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. 2023 Aug 11;11:1242481. doi: 10.3389/fcell.2023.1242481

FIGURE 4.

FIGURE 4

The N-terminus of SATB1 forms phase separated droplets. (A) Graphical representation of all CRY2-mCherry constructs generated. The graph displays the IDR prediction by VL3 PONDR score in magenta and the prediction of prion-like domains by PLAAC algorithm in green. (B) Formation of optoDroplets upon 488 nm pulse illumination in live cells transiently transfected with CRY2-mCherry-N-terminus_SATB1 constructs as well as with negative (vector) and positive control constructs (N-terminal part of FUS protein). Two groups of cells were identified based on the original relative expression levels of the recombinant proteins. Representative of two to three biological replicates. Scale bar 5 μm. (C) OptoDroplets displayed a round shape and the ability to coalesce. (D) Number of optoDroplets normalized to the cell size at each time point upon activation, to visualize the speed of droplet assembly and its dependence on the original concentration of the protein. (E) Number of optoDroplets normalized to the cell size at each time point of inactivation (488 nm laser was off) to visualize the speed of droplet disassembly and its dependence on the original concentration of the protein. (F) FRAP experiment utilizing either the N-terminus of SATB1 construct or the FUS positive control construct. The fitted curves were generated by the exponential recovery curve fitting function of Fiji. In (D, E, F), the error bars represent the s.e.m.