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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

Cumulative distribution function analysis of ribosome diffusion.(A) The cumulative distribution function of ribosomes for cells in steady state (solid curve) is best fit by two-components (dashed curve) composed of a slow (56%) and a fast (44%) diffusion component (dash-dotted curves). A one-component single-exponential fit (dotted curve) does not adequately fit the experimentally obtained CDFs. (B) The cumulative distribution function of ribosomes after addition of puromycin is best fit by a two-component fit with a shift towards the faster component (73% fast). (C) The cumulative distribution function of ribosomes with β-actin mRNA tethered to focal adhesions (see Figure 3—figure supplement 1) is best fit by a two-component fit with a shift towards the slower component (63% slow). (D) The cumulative distribution function of ribosomes co-moving with β-actin mRNA (green curve) is best fit by a two-component fit (62% slow). Ribosomes that were not co-moving with ribosomes are also best fit by a two-component fit (in purple), but the ratio shifts towards the faster diffusion component (54% slow). (E) MSD curves of co-moving ribosomes (in green) depict increased corralling and an exploration area of 0.06 μm2. MSD curves of non-co-moving ribosomes depict faster diffusion behavior and are less corralled (purple curve).DOI:http://dx.doi.org/10.7554/eLife.10415.012
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fig2s2: Cumulative distribution function analysis of ribosome diffusion.(A) The cumulative distribution function of ribosomes for cells in steady state (solid curve) is best fit by two-components (dashed curve) composed of a slow (56%) and a fast (44%) diffusion component (dash-dotted curves). A one-component single-exponential fit (dotted curve) does not adequately fit the experimentally obtained CDFs. (B) The cumulative distribution function of ribosomes after addition of puromycin is best fit by a two-component fit with a shift towards the faster component (73% fast). (C) The cumulative distribution function of ribosomes with β-actin mRNA tethered to focal adhesions (see Figure 3—figure supplement 1) is best fit by a two-component fit with a shift towards the slower component (63% slow). (D) The cumulative distribution function of ribosomes co-moving with β-actin mRNA (green curve) is best fit by a two-component fit (62% slow). Ribosomes that were not co-moving with ribosomes are also best fit by a two-component fit (in purple), but the ratio shifts towards the faster diffusion component (54% slow). (E) MSD curves of co-moving ribosomes (in green) depict increased corralling and an exploration area of 0.06 μm2. MSD curves of non-co-moving ribosomes depict faster diffusion behavior and are less corralled (purple curve).DOI:http://dx.doi.org/10.7554/eLife.10415.012

Mentions: To image single ribosomes, the 60S large subunit protein L10A was labeled with PATagRFP for single particle tracking using photo-activated localization microscopy (sptPALM). Stable expression of fluorescently labeled L10A has been shown to label ~40% of ribosomes (Wu et al., 2015). Ribosomes could be tracked for an average lifetime of ~200 ms (6.2 frames) after applying 405 nm activation energy. Since a small amount of activation was used in order to track single ribosome molecules, only a small fraction of total ribosomes were tracked. Ribosomes exhibited heterogeneity in diffusive motion, much as with β-actin mRNA (Video 5). Ribosome movement in cells at steady-state is best fit with the same two-component fit as mRNA: with a linear combination of a fast apparent diffusion coefficient of 0.4 μm2/s, and a slower one with a coefficient of 0.1 μm2/s. A two-component fit of experimentally obtained CDFs of ribosome trajectories reveals that ribosomes collectively show a shift towards a slower diffusion component as compared to β-actin mRNA (56% vs. 42%, respectively) and suggests most ribosomes actively engage in translation throughout the cytoplasm (compare Figure 2A vs. 2F). With the addition of puromycin, a significant increase in ribosome diffusion was observed just as with β-actin mRNA (Figure 2F), and the population of faster ribosomes increased to 73% (Figure 2—figure supplement 2B, Video 6).Video 5.Ribosomes are visualized with photo-activated localization microscopy (PALM) in fibroblasts stably expressing the L10A 60s large subunit tagged with photo-activated TagRFP (PATagRFP).


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)

Cumulative distribution function analysis of ribosome diffusion.(A) The cumulative distribution function of ribosomes for cells in steady state (solid curve) is best fit by two-components (dashed curve) composed of a slow (56%) and a fast (44%) diffusion component (dash-dotted curves). A one-component single-exponential fit (dotted curve) does not adequately fit the experimentally obtained CDFs. (B) The cumulative distribution function of ribosomes after addition of puromycin is best fit by a two-component fit with a shift towards the faster component (73% fast). (C) The cumulative distribution function of ribosomes with β-actin mRNA tethered to focal adhesions (see Figure 3—figure supplement 1) is best fit by a two-component fit with a shift towards the slower component (63% slow). (D) The cumulative distribution function of ribosomes co-moving with β-actin mRNA (green curve) is best fit by a two-component fit (62% slow). Ribosomes that were not co-moving with ribosomes are also best fit by a two-component fit (in purple), but the ratio shifts towards the faster diffusion component (54% slow). (E) MSD curves of co-moving ribosomes (in green) depict increased corralling and an exploration area of 0.06 μm2. MSD curves of non-co-moving ribosomes depict faster diffusion behavior and are less corralled (purple curve).DOI:http://dx.doi.org/10.7554/eLife.10415.012
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Related In: Results  -  Collection

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fig2s2: Cumulative distribution function analysis of ribosome diffusion.(A) The cumulative distribution function of ribosomes for cells in steady state (solid curve) is best fit by two-components (dashed curve) composed of a slow (56%) and a fast (44%) diffusion component (dash-dotted curves). A one-component single-exponential fit (dotted curve) does not adequately fit the experimentally obtained CDFs. (B) The cumulative distribution function of ribosomes after addition of puromycin is best fit by a two-component fit with a shift towards the faster component (73% fast). (C) The cumulative distribution function of ribosomes with β-actin mRNA tethered to focal adhesions (see Figure 3—figure supplement 1) is best fit by a two-component fit with a shift towards the slower component (63% slow). (D) The cumulative distribution function of ribosomes co-moving with β-actin mRNA (green curve) is best fit by a two-component fit (62% slow). Ribosomes that were not co-moving with ribosomes are also best fit by a two-component fit (in purple), but the ratio shifts towards the faster diffusion component (54% slow). (E) MSD curves of co-moving ribosomes (in green) depict increased corralling and an exploration area of 0.06 μm2. MSD curves of non-co-moving ribosomes depict faster diffusion behavior and are less corralled (purple curve).DOI:http://dx.doi.org/10.7554/eLife.10415.012
Mentions: To image single ribosomes, the 60S large subunit protein L10A was labeled with PATagRFP for single particle tracking using photo-activated localization microscopy (sptPALM). Stable expression of fluorescently labeled L10A has been shown to label ~40% of ribosomes (Wu et al., 2015). Ribosomes could be tracked for an average lifetime of ~200 ms (6.2 frames) after applying 405 nm activation energy. Since a small amount of activation was used in order to track single ribosome molecules, only a small fraction of total ribosomes were tracked. Ribosomes exhibited heterogeneity in diffusive motion, much as with β-actin mRNA (Video 5). Ribosome movement in cells at steady-state is best fit with the same two-component fit as mRNA: with a linear combination of a fast apparent diffusion coefficient of 0.4 μm2/s, and a slower one with a coefficient of 0.1 μm2/s. A two-component fit of experimentally obtained CDFs of ribosome trajectories reveals that ribosomes collectively show a shift towards a slower diffusion component as compared to β-actin mRNA (56% vs. 42%, respectively) and suggests most ribosomes actively engage in translation throughout the cytoplasm (compare Figure 2A vs. 2F). With the addition of puromycin, a significant increase in ribosome diffusion was observed just as with β-actin mRNA (Figure 2F), and the population of faster ribosomes increased to 73% (Figure 2—figure supplement 2B, Video 6).Video 5.Ribosomes are visualized with photo-activated localization microscopy (PALM) in fibroblasts stably expressing the L10A 60s large subunit tagged with photo-activated TagRFP (PATagRFP).

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