moving mRNA population (Figure 3D; Figure 3—figure supplement 2B). mRNAs found to co-move with ribosomes shifted towards slower (Figure 3—figure supplement 2A, Figure 4B) and more corralled movement (Figure 3—figure supplement 2B), an indication that these mRNAs were in polysomes and were more likely to dwell locally. Likewise, ribosomes found to co-move with β-actin mRNA trended slower and more corralled, reminiscent of mRNA localized to focal adhesions (Figure 2—figure supplement 2E compared to Figure 1C). Together, the analysis of the mobility and co-movement of mRNA and ribosomes provide a detailed picture of the translational landscape inside live cells, capable of identifying translational hotspots (Figure 3D,G).10.7554/eLife.10415.020Figure 4.Multi-color live cell imaging and simultaneous tracking of β-actin mRNA and ribosomes at focal adhesions.


Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes.

Katz ZB, English BP, Lionnet T, Yoon YJ, Monnier N, Ovryn B, Bathe M, Singer RH - Elife (2016)

HMM-Bayesian analysis of non-co-moving mRNAs.(A) Non-co-moving mRNA trajectories are color-coded according to their diffusive state, as determined by HMM-Bayesian analysis (7931 visits to the fast state D1 are displayed in shades of blue, and 2623 visits to the slow state D2 are displayed in shades of green). Focal adhesions are depicted in white. Scale bar: 5 μm. (B) State-duration histogram for all fast states D1 as determined from the trajectories displayed in (A) Insert: Histogram of only internal state durations of D1 (excluding states at start and end of each track). (C) Corresponding state-duration histogram of all slow state (D2) durations as determined from the trajectories displayed in (A). The insert depicts the internal state durations (excluding states at start and end of each track) for D2.DOI:http://dx.doi.org/10.7554/eLife.10415.023
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Related In: Results  -  Collection

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fig4s3: HMM-Bayesian analysis of non-co-moving mRNAs.(A) Non-co-moving mRNA trajectories are color-coded according to their diffusive state, as determined by HMM-Bayesian analysis (7931 visits to the fast state D1 are displayed in shades of blue, and 2623 visits to the slow state D2 are displayed in shades of green). Focal adhesions are depicted in white. Scale bar: 5 μm. (B) State-duration histogram for all fast states D1 as determined from the trajectories displayed in (A) Insert: Histogram of only internal state durations of D1 (excluding states at start and end of each track). (C) Corresponding state-duration histogram of all slow state (D2) durations as determined from the trajectories displayed in (A). The insert depicts the internal state durations (excluding states at start and end of each track) for D2.DOI:http://dx.doi.org/10.7554/eLife.10415.023
Mentions: Simultaneous TIRF excitation of GFP labeled β-actin mRNA and PATagRFP labeled ribosomes (fluorescently activated by a 405 nm laser) enabled dual-color single molecule tracking using two synchronized EMCCD cameras (Figure 3A–B). mRNA trajectories were spatially and temporally correlated with ribosome trajectories to identify a co-moving population for diffusion analysis (see Methods and Figure 3—figure supplement 3). Whereas all endogenous β-actin mRNA molecules in the cell are detected using our tdMCP-GFP labeled reporter system, only a few ribosomes (on average 10 molecules/frame) can be tracked at each given time using the sptPALM technique. Consistent with this experimental limit, only a subset of β-actin mRNA were identified as co-moving with ribosomes (Figure 3C–D; Figure 4—figure supplement 3A). We hypothesized those mRNAs that co-moved with ribosomes were likely loaded with multiple ribosomes and may exhibit slower movement. Consistent with this, diffusion analysis showed a shift towards slower movement in the co-moving mRNA population (Figure 3D; Figure 3—figure supplement 2B). mRNAs found to co-move with ribosomes shifted towards slower (Figure 3—figure supplement 2A, Figure 4B) and more corralled movement (Figure 3—figure supplement 2B), an indication that these mRNAs were in polysomes and were more likely to dwell locally. Likewise, ribosomes found to co-move with β-actin mRNA trended slower and more corralled, reminiscent of mRNA localized to focal adhesions (Figure 2—figure supplement 2E compared to Figure 1C). Together, the analysis of the mobility and co-movement of mRNA and ribosomes provide a detailed picture of the translational landscape inside live cells, capable of identifying translational hotspots (Figure 3D,G).10.7554/eLife.10415.020Figure 4.Multi-color live cell imaging and simultaneous tracking of β-actin mRNA and ribosomes at focal adhesions.

Bottom Line: A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors.These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower.This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, United States.

ABSTRACT
Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

No MeSH data available.


Related in: MedlinePlus