Fig. 4: Light-activated CRY2olig condensates can be disrupted using DisCo.

a Schematic of DisCo approach with CRY2olig. Light triggers condensate formation, while rapamycin-mediated recruitment of C-BLOCK to a FRB ‘hook’ on CRY2olig scaffold disrupts condensates. b, c CRY2olig condensates are disrupted equally regardless of recruitment orientation. DisCo was performed on HEK293T cells expressing CRY2olig-FRB-mCh and mCh(K70N)-FKBP, or EGFP-FRB-CRY2olig and mCh-FKBP. Cells were illuminated with light (488 nm, 100 ms every 30 s) at 18–22 h post-transfection, then 333 nM rapamycin was added 5 min after light onset. Representative images shown in (b), with quantification of cytosolic disruption in (c). Data were quantified as in Fig. 1c and represent average and error (s.d., n = 10 cells examined from three independent experiments). Scale bars, 10 µm. d Kinetics of condensate disassembly. HEK293T cells expressing CRY2olig-FRB-mCh and mCh(K70N)-FKBP were illuminated 5 min (488 nm, 100 ms pulse every 20–30 s), 18–22 h after transfection. Black triangles, dark, no rapamycin. Blue squares, light (488 nm, 100 ms pulse every 30 s), 333 nM rapamycin. Red circles, control cells expressing CRY2olig-mCh (no FRB) and mCh(K70N)-FKB, with 333 nM rapamycin in dark. All experiments were performed at 33.5 °C. Data shows average and error (s.d., n = 5 cells examined from three independent experiments). e FRAP experiments with EGFP-FRB-CRY2olig condensates formed after 3 min light treatment. Data shows average and error (s.e.m, n = 6 cells examined from three independent experiments). Representative images of a photobleached condensate are shown at bottom. Scale bars, 10 µm. f Ca²⁺-dependent DisCo. HEK293T cells expressing EGFP-CaM-CRY2oligC9 and mCh-CBP formed condensates with light (488, 100 ms pulse every 30 s for 6 min). Addition of 2.5 mM CaCl₂ and 3 µM ionomycin resulted in condensate disruption. Image at far right shows control experiments subject to the same light and Ca²⁺ treatments, but without C-BLOCK. The experiment was repeated a second time with similar results. Scale bars, 10 µm. g Quantification of kinetics and extent of Ca²⁺-dependent disruption of CRY2olig condensates in experiments shown in (f).