Figure 5.
Figure 5. 3D distribution of β-actin mRNA within the nucleus of living cells. (A) 3D projection of a nucleus showing β-actin mRNA carrying a 24× MS2 stem-loop cassette labeled with eYFP-MS2 coat protein (green), NPCs labeled with POM121-tdTomato (red), and heterochromatin DNA labeled with the vital dye Vybrant DyeCycle Violet (blue). The view is a time projection (β-actin mRNA, 5 s; NPC, 0.3 s; DNA, 1 s) rotated 10° around the y axis. The less intense clouds of signal represent mobile mRNA over this time span and spots represent immobile mRNAs (Video 2). The white box indicates the region of interest of the nucleus shown in B–D. (B) Zoomed in rotational view of β-actin mRNA (green) and heterochromatin (blue). Numbers below the images indicate the rotation angle. Arrowheads point at two mRNA clusters that appear to colocalize with heterochromatin. Whereas the upper cluster sits on top of the chromatin (40°–120°), the lower cluster is located within the heterochromatin. Projection was done without interpolation resulting in line patterns close to the 90° view (e.g., see 60°) and are therefore not displayed. The full 360° rotation is shown in Video 3. (C) Rotational view of β-actin mRNA and NPCs analogue to B. At all angles the β-actin mRNA partially overlaps with an NPC (bottom arrow). The full 360° rotation is shown in Video 4. B and C are static projections of the first frame of the videos. (D) Time course of β-actin mRNA and NPCs from three rotational perspectives. The 40° and 160° views for the 0-s time point are shown in C with blue frames. The mRNA–NPC interaction is most visible at an angle of 0° and invisible from the opposite side of the cell (160°) and ultimately nonproductive (mRNA arrives and leaves NPC on the same side). The last three views are from the first slices of the stack at an angle of 0° at later time points of the same video, illustrating repeated activity of a specific nuclear pore over time. Cyan bars under images indicate identical time groups. (E) Nuclear pore 3D projection from A. Arrowheads point to nuclear pores at the top/plane +4 (a) and bottom/plane −4 (b) of the nucleus. (F) 3D projection of the RNA channel from A. Arrowheads point to examples of a mobile β-actin mRNA resulting in a blurred projection (c) and a stationary β-actin mRNA resulting in a sharp projection (d). (G) 3D projection of the DNA channel from A. (H) Intensity frequency histogram of the cell (n = 1) in F showing three distributions: to the left, background resulting from rotation of the deconvolved image (arrowhead e); a lower intensity peak resulting from mobile mRNAs in the time projection (arrowhead f); and, to the right, a higher intensity peak resulting from stationary mRNAs in the time projection (arrowhead g). (I) Bar graph showing the level of β-actin mRNA localized to heterochromatin and heterochromatin periphery relative to nucleoplasm. On average a slight reduction of mRNA occupation in the heterochromatin was found, but was not statistically relevant (two-sided test). *, significant within 2σ confidence interval on the standard deviation; ns, not significant. Images were processed as described in Materials and methods and contrast was adjusted for visibility.

3D distribution of β-actin mRNA within the nucleus of living cells. (A) 3D projection of a nucleus showing β-actin mRNA carrying a 24× MS2 stem-loop cassette labeled with eYFP-MS2 coat protein (green), NPCs labeled with POM121-tdTomato (red), and heterochromatin DNA labeled with the vital dye Vybrant DyeCycle Violet (blue). The view is a time projection (β-actin mRNA, 5 s; NPC, 0.3 s; DNA, 1 s) rotated 10° around the y axis. The less intense clouds of signal represent mobile mRNA over this time span and spots represent immobile mRNAs (Video 2). The white box indicates the region of interest of the nucleus shown in B–D. (B) Zoomed in rotational view of β-actin mRNA (green) and heterochromatin (blue). Numbers below the images indicate the rotation angle. Arrowheads point at two mRNA clusters that appear to colocalize with heterochromatin. Whereas the upper cluster sits on top of the chromatin (40°–120°), the lower cluster is located within the heterochromatin. Projection was done without interpolation resulting in line patterns close to the 90° view (e.g., see 60°) and are therefore not displayed. The full 360° rotation is shown in Video 3. (C) Rotational view of β-actin mRNA and NPCs analogue to B. At all angles the β-actin mRNA partially overlaps with an NPC (bottom arrow). The full 360° rotation is shown in Video 4. B and C are static projections of the first frame of the videos. (D) Time course of β-actin mRNA and NPCs from three rotational perspectives. The 40° and 160° views for the 0-s time point are shown in C with blue frames. The mRNA–NPC interaction is most visible at an angle of 0° and invisible from the opposite side of the cell (160°) and ultimately nonproductive (mRNA arrives and leaves NPC on the same side). The last three views are from the first slices of the stack at an angle of 0° at later time points of the same video, illustrating repeated activity of a specific nuclear pore over time. Cyan bars under images indicate identical time groups. (E) Nuclear pore 3D projection from A. Arrowheads point to nuclear pores at the top/plane +4 (a) and bottom/plane −4 (b) of the nucleus. (F) 3D projection of the RNA channel from A. Arrowheads point to examples of a mobile β-actin mRNA resulting in a blurred projection (c) and a stationary β-actin mRNA resulting in a sharp projection (d). (G) 3D projection of the DNA channel from A. (H) Intensity frequency histogram of the cell (n = 1) in F showing three distributions: to the left, background resulting from rotation of the deconvolved image (arrowhead e); a lower intensity peak resulting from mobile mRNAs in the time projection (arrowhead f); and, to the right, a higher intensity peak resulting from stationary mRNAs in the time projection (arrowhead g). (I) Bar graph showing the level of β-actin mRNA localized to heterochromatin and heterochromatin periphery relative to nucleoplasm. On average a slight reduction of mRNA occupation in the heterochromatin was found, but was not statistically relevant (two-sided test). *, significant within 2σ confidence interval on the standard deviation; ns, not significant. Images were processed as described in Materials and methods and contrast was adjusted for visibility.

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