Single-molecule mNeonGreen-Plk4 quantifications in the cytosol of the syncytial fly embryo by FCS. (A) Normalized fitted ACF (Fit, light blue dashed line), with SD (shaded area) and MEMfit distributions (Distribution, red line) for mNeonGreen-Plk4 in the cytoplasm. Based on the two fitting methods, three times scales were determined: the fastest time scale peak corresponds to the triplet state of the fluorophore (7.85 × 10−6 s); whereas the second and third slower time scales correspond to distinct 3D diffusional mobility of mNeonGreen-Plk4 in the cytoplasm, from which the diffusion coefficients (D) were calculated (fastest fraction: 7.89 × 10−4 s, D = 17.2 µm2/s; slower fraction: 9.11 × 10−3 s, D = 1.49 µm2/s). The residuals from the fitted data (Fit) are shown below the graphs. (B) Plk4 undergoes limited oligomerization in the cytosol of the Drosophila blastoderm embryo. The mNeonGreen distribution was fitted to a Weibull distribution, which has a peak value of 4,100 Hz. Next, the mNeonGreen–Plk4 data were fitted with an additional Weibull distribution (one for monomer-like and another for oligomer-like). The second mNeonGreen-Plk4 distribution peaks at 18,450 Hz. From this analysis, it follows that the overall normalized brightness (intensity per particle, mean ± SD) for mNeonGreen–Plk4 in the cytoplasm is higher than for the single mNeonGreen monomer injected into the cytoplasm at a similar concentration, indicating that Plk4 is present both as a monomer (around 30.1% of its diffusing pool) and as low-order oligomers (69.9% of diffusing mNeonGreen–Plk4 pool).
