Figure 1.
Global variability in transcription.
(A) Variation in BrU incorporation. Hela cells were incubated for 30 min with 5 mM BrU. Br-RNA signal shows a large variation between cells (confocal section). (B). Kinetics of RNA pol II in vivo. FLIP analysis of GFP–RNA pol II. Half of the nucleus was bleached continuously as confocal images were collected approximately every 5 s. (C) Decay in fluorescence intensity of the unbleached area of the nucleus in the FLIP experiment (intensity, arbitrary units) (n = 60). (D) Log plot analysis of FLIP data showing two populations of RNA pol II molecules, as described in Hieda et al [23]. (E) Distribution of t1/2 for the DNA-associated form of RNA pol II in the cell population (n = 60). (F) BrU incorporation is correlated with the half-life of the DNA-associated RNA pol II population. After FLIP, cells were incubated with BrU, fixed, and immunolabelled for Br-RNA. Individual cells were identified, and the values of t1/2 for the DNA-associated form of RNA pol II and the corresponding Br-RNA intensity were plotted. (G–I) Histone H2B–GFP fast-exchanging population is a reporter of transcription elongation. (G) Hela cells expressing histone H2B–GFP were used for FLIP analysis (left panel). These cells were subsequently subjected to BrU incorporation. After identification of the same cells (middle panel), we measured Br-RNA production (right panel), which shows a good correlation (I). In (H) we show the variability in t1/2 for the rapid-exchangeable histone H2B–GFP (transcription-dependent fraction, Figure S4) (n = 100). Again, this shows a high variability between cells in a population. (J and K) RNA pol II molecules track at similar speed inside a given cell. High-power images of Br-RNA foci in two nuclei with different mean intensity of Br-RNA after 15 min of incorporation of BrU. The images have been pseudo-coloured to improve visualisation (purple = low, red = high). All the foci in an individual nucleus show a consistent level of Br-RNA intensity. (L) Analysis of the noise in Br-RNA production in transcription foci in individual cells. The plot shows 40 cells (>200 foci were analysed per cell). Transcription rates of Br-RNA foci appear to be correlated. Bars: (A), 10 µm; (K), 0.200 µm.
Figure 2.
Kinetics of RNA pol II in situ: effect of NTP concentration.
(A) Transcription in the nucleus and mitochondria are related. Br-RNA was immunodetected in cells after BrU incorporation. Cells with a very actively transcribing nucleus also have high levels of transcription in the mitochondria. (B) Analysis of Br-RNA signals in nucleoplasm versus mitochondria (arbitrary units) in images like that in (A) (confocal images). (C) Cells were fused using polyethylene glycol and after 2.5 h were exposed for 30 min to 2.5 mM BrU. Arrows point to fused cells, where signals in nuclei sharing the same cytoplasm present very similar intensities. (D) Analysis of the CV between nuclei of neighbouring cells (mono) and nuclei sharing the same cytoplasm (multi); CV of Br-RNA incorporation (red columns), CV of RNA pol II elongating (blue columns), and CV of the dynamics of exchange of histone H2B (green columns). (E) Study of the enzymatic kinetic properties of RNA pol II with respect to [NTP]. The assay was performed as described in Figure S5, using as a tracer the incorporation of BrUTP into the nascent transcripts of Hela cells. This panel shows the dependence of transcription rate, V (arbitrary units/minute) on NTP concentration (micromoles). The experimental data fit a hyperbolic curve consistent with Michaelis-Menten kinetics. (F) Plot of [NTP]/V versus [NTP]; the straight line obtained suggests that RNA pol II has Michaelis-Menten-like kinetics [28]. (G) Dependence of V on ATP concentration (micromoles) using normotonic conditions. This curve shows a sigmoidal pattern. (H) Plot of [ATP]/V versus [ATP] suggests that RNA pol II is allosteric with respect to ATP. The sigmoidal curve of V versus [ATP] suggests that the rate of BrUTP incorporation has a quadratic dependence on [ATP]. (I) Repetition of (G) but using swollen cells (hypotonic shock) in order to decondense the chromatin. Under these conditions, the kinetics of RNA pol II with respect to [ATP] appears hyperbolic. (J) Effect of intracellular [ATP] on BrU incorporation. The intracellular [ATP] was perturbed by incubation with DG (25 mM) and no glucose for 12 h, which decreases [ATP], or incubation with succinate (5 and 10 mM) for 30 min, which increases [ATP]. Green dotted line represents BrU incorporation under ATP control conditions (high glucose, 4.5 g/l). Bars = 10 µm.
Figure 3.
Mitochondria determine transcription elongation speed.
(A) Hela cells were sorted according to the mitochondrial content after staining with MitoTracker Green FM. Two populations of cells were sorted, with a difference in mitochondrial content of around 5-fold (R1 and R2). (B) Four hours after plating, cells were incubated with BrU. These experiments show a direct relationship between mitochondrial content and both RNA production and mRNA content (quantification in S8A and S8B). (C) Cells stained with the redox-sensitive mitochodrial probe CMXRos (middle panel) and Br-RNA (left panel). (D) Quantitative analysis of images like that in (C) (arbitrary units). There is a direct relationship between CMXRos and BrU incorporation signals (confocal images). (E) Co-staining of Br-RNA and P-S6 in Hela cells. (F) Nuclear Br-RNA production under conditions of ATP depletion. Cells were incubated with DG for 12 h, and at the end of incubation 5 mM BrU was added. In this plot we superimposed the data points of three different conditions used: glucose (average: blue dot), 25 mM DG (average: green dot), or 50 mM DG (average: red dot). (G) ATP affects CV in BrU incorporation. Intracellular ATP concentration was raised by succinate incubation (5 and 10 mM) for 30 min. The CV of Br-RNA is reduced in a manner proportional to ATP (blue bars), and BrU incorporation increases in parallel to intracellular ATP increase (red dotted line). (H) CV of the speed of exchange of histone H2B–GFP. This reporter behaves very similarly to BrU, decreasing as intracellular ATP increases. Bars = 10 µm.
Figure 4.
Asymmetric segregation of mitochondria.
(A) Visualisation of mitochondria with MitoTracker Green FM. (B) Quantitative analysis of the mitochondrial content in samples stained as in (A) (n = 800). (C) Asymmetric segregation of mitochondria in mitotic cells. Cells in telophase show asymmetric segregation of mitochondria (green) and DNA (red). (D) Quantitative analysis of the ratio (rat.) of mitochondrial content between daughter cells (n = 300). (E) Mitochondrial content is correlated with the cell cycle length, as demonstrated by the plot of the ratio of cell cycle length versus the ratio of mitochondrial content between daughter cells at division (R2 = 0.8). (F) A weak correlation was found between the ratio of daughter cell volumes at birth (measured by the soluble protein DsRed) and their relative cell cycle lengths (R2 = 0.06). (G) Analysis of the difference in mitochondrial content between sister cells after the first division (ratio of intensities of mito-YFP) and the second division. No relationship could be observed between segregation of mitochondria in two consecutive mitotic events. (H) Analysis of the interdivision time between consecutive cell cycles in individual cells. Bars: (A), 10 µm; (C), 5 µm.