| dc.description.abstract | Calcium (Ca^2+) acts as a universal second messenger to regulate a myriad of biological
processes, including gene expression, cell metabolism, lymphocyte activation and cell growth. In
non-excitable cells such as cells of the immune system, store-operated Ca2+ entry (SOCE)
through the Ca^2+ release-activated Ca^2+ (CRAC) channel is primarily coordinated by two
components: the ER-resident Ca2+ sensor protein stromal interaction molecule 1 (STIM1) and the
pore subunit ORAI1 on the plasma membrane. To meet the demand of chemical biology tools
for remote control of Ca^2+ signaling in mammals with high precision, we set out to engineer
photo-sensitivities into either STIM1 (OptoSTIM1) or ORAI1 (OptoORAI1) to generate
photoswitchable CRAC channels.
OptoSTIM1 was engineered by combining STIM1-ORAI1 activation region (SOAR) of
STIM1 with the light-reactive light-oxygen-voltage (LOV2) domain. The light-inducible effects
were assessed by Ca^2+ influx with genetically-encoded calcium indicators, degrees of NFAT
translocation, and expression levels of Ca^2+/NFAT downstream targets. To generate
OptoORAI1, LOV2 was inserted into the loop region of ORAI1 and thus acted as an allosteric
switch to induce structural rearrangement within ORAI1 to open the channel. Through several
rounds of randomized screening and optimization, we identified one OptoORAI1 variant
exhibiting a high dynamic change in the light-induced Ca^2+ response without noticeable dark
activity. In parallel, to enable more flexible and versatile optogenetic engineering of proteins, we
developed a series of engineered LOV2 variants (cpLOV2) through circular permutation.
cpLOV2 creates new interfaces to cage protein function, thus enabling broader applications of
LOV2-based optogenetic tools to targets that are otherwise not photo-controllable.
In summary, our single-component OptoCRAC tools provide new opportunities to
remotely and precisely control the Ca^2+ signaling at high spatial and temporal resolution. We
have successfully demonstrated the use of OptoCRAC to photo-tune Ca^2+/NFAT-dependent gene
expression, as well as transcriptional reprogramming of endogenous genes when coupled with
the CRISPR/Cas9 genome editing technique. | en |
