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. 2021 Feb 23;40(6):e107165. doi: 10.15252/embj.2020107165

Figure 3. TFAM and mtDNA form heterogenous droplets whose relaxation timescale is set by mtDNA in vitro .

Figure 3

  • A
    Phase diagram of mtDNA versus TFAM denoting single/soluble phase (gray) or two phases/droplets (green). Each point on the phase diagram representing a unique DNA and protein concentration was measured from n = 2–12 independent experiments. Black solid line delineates deduced phase boundary.
  • B
    Aspect ratio as a function of dimensionless concentration (molar concentration mtDNA/molar concentration TFAM). Values represent binned mtDNA/TFAM conditions as measured in (A), and error bars are SEM. Green dashed line is an exponential fit to the data where y=1.5–0.2e(−x/(3.8×10−4)). Inset: number of TFAM‐mtDNA droplets per field of view as a function of dimensionless concentration. Values represent binned conditions from mtDNA/TFAM conditions measured in (A) and error bars are SEM. Green dashed line is an exponential fit to the data where y = 5,600 – 2,100e (−x/2.1).
  • C–G
    SIM images of droplets 30 min after mixing with various amounts of mtDNA: 0 nM (C), 0.1 nM (D), 1 nM (E), 4 nM (F), and 10 nM (G). Top row is of TFAM‐DyLight‐594 (red), middle row is of mtDNA‐Alexa 488 (green) and bottom row is the merged image. Scale bar = 2 µm.
  • H, I
    FRAP experiments on TFAM‐mtDNA droplets at 25 µM TFAM (H, red) and 10 nM mtDNA (I, green). Scale bar = 1 µm.
  • J
    FRAP recovery curve showing intensity as a function of time for TFAM (red) and mtDNA (green). Black line are single exponential fits, where mobile fraction ≈ 0.60 and characteristic recovery time t ≈ 350 ± 30 s for TFAM (y = 0.73–0.38exp(‐t/350)), and recovery times t >> 15 min for mtDNA (error = 95% CI). Values represent averages ± SD from n = 16 droplets.