Figure S5.
In support of Fig. 5: FCS analysis of Drosophila embryos. (A) Maximum intensity z projections from a time-lapse video of a syncytial D. melanogaster embryo expressing endogenous mNeonGreen-Plk4 (green) and MT reporter RFP–β-tubulin (magenta). Plk4 localizes at the centrosomes (high-intensity tubulin spots) in interphase. Larger green dots result from yolk auto-fluorescence. At time point t = 00:00, the embryo is in metaphase of nuclear cycle 11. The insets show the progression of a single nucleus and its daughters throughout one cell cycle. The cell cycle stage is indicated above each image. Time is reported as minutes:seconds. The asterisk indicates an abnormal mitotic spindle. (B) FCS measurements of purified mNeonGreen fluorophore in a buffer supporting viability of the cytoplasm (Telley et al., 2013). (C) FCS measurements of mNeonGreen after injection into the cytosol of syncytial embryos expressing RFP–β-tubulin. The graphs show the normalized, fitted ACFs (blue dots and light blue curve), with SD (shaded area) and MEMfit (red line). The time lags (diffusion times) determined using the two fitting methods shown next to the MEMfit curves are in agreement. The peak at the fast time scale corresponds to the triplet state of the fluorophore (9.48 × 10−6 s in solution; 22 × 10−6 s in the cytoplasm), whereas the second peak in the slower time scale corresponds to the 3D diffusion of mNeonGreen, from which a diffusion coefficient D was calculated (1.59 × 10−4 s, D = 85.21 µm2/s in solution; 6.54 × 10−4 s, D = 20.72 µm2/s in the cytoplasm). The residuals obtained from the best fit are shown below the ACF graphs. (D) Single-molecule mNeonGreen–Plk4 quantifications in the cytosol of the syncytial fly embryo. i: Intensity traces of mNeonGreen–Plk4 (black) and background noise (gray). Of note, intensity bursts of mNeonGreen–Plk4 are well distinguishable from background noise (inset). ii: Raw ACFs from multiple independent FCS measurements. While the intensity of background acquisitions as measured in RFP–tubulin expressing embryos does not auto-correlate, traces from mNeonGreen–Plk4 expressing embryos exhibit significant autocorrelation.

In support of Fig. 5: FCS analysis of Drosophila embryos. (A) Maximum intensity z projections from a time-lapse video of a syncytial D. melanogaster embryo expressing endogenous mNeonGreen-Plk4 (green) and MT reporter RFP–β-tubulin (magenta). Plk4 localizes at the centrosomes (high-intensity tubulin spots) in interphase. Larger green dots result from yolk auto-fluorescence. At time point t = 00:00, the embryo is in metaphase of nuclear cycle 11. The insets show the progression of a single nucleus and its daughters throughout one cell cycle. The cell cycle stage is indicated above each image. Time is reported as minutes:seconds. The asterisk indicates an abnormal mitotic spindle. (B) FCS measurements of purified mNeonGreen fluorophore in a buffer supporting viability of the cytoplasm (Telley et al., 2013). (C) FCS measurements of mNeonGreen after injection into the cytosol of syncytial embryos expressing RFP–β-tubulin. The graphs show the normalized, fitted ACFs (blue dots and light blue curve), with SD (shaded area) and MEMfit (red line). The time lags (diffusion times) determined using the two fitting methods shown next to the MEMfit curves are in agreement. The peak at the fast time scale corresponds to the triplet state of the fluorophore (9.48 × 10−6 s in solution; 22 × 10−6 s in the cytoplasm), whereas the second peak in the slower time scale corresponds to the 3D diffusion of mNeonGreen, from which a diffusion coefficient D was calculated (1.59 × 10−4 s, D = 85.21 µm2/s in solution; 6.54 × 10−4 s, D = 20.72 µm2/s in the cytoplasm). The residuals obtained from the best fit are shown below the ACF graphs. (D) Single-molecule mNeonGreen–Plk4 quantifications in the cytosol of the syncytial fly embryo. i: Intensity traces of mNeonGreen–Plk4 (black) and background noise (gray). Of note, intensity bursts of mNeonGreen–Plk4 are well distinguishable from background noise (inset). ii: Raw ACFs from multiple independent FCS measurements. While the intensity of background acquisitions as measured in RFP–tubulin expressing embryos does not auto-correlate, traces from mNeonGreen–Plk4 expressing embryos exhibit significant autocorrelation.

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