Abstract
Purpose :
The mechanisms underlying an increased lens size due to lens epithelial cell proliferation in presbyopia remain unknown. Biomechanical strain is hypothesized to be a contributor, as the zonules attach to the lens germinative zone. Shroom3, a cytoskeletal protein, is necessary for ocular development and facilitates lens placode invagination. Preliminary studies demonstrate that Shroom3 mutants lack ventral optic cup closure, illustrating the significance of Shroom3 in epithelial folding, fusion, and potentially, proliferation. We hypothesize inducing intercellular strain through Shroom3 overexpression increases proliferation in vitro.
Methods :
Model epithelia, Madin-Darby canine kidney (MDCK) cells, were transfected with Shroom3 to create a stable line (pTre3-Shroom3-mCherry) inducible with doxycycline with dual selection hygromycin/G418. Once cells reached confluence on glass coverslips, EdU was incorporated into cells for 18 hours and fixed with paraformaldehyde. Cells were imaged throughout the coverslip centrally and peripherally (n=5) using Zeiss Axio Observer (N=3). MDCK wildtype (wt) cells were used to control for the effect of doxycycline. The number of nuclei and EdU+ cells were counted using ImageJ. Statistical analysis was performed using two-tailed Student’s t-test.
Results :
SHROOM3 overexpression resulted in significantly reduced mitosis, as shown through nuclei counts (p≤0.001) and percentage of EdU+ cells (p≤0.001). The addition of doxycycline affected proliferation differently depending on the location of cells on the coverslip. Peripheral doxycycline+ wt cells showed decreased proliferation (p≤0.05) compared to pTre3g-Shroom3-mCherry doxycycline- cells, but not compared to peripheral doxycycline- wt cells. Central doxycycline+ wt cells showed decreased proliferation (p≤0.05) compared to doxycycline- wt cells but not to pTre3g-Shroom3-mCherry doxycycline- cells.
Conclusions :
Our results contradict our hypothesis and instead show that Shroom3 decreases proliferation. Although doxycycline affected proliferation both centrally and peripherally, results were not consistent between cells with/without doxycycline; our results can be attributed to SHROOM3 expression levels. Future experiments will apply these methods to in vivo models.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.