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Biomechanics

Biomechanics - Poster Session B

Poster E7 - Eversion of Epithelial Apical-basal Polarity Requires α6β4 Integrins

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  An important aspect of epithelial cells is apical-basal polarity, which segregates different proteins to the top (apical) and bottom (basal) surfaces of the cell. For reference, in 3D culture, the apical surface faces a fluid-filled center or lumen whereas the basal surface faces the outer surface that is in contact with ECM. Alterations to apical-basal polarity, such as “apical-out” or inverted polarity, have been observed in many epithelial cancers, suggesting that changes in apical-basal polarity can be pathogenic. Previously we showed that epithelial cysts can change from an apical-in polarity to an apical-out polarity through a physical rearrangement of cells, a process we and others have termed eversion (Narayanan, Biophys J 2023). The objective of this project is to understand the biophysical and biochemical mechanisms that drive epithelial eversion and maintain the apical-out phenotype.

Materials and

Methods:  To study eversion, we used MDCK cells (a non-cancerous dog kidney cell line that is frequently used as a model for normal epithelium) and Caco-2 cells (a cancerous, noninvasive cell line derived from a human colorectal adenocarcinoma). Both cell lines were grown in Matrigel or Matrigel-Collagen mixtures and allowed to develop into a spheroid with a single, hollow lumen. Following the formation of the spheroid, eversion was stimulated using Rho Activator II (Cytoskeleton) as previously described in Narayanan, Biophys J 2023. We compared the effects of Rho Activator II on wild-type cells or cells in which α6, α3, αV, β1, or β4 were knocked out. In additional experiments, we used a soft agar assay to assess anchorage independence.

Results, Conclusions, and Discussions: To follow up on our prior work studying eversion, we sought to understand if eversion is an integrin-dependent process. Screening MDCK cells in which individual integrin genes had been knocked out, we observed that MDCK cells lacking α6 or β4 integrin did not evert. Furthermore, wild-type cells had upregulated β4 protein expression following eversion. Additional experiments showed that an α6 blocking antibody inhibited eversion in MDCK and Caco-2 cells. Because α6β4 integrins bind to laminin, we investigated if laminin was important for eversion. MDCK cells were seeded into collagen or Matrigel (Matrigel contains laminin whereas collagen does not). MDCK spheroids in collagen did not contain a basal laminin ECM network and did not evert apical-basal polarity upon stimulation by Rho Activator II.
Additionally, to further explore eversion, we sought to understand if eversion to apical-out polarity is permeant. To test the ability of spheroids to ‘revert’ to apical-in polarity, we treated spheroids with Rho Activator II for 48 hours. Nearly 100% of spheroids everted. Next, we changed the media to remove Rho Activator II for 4 days. Upon removal of rho a significant number of cysts returned to normal polarity (apical-in). In some cysts where rho was removed, it appeared that cells in the lumen were undergoing apoptosis, suggesting that the lumen is re-formed by apoptosis of cells at the center of the cyst. We hypothesized that cells in everted spheroids may be anchorage-independent. To test this, we used a soft agar assay and observed that cells treated with Rho Activator II were anchorage-independent.
Eversion is a new physical mechanism for how 3D epithelial cells can rapidly invert apical-basal polarity. Our studies have shown that spheroids can return to normal apical-in polarity following removal of the eversion stimulus, indicating that eversion is not permeant. Furthermore, apical-out polarity may be driven by cells becoming anchorage-independent. Additionally, we have identified α6β4 integrins, which comprise laminin-binding hemidesmosomes, as important for the eversion process. In summary, our work further identifies biophysical mechanisms for how epithelial cells can rapidly switch apical-basal polarity, which may be of relevance to the switching of polarity during cancer progression.

Acknowledgements (Optional):