Correlating <i>in Vitro</i> and <i>in Vivo</i> Activities of Light-Inducible Dimers: A Cellular
Optogenetics Guide
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Abstract
Light-inducible
dimers are powerful tools for cellular optogenetics,
as they can be used to control the localization and activity of proteins
with high spatial and temporal resolution. Despite the generality
of the approach, application of light-inducible dimers is not always
straightforward, as it is frequently necessary to test alternative
dimer systems and fusion strategies before the desired biological
activity is achieved. This process is further hindered by an incomplete
understanding of the biophysical/biochemical mechanisms by which available
dimers behave and how this correlates to <i>in vivo</i> function.
To better inform the engineering process, we examined the biophysical
and biochemical properties of three blue-light-inducible dimer variants
(cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated
these characteristics to <i>in vivo</i> colocalization and
functional assays. We find that the switches vary dramatically in
their dark and lit state binding affinities and that these affinities
correlate with activity changes in a variety of <i>in vivo</i> assays, including transcription control, intracellular localization
studies, and control of GTPase signaling. Additionally, for CRY2,
we observe that light-induced changes in homo-oligomerization can
have significant effects on activity that are sensitive to alternative
fusion strategies