Poster V4 - Estradiol influences epithelial cell actin and shape dynamics imaged with 3D lattice lightsheet microscopy

Introduction: The ability of cells to dynamically alter their structure and shape plays a critical role in numerous healthy and disease processes, such as cell division, migration, and invasion. Estrogens can play a key role in regulating these cellular activities. For example, estrogens are known to mediate cell invasiveness in estrogen-dependent conditions, such as endometriosis, estrogen receptor-positive breast cancers, and gynecological cancers. Moreover, estrogens have been linked to cytoskeletal remodeling, which likely influences cell motility and morphodynamic flexibility – a trait which is critical for cells to be able to squeeze through tissue microtracks and vascular barriers. Actin dynamics are a key driver of cell migration and the formation of filopodia, lamellipodia, and invadopodia. Protrusions are not created by upregulating actin polymerization alone, but changing cytoskeleton dynamics can drive their formation and suppression. The impact of estrogen on cytoskeletal arrangement over time remains unclear. In the present study, we sought to clarify the effect of 17β-estradiol (E2), a dominant and potent estrogen molecule, on cytoskeleton and shape dynamics in the 12Z human endometriotic epithelial cell line. We provide what we believe is the first characterization of live 2D and 3D cellular dynamics in 12Z cells.

Materials and

Methods: 12Z cells (ABM) were treated with a vehicle control (0.001% ethanol) or 10 nM E2 (Sigma Aldrich) in phenol-free culture media for 24 or 48 hours prior to imaging. 12Z cells were transfected with a LifeAct-GFP probe for G- and F-actin using electroporation technology. Fluorescent actin time-lapses were captured using the Zeiss Lattice Light Sheet 7 (LLS7) microscope, then image stacks were loaded into ImageJ and individually thresholded for optimal object segmentation. Morphology parameters were obtained from binarized images. Protrusions were derived from the binarized image stacks via morphological operations and actin dynamics were analyzed using optical flow.

Results, Conclusions, and Discussions: We showed that lattice lightsheet microscopy can be used to visualize a variety of actin structures in 3D, including ruffles, filopodia, lamellipodia, and invadopodia-like protrusions over time (Figure 2). E2, when applied for 24 hours, significantly decreased cell circularity, solidity, and rate of change of circularity (Figure 3), with relative morphological changes up to ~20% occuring over a 30-60 min time scale for control and E2 groups (Figure 4). 24-hour E2 treatment notably induced the formation of large membrane protrusions with invadopodia-like dynamics (Figure 5). Moreover, E2 treatment led to a more disordered flow of actin within those protrusions (Figure 6), which would be beneficial in sensing non-uniform local topography. Together, these results suggest that E2 may prime cells for invasion in estrogen-dependent diseases by endowing cells with a migratory morphology and active invadopodia- like membrane protrusions.

Acknowledgements (Optional): We thank the Biosciences Core Imaging Incubator at the University of Maryland for providing access to the Zeiss LLS7 microscope. Schematics in Figure 1 were created with Biorender.com.