(H-308) Optogenetic Tools for Studying Endothelial Cell Mechanobiology

Human Embryonic Kidney (HEK 293T) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine (FBS) and 1% of penicillin-streptomycin in T75  polystyrene flasks. Cells were maintained in a 5% CO2 water-jacketed incubator (Thermo/Forma 3110) at 37°C. Cells were seeded at 25% confluency in 35 mm glass bottom dishes (MatTek) and transfected at ~50% confluency 24 hours later using the TransIT-293 transfection reagent (Mirus Bio) following manufacturer instructions. Cells were imaged 24 hours after transfection.

Endothelial colony forming cells (ECFC) were cultured in Endothelial Cell Growth Media (EGM-2, Lonza) with 10% defined FBS in gelatin treated T75 polystyrene flasks. Cells were seeded at 25% confluency in gelatin treated 35 mm glass bottom dishes (MatTek) and infected with second-generation lentivirus produced using the pHR transfer vector, pCMV-dR8.91, and pMD2.G from Lenti-X HEK 293T cells at 50% confluency 24 hours later. Cells were imaged 48-72 hours after infection.

Cells were imaged with the Leica DMI6000B fluorescence microscope under Leica MetaMorph control, with a sCMOS camera (pco.edge), an LED illuminator (Lumencor Spectra-X), and a 63× oil immersion objective. Cell area was established using ImageJ software to outline the cell shape and calculate area. Each cell area was calculated at the initial 0 minute time point (T0) and final 10 minute time point (T10). The change in cell area percentage was determined by calculating the difference in change of the area at T0 versus T10.  

Our findings in HEK 293T cells align with previous reported results: both BcLOV4-mCherry and opto-RhoA-mCherry express in the cytosol prior to blue light illumination of the cell, which triggers rapid membrane translocation of the protein. Over a 10 minute time course in which cells were stimulated with a total of 1 second of blue light stimulation per minute (1.67% stimulation duty cycle), we observed that opto-RhoA-expressing cells decreased in area, while BcLOV4 control cells stayed roughly the same in size. In ECFCs, we observed cytosolic mCherry fluorescence in cells transduced with BcLOV4 (control) and opto-RhoA, indicating successful infection and protein expression. We observed greater fluctuation in cell shape over the imaging time course in ECFCs compared with HEK 293T cells, which we attribute to the larger size and motility level of ECFCs. Over 10 minutes of imaging mCherry fluorescence while stimulating cells with a 1.67% duty cycle of blue light, we observed a greater average decrease in area for opto-RhoA-expressing cells than for BcLOV4 cells. This result suggests that, like in other cell types we have studied, membrane recruitment of the RhoA GTPase in ECFCs drives actomyosin contractility within minutes and demonstrates the viability of using light to control the mechanical state of endothelial cells.

This study establishes expression of a single-component optogenetic tool in primary endothelial cells and provides initial characterization data of tool function. While these pilot experiments generated insufficient transduced cells for assessing statistical significance of this data, viral infection and imaging conditions determined in these experiments will inform our future work with optogenetic control of RhoA signaling in endothelial cells. Immediate future directions from this study include direct observation of the actin cytoskeleton in response to opto-RhoA stimulation using phalloidin staining and measurement of YAP/TAZ nuclear localization and transcription. 

This project was funded by the NSF-funded Science and Technology Center, Center for Engineering Mechanobiology (CEMB), award number CMMI-1548571.