Rapid blue-light-mediated induction of protein interactions in living cells

Abstract

Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. We demonstrate the utility of this system by inducing protein translocation, transcription and Cre recombinase-mediated DNA recombination using light.

Figure 1

Mapping of interacting domains of CRY2 and CIB1. (a) Schematic showing full-length CRY2 and CIB1 constructs used in experiments. The numbers below the proteins indicate amino acid residue position. (b)β-galactosidase activity of CRY2 and CIB1 constructs tested for interaction in the dark or in blue light (461 nm, 1.9 mW, 4 hr). The Gal4 binding domain (Gal4BD-X) and Gal4 activation domain (Gal4AD-Y) fusions used are indicated. The control vector was pGBKT7rec containing no insert. Error bars represent standard deviation (n = 3 samples). The inset panel shows immunoblot analysis of Gal4BD fusion proteins in yeast.

Figure 2

Light-triggered translocation of CRY2 in mammalian cells. (a) Schematic showing fusion proteins. CIBN-pmGFP contains a CaaX box prenylation motif for targeting to the plasma membrane. (b) Images of CIBN-pmGFP (left panel) and CRY2-mCh (right two panels) coexpressed in HEK293T cells. The localization of CRY2-mCh is shown prior to light excitation (middle panel) and 20 s following a 100 ms pulse of blue light (488 nm, 25μW) (right panel). Scale bar 5 μm. (c) Time course of CRY2-mCh recruitment to the plasma membrane following a single 100 ms pulse of 488 nm light (25 μW). CIBN-pmGFP localization is shown in the left panel. Scale bar 2 μm. (d) CRY2-mCh translocation kinetics following a 100 ms pulse of 488 nm light (arrow). The distribution of CIBN-pmGFP and the line used to generate the CRY2-mCh kymograph is shown in the upper left panel. Scale bar 1 μm. The bottom panel shows quantification of CRY2-mCh in the cytoplasm and at the plasma membrane, using the regions shown in (c) by the dotted and solid lines respectively. Each fraction was normalized between 0 and 1. (e) Activation and reversal time course of the CRY2_PHR-CIBN interaction. The top panel shows cells coexpressing the indicated constructs before and after delivery of two 100 ms pulses of blue light (25 μW) spaced 12.5 min apart. The bottom panel shows quantification of cytoplasmic CRY2_PHR-mCh, with light pulses delivered at t = 0 and t = 12.5 min (arrows).

Figure 3

Light induced activation of transcription and DNA recombination. (a) Schematic showing split Gal4 modules (Gal4BD-CRY2 and Gal4AD-CIB1) expressed in yeast cells containing an HA-tagged reporter protein under control of a galactose-inducible promoter. (b) Immunoblot analysis of the HA-tagged reporter (top panel) in response to blue light pulses (10 s pulses, 1.7 mW, 8 min apart). The control lane contains lysates from cells expressing only the reporter. The graph at bottom shows the quantification of western blot bands. (c) Schematic showing the two split Cre constructs (CIBN-CreC and CRY2-CreN) and the Cre reporter. (d) The plot shows % Cre reporter recombination (# GFP expressing cells / # mCherry expressing cells) measured 48 hours after transfection of HEK293T cells with the Cre reporter and indicated constructs. Cells were exposed to blue light pulses (450 nm, 4.5 mW) for indicated times (15 min, 1 hr, or 24 hrs), or kept in the dark for the duration (−). The error bars represent standard deviation for 3 samples from 3 independent experiments. (e) GFP fluorescence images from samples containing both CRY2-CreN and CIBN-CreC that were exposed to 24 hours of blue light or dark. Scale bar 20 μm.