Nature Methods Rapid blue-light mediated induction of protein interactions in living cells Matthew J Kennedy, Robert M Hughes, Leslie A Peteya, Joel W Schwartz, Michael D Ehlers & Chandra L Tucker Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Table 1 Mutation of putative NLS sequences in CRY2 and CIB1. Reversibility and repeated induction of CRY2-CIBN interaction. CRY2 PHR -CIBN interaction can be activated by two-photon excitation. YFP can be imaged without activating CRY2-CIBN interaction. CRY2-CIB modules and light treatments show no toxicity in cultured cells. Note: Supplementary Table 2 and Supplementary Video 1 are available on the Nature Methods website.
CIB1 predicted NLS 93 19 K R K F D T E T K D C N E K K K K aaa cgg aag ttt gat aca gag act aag gat tgt aat gag aag aag aag aag GCa GCg GCg GCg A A A A CRY2 predicted NLS 541 557 K R V K P E E E E E R D M K K S R aag aga gtg aaa cct gag gaa gaa gaa gag aga gac atg aag aaa tct agg GCg GCa A A Supplementary Figure 1. Mutation of putative NLS sequences in CRY2 and CIB1. Lysine residues contained in putative NLS sequences identified by PSORT (http://psort.ims.u-tokyo.ac.jp/) were mutated to alanine. The numbers indicate amino acid residue position.
a 488 nm CRY2 CRY2PHR Fraction cytoplasmic CRY2.9.7 -.2..2.4 b 488 nm 2 4 6 Time (min) 8 1 Fraction cytoplasmic CRY2PHR.4 5 1 Time (min) 15 Supplementary Figure 2. Reversibility and repeated induction of CRY2-CIBN interaction. (a) CRY2-CIBN and CRY2PHR-CIBN interactions show nearly identical activation (inset) and reversal kinetics. A blue light pulse (1 ms, 488 nm, 25 μw) was delivered at t = (arrow). Cytoplasmic mch signal was quantified at several 2 x 2 pixel regions drawn over cytosolic portions of the cell being careful to exclude the plasma membrane. Measurements from these regions were averaged for each cell. Measurements were repeated for at least 3 different cells and averaged to generate the traces shown. The data represents the mch signal at time (t) divided by the average mch signal prior to blue light illumination. (b) Translocation of CRY2PHR-mCh to the plasma membrane can be repeatedly induced with blue light stimulation. Cytoplasmic CRY2-mCh intensity was measured in response to a train of blue light pulses (1 ms, 488 nm, 25 μw) delivered every 2 s (arrows).
a CRY2PHR-mCh Before excitation b -1s c Fraction CRY2PHR (normalized intensity) After excitation After 488 nm excitation 1s 25 s 26 s 5 s d CIBN-pmGFP 51 s 75 s CRY2PHR-mCh CIBN-pmGFP 76 s Merge Plasma membrane.4 Dark Cytosol.2. 2 4 Time (s) 6 stimulated Supplementary Figure 3. CRY2PHR-CIBN interaction can be activated by two-photon excitation. (a) HEK293T cells expressing CRY2PHR-mCh and CIBN-pmGFP were excited with two-photon illumination at to induce translocation of CRY2PHR-mCh to the plasma membrane (second panel). Following spontaneous dissociation of CRY2PHR-mCh from the plasma membrane, the same cells were excited with 488 nm light for comparison (third panel). The distribution of CIBN-pmGFP is shown in the far right panel. Scale bar 5 μm. (b) Plasma membrane localization of CRY2PHR-mCh following two-photon excitation at every 25 s (arrows). Scale bar 1 μm. (c) Quantification of CRY2PHR-mCh redistribution from the cytosol to the plasma membrane in response to two-photon excitation at (arrows). (d) Translocation of CRY2PHR-mCh triggered by two-photon excitation in organotypic hippocampal slices. Hippocampal neurons from organotypic slice cultures expressing CRY2PHR-mCh and CIBN-pmGFP were either maintained in darkness (top panels) or stimulated with two-photon excitation at (bottom panels). Note the colocalization of red (CRY2PHR-mCh) and green (CIBN-pmGFP) signals in the photoexcited sample. Scale bar 5 μm.
a CIBN-pmCitrine b 514 nm.14 µw 1.3 µw 4.4 µw 6.7 µw 1.1 µw CRY2-mCh Fraction cytoplasmic CRY2-mCh.9.7.5 2 4 6 8 1 514 nm power (µw) Dark Supplementary Fig. 4. YFP can be imaged without activating CRY2-CIBN interaction. (a) CIBN fused to the YFP variant citrine (CIBN-pmCitrine) was imaged with increasing intensities of 514 nm light (indicated above top panel). The bottom panel shows the CRY2PHR-mCh distribution following 2 frames of CIBN-pmCitrine imaging (514 nm, 1 ms integration time / frame). Note that at lower intensities, YFP can be visualized without triggering the CRY2-CIBN interaction. Higher intensities of 514 nm illumination triggered translocation of CRY2PHR-mCh to the plasma membrane. (b) Quantification of CRY2PHR-mCh translocation following 2 frames of 514 nm illumination at the indicated powers. The dashed line represents the level of cytoplasmic CRY2PHR-mCh following a saturating pulse of 488 nm light.
Supplementary Table 1 Construct(s) expressed % viable cells (dark) % viable cells (blue light) Untransfected cells 95 ±.4 98 ± 2.6 egfp-n1 (control) 95 ± 2.3 92 ±.7 CRY2-CreN + CIBN-CreC 92 ± 1.6 92 ± 1.7 Supplementary Table 1. CRY2/CIB modules and light treatments show no toxicity in cultured cells. HEK293T cells were transfected with indicated plasmids and kept in the dark for the duration or exposed to blue light pulses (1 hr, 45 nm, 4.5 mw) at 2 hours post transfection. At 24 hours post transfection, samples were harvested and stained with trypan blue, to allow identification of viable vs. dead cells. Percentages of viable cells are reported in the table, based on three independent counts.