Fig. 4: Biophysical characteristics underlying the anchor function of PQBP5.

a Purified PQBP5, nucleolin, and fibrillarin proteins were fluorescently labeled at different wavelengths, and the frequency and size of droplets were investigated in the absence or presence of ribosomal RNA. The lower graphs show quantitative analyses of eight chips (observed area 100 μm × 100 μm). The right agar gel confirms the quality of ribosomal RNA after purification. In the absence of RNA (left panels and graphs), small numbers of PQBP5 droplets were observed 6 and 9 h after mixing the fluorescent protein with buffer. A few fibrillarin droplets were observed at 3 and 6 h, whereas no nucleolin droplets were detected. In the presence of RNA (right panels and graphs), PQBP5 droplets appeared immediately after mixing, with the number peaking at 3 hours, and the sizes continuing to increase for up to 9 h. The number and size of fibrillarin droplets both peaked at 3 h, but these droplets disappeared thereafter. A few nucleolin droplets were observed only at 3 h. Data are presented as mean values ± SEM. b Mixtures of two nucleolar proteins under isotonic conditions. Neither PQBP5 nor fibrillarin cooperatively formed droplets with nucleolin (left and middle panels), whereas PQBP5 and fibrillarin formed droplets cooperatively (right panels). The similar experiment was repeated more than five times. c Formation of co-droplets by the three nucleolar proteins. Initially, PQBP5 and nucleolin surrounded fibrillarin, but their lamellar relationship was reversed at 3 h. The similar experiment was repeated more than five times. d Effect of osmolarity on droplet formation by the three nucleolar proteins. PQBP5 droplets were most resistant to changes in osmolarity. Fibrillarin droplets did not form under hypertonic and hypotonic conditions, whereas few nucleolin droplets were observed under all conditions. The similar experiment was repeated more than five times.