Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes
- Albert Einstein College of Medicine, United States
- Salk Institute for Biological Studies, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Massachusetts Institute of Technology, United States
Figures
Figure 1 with 2 supplements
The diffusion properties of β-actin mRNA molecules around focal adhesions are slower and more corralled.
(A) Overlay of a single frame from Video 1 of β-actin mRNA labeled with MS2-GFP and focal adhesions labeled with paxillin-mCherry. (B) The cumulative distribution function of all single molecule …
Figure 1—figure supplement 1
Tracking of individual β-actin mRNA molecules at adhesion compartments.
(A) The schematic depicts the side view of a fibroblast leading edge with focal adhesions labeled with paxillin-mCherry (red stars) and β-actin mRNA labeled with GFP tagged MS2 capsid proteins on 24 …
Figure 1—figure supplement 2
Cumulative distribution function analysis of β-actin mRNA localized to focal adhesions.
(A) β-actin mRNA localized to focal adhesions: The cumulative distribution function of all single molecule displacements for β-actin mRNA localized to focal adhesions is fit to a one component fit: . An apparent diffusion coefficient is obtained from the fit. (B) Cytoplasmic β-actin mRNA not localized to focal adhesions: The cumulative distribution function of all cytoplasmic β-actin mRNA not localized to focal adhesions is fit to a one component fit, resulting in an apparent diffusion coefficient . (C) β-actin mRNA localized to focal adhesions: A two-component fit of (dashed curve) fits the experimentally obtained CDFs (solid curve) much better. A=0.51 is determined from the two-component fit (51% slow component and 49% fast component, dash-dotted curves). (D) Cytoplasmic β-actin mRNA not localized to focal adhesions: A two-component fit (dotted curve) fits the experimentally obtained CDFs (solid curve) much better. A=0.42 is determined from the two-component fit (42% slow component and 58% fast component, dash-dotted curves).
Figure 2 with 2 supplements
β-actin mRNA trajectories are faster and less-corralled after ribosome dissociation via puromycin treatment.
(A) Treatment with puromycin dissociates ribosomes from mRNA and shifts β-actin mRNA trajectories significantly towards faster diffusion (arrow). (B) Mean square displacement curves also indicate a …
Figure 2—figure supplement 1
β-actin mRNA trajectories are faster and less-corralled after addition of hippuristanol.
(A) Treatment with hippuristanol inhibits translational initiation and shifts β-actin mRNA trajectories significantly towards faster diffusion (arrow). The cumulative distribution function of all …
Figure 2—figure supplement 2
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 …
Figure 3 with 3 supplements
Cellular maps of areas with increased β-actin translation.
(A) Super-resolution PALM density map composed of all 63,940 β-actin mRNA localizations from 6,235 trajectories. The PALM density plot was generated with VISP (El Beheiry and Dahan, 2013) from a …
Figure 3—figure supplement 1
Tracking of individual ribosome molecules with β-actin mRNA tethered to focal adhesions.
(A) The schematic depicts the side view of a fibroblast leading edge with focal adhesions integrating the mRNA tethering construct (vinculin fused to the MS2 capsid protein). All β-actin mRNA tagged …
Figure 3—figure supplement 2
Diffusion characteristics of β-actin mRNA co-moving with ribosomes and without ribosomes.
(A) β-actin mRNAs that co-move with ribosomes exhibitslower apparent diffusion coefficients. (B) The mean square displacement curve of co-moving β-actin mRNA trajectories displays a shift towards …
Figure 3—figure supplement 3
mRNA/ribosome colocalization statistics in the absence and presence of puromycin.
We generated a matrix of the distances between all detected mRNA particles and all detected ribosome particles, and computed the corresponding histogram . We then normalized the distance …
Figure 4 with 3 supplements
Multi-color live cell imaging and simultaneous tracking of β-actin mRNA and ribosomes at focal adhesions.
(A) The image from a single frame from Video 10 depicts simultaneous tracking of β-actin mRNA (green) and ribosomes (magenta) in live mouse embryonic fibroblasts with focal adhesions labeled with …
Figure 4—figure supplement 1
HMM-Bayesian analysis of co-moving mRNAs.
(A) mRNAs that co-move with ribosomes are color-coded according to their diffusive state, as determined by HMM-Bayesian analysis (co-moving ribosomes not shown). The fast state D1 (individual states …
Figure 4—figure supplement 2
HMM-Bayesian analysis of co-moving ribosomes.
(A) Ribosomes that co-move with β-actin mRNA are color-coded according to their diffusive state, as determined by HMM-Bayesian analysis. The fast state D1 (individual trajectories in shades of …
Figure 4—figure supplement 3
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 …
Videos
Video 1
Mouse embryonic fibroblasts (MEFs) from the MBS mouse with 24 MS2 stem-loop binding sites in the β-actin 3'UTR are labeled with tdMCP-GFP.
Focal adhesions are labeled with paxillin-mCherry. Live cell mRNA imaging was performed with TIRF excitation for 500 streaming frames at a frame exposure time of 35 ms. The movie is played at 30 …
Video 2
The MBS MEF in Video 1 cropped and enhanced to highlight several β-actin mRNA molecules that persist at adhesions.
The scale bar is 5 µm and the movie is played at 30 fps.
Video 3
β-actin mRNA is visualized 60 min after addition of puromycin.
Compared to steady-state movement, mRNA without ribosomes is much faster and homogenous throughout the cytoplasm.
Video 4
β-actin mRNA is visualized 20 min after addition of hippuristanol.
Compared to the control (left), the movement of mRNA without ribosomes (right, same cell 20 min later) is much faster and homogeneous throughout the cytoplasm.
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).
405-nm activation energy is controlled manually to limit the number of fluorescent molecules, enabling single particle tracking of ribosomes. Focal adhesions are labeled with vinculin-GFP. Ribosomes …
Video 6
Ribosomes visualized with L10A-PATagRFP after puromycin addition.
Focal adhesions are labeled with vinculin-GFP. The stack was acquired at 35 ms per frame and played at 30 fps with a 5 µm scale bar.
Video 7
Ribosomes visualized in cells with β-actin mRNA tethered to focal adhesions.
Non-fluorescent β-actin mRNA with MS2 stem-loops in the 3'UTR becomes tethered to vinculin that is tagged with tdMCP-GFP. Ribosomes can be seen preferentially immobilizing at larger focal adhesions …
Video 8
Ribosomes in cells with β-actin mRNA tethered to adhesions after the addition of puromycin.
Compared to the ribosomes in Video 7, it is easy to see that puromycin dissociates ribosomes from mRNA tethered at focal adhesions. The stack was acquired at 35 ms per frame and played at 30 fps …
Video 9
β-actin mRNA and ribosomes are simultaneously visualized and tracked after puromycin addition.
The stack was acquired at 35 ms per frame and played at 30 fps with a 10 µm scale bar.
Video 10
β-actin mRNA and ribosomes are simultaneously visualized and tracked after the cell’s focal adhesions were identified with paxillin-mCherry and bleached.
The stack was acquired at 35 ms per frame and played at 30 fps with a 5 µm scale bar.
