Photocontrol of intranuclear SATB1 cleavage. (a) Photocontrolled
delivery of the TEV protease into the nucleus of MCF10A cells and
subsequent specific cleavage of a transcription factor. The TEV protease
has a prepended OptoNLS and two attached CFPs, to block passive diffusion
into the nucleus. A TEV protease site was engineered into the SATB1
transcription factor, which was also flanked by two fluorophores to
allow intranuclear cleavage to be visualized. (b) TEV-protease (blue)
is kept in the cytoplasm and enters the nucleus after photoactivation.
SATB1 in the nucleus (yellow) containing a TEV target site will remain
intact until the protease enters the nucleus and starts cleavage.
Upon cleavage of the protein, its part containing the NLS will remain
nuclear whereas the other part (red) will distribute all over the
cell. (c) Fluorescence images: before photorelease of the TEV protease,
GFP (green), and mCherry (red) are colocalized and fully contained
in the nucleus (left panel), confirming that an intact SATB1 is expressed
and localized in the nucleus. After photorelease of TEV, it enters
the nucleus and there it cleaves SATB1. The green N-terminal fragment
stays in the nucleus since it has a functional NLS; the red C-terminal
fragment transitions to the cytoplasm (middle). (right) Images split
into single color channels.