T1 - Shining Light on Calcium-mediated Growth Control and Tumor Progression With Optogenetics

Introduction: : Calcium (Ca²⁺) is a crucial second messenger that coordinates cellular processes across tissues. However, direct control over Ca²⁺-mediated processes remains limited. Here, we use the optogenetic channelrhodopsin, CsChrimson, to investigate how Ca²⁺ signaling impacts epithelial growth, morphogenesis, and tumor progression in Drosophila melanogaster. Optogenetic activation of CsChrimson modulates cytosolic Ca²⁺ levels, influencing proliferation, apoptosis, and integrin-mediated adhesion. A biphasic Ca²⁺ response was observed, wherein moderate activation increased final organ size, while prolonged stimulation (>6 hours) elevated apoptosis and led to morphological defects like epithelial blistering. To probe oncogenic interactions, we developed a transgenic line co-expressing CsChrimson and Ras-V12, enabling investigation into how specific Ca²⁺ dynamics impact with tumor growth and cancer cell mechanics. Together, these results demonstrate how dynamic programming of Ca²⁺ signaling can regulate tissue-level growth control and morphology, with translational applications for individualized cancer therapies.

Materials and

Methods: : Genetic Tools and Fly Lines: We used the GAL4-UAS system to express CsChrimson, GCaMP6 and/or Ras-V12 in developing Drosophila tissues.
Imaging and Microfluidics: To support live imaging, a PETL (polyethylene terephthalate laminate) microfluidic device was used to enable long-term imaging and continuous media flow. Wing imaginal discs were imaged using a Nikon A1R-MP confocal microscope, with step sizes of 0.8 μm across z-planes using 40x and 60x oil objectives.
Optogenetic Activation: Embryos were raised in darkness with 1 mM All-Trans-Retinal supplemented for CsChrimson functionality. Activation was performed using red-light LED strips (~600 nm) controlled by an Arduino microcontroller. A digital illuminance meter was used to calibrate light intensity.

Results, Conclusions, and Discussions:: Ca²⁺-Mediated Growth Regulation via CsChrimson. Optogenetic activation increased cytosolic Ca²⁺ and affected multiple cellular processes in a dose- and time-dependent manner. Intermediate light intensity (5 lux) maximized wing disc size, while higher intensities caused apoptosis and epithelial defects. Prolonged activation led to blistering and disrupted integrin localization, compromising adhesion. Lower-frequency pulses promoted proliferation and tissue-wide compensatory responses. Co-expressing CsChrimson and Ras-V12 enables evaluation of how Ca²⁺ signaling intersects with oncogenic growth and cancer therapeutics.

Our results show that Ca²⁺ dynamics are tightly interwoven to regulate epithelial growth and final size control. Optogenetic tools enable precise Ca²⁺ manipulation, revealing a biphasic growth response with an optimal activation range. The optogenetic Ca²⁺ control combined with the integrative capabilities of Drosophila opens new avenues for preclinical testing of cancer wound healing modulation therapies.

Acknowledgements and/or References (Optional):: Imaging was performed at the Notre Dame Integrated Imaging Facility. This work was supported by the EMBRIO Institute (NSF contract 2120200), NIH R35 GM156615 & GM124935.