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Fig. 2 A CRY2-fused dimerization domain is required for light-dependent nuclear clearing and functional loss of activity. (A) Live cell imaging showing formation of CRY2(?NLS)-mCh-tTA clusters in the nucleus upon initial light exposure, which coalesce into larger puncta over the 90 min timecourse. Scale bar, 10 ?m. (B) Schematic showing constructs used in (C) and (D). CRY-Gal?DD-VP16 contains Gal4BD residues 1?147 but is missing residues 66?95, which are important for dimerization. (C) Luciferase activity of cells expressing indicated constructs exposed to 18 h dark or blue light pulses. Data represents average and error (s.e.m.) from four independent experiments. Inset shows the ratio of activity in dark to activity in light. **P-value < .05. (D) Localization of protein fusions in HEK293T cells, assayed by immunohistochemistry using an anti-Gal4BD antibody. Cells were treated as in (C). (E?G) Addition of a tetramerizing dsRed domain restores light response to CRY-Gal?DD-VP16. (E) Schematic of construct used in (F) and (G). (F) Representative HEK293T cells expressing CRY-dsRed-Gal?DD-VP16, exposed to 18 h dark or blue light pulses, and assayed for localization as in (C). (G) Luciferase activity of cells expressing CRY-Gal?DD-VP16 alone or with an added back multivalent domain (CRY-dsRed-Gal?DD-VP16) assayed as in (C). Data represents average and error (s.e.m.) for three independent experiments. Inset shows the ratio of activity in dark to activity in light. ***P-value < .005. (H) Comparison of effect of adding back a tetramerizing dsRed domain versus a monomeric mCherry domain to CRY-Gal?DD-VP16. Luciferase assay was carried out as in (C). Data represents average and error (s.d., n = 3) from one experiment, and experiments were repeated two times with similar results.