Fig. 7: Heterotypic PLDs interact at sub-saturation concentrations.

a A schematic representation of the bead halo assay. Created with BioRender.com. b 250 nM of AlexaFluor488-labeled His₆-MBP-Scaffold constructs \(-\) where scaffold signifies either SS18^PLD, BRG1^PLD, FOXG1^N-IDR, or control His₆-MBP-GGGCGGG, were attached to Ni-NTA beads. 250 nM of FUS^PLD labeled with AlexaFluor594 was then added to the above beads (see Materials and Methods for further details). Binding was quantified using the ratio of fluorescence intensities (fluorescence signal from the client FUS^PLD/fluorescence signal from the scaffold) on the surface of the bead. c A box-and-whisker chart of the intensity ratios is plotted with the mean and standard deviation (n = 8 beads/trial). Significance is shown in Fig. S16. d HEK293T cells co-expressing OptoFUS^PLD-NLS (Cry2-mCherry-FUS^PLD-NLS) and GFP-SS18^PLD below their saturation concentrations. Upon blue light activation, OptoFUS^PLD co-condenses with GFP-SS18^PLD. Mean intensity profiles of the co-condensates formed are shown as a function of time for condensates within the inset image. Green represents the intensity profile of GFP-SS18^PLD and red represents the intensity profile for OptoFUS^PLD. The corresponding movie is shown in supplementary movie 1. e Pre-existing GFP-SS18^PLD clusters act as nucleation sites for OptoFUS^PLD condensates upon blue light activation (also see supplementary movie 2 and supplementary movie 3). The scale bar is 5 μm for all images.