Apicobasal Polarity Protein PARD3 Plays an Essential Role in Human Corneal Epithelial Cells

Sep 30, 2026

Apicobasal polarity-determinant Pard3 is examined in human corneal epithelial (CE) cells using a complementary cell culture model. PARD3-knockout (PARD3KO) cells were generated in hTCEpi by CRISPR/Cas9 and compared with the parental line using proliferation assays, adhesion and detachment tests, actin polymerization imaging, and real-time barrier and FITC-dextran permeability readouts. Sequencing deconvolution, immunoblots, and immunofluorescence confirm PARD3 loss, faster S-phase stalling, weaker ECM adhesion, cytoskeletal disruption, EMT marker upregulation, and defective junctional barrier function.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages15

Reading Time38 min

AccessFree to download

Purpose

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Conclusions

Cornea Apicobasal Polarity Protein PARD3 Plays an Essential Role in Human Corneal Epithelial Cells Simran Kumar,1 Peri Sohnen,1 Satinder Kaur,1 ,2 Mehak Vohra,1 ,3 Danielle M. Robertson,4 Sudha Swamynathan,1 and Shivalingappa K. Swamynathan1 1Department of Ophthalmology, University of South Florida, Tampa, Florida, United States 2Department of Ophthalmology, Stanford University, California, United States 3 Shroff Charity Eye Hospital, New Delhi, India 4Department of Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States Correspondence: Shivalingappa K. Swamynathan, Department of Ophthalmology, University of South Florida, 12901 Bruce B. Downs Blvd, Room 2115 Tampa, FL 33612, USA; [ss65@usf.edu](ss65@usf.edu). Received: August 19, 2025 Accepted: December 9, 2025 Published: January 5, 2026 Citation: Kumar S, Sohnen P, Kaur S, et al. Apicobasal polarity protein PARD3 plays an essential role inhuman corneal epithelial cells. Invest Ophthalmol Vis Sci. 2026;67(1):3 . [https://doi.org/10.1167/iovs.67.1.3](https://doi.org/10.1167/iovs.67.1.3) PURPOSE. Previously, we reported that the apicobasal polarity-determinant Pard3 regulates mouse corneal epithelial (CE) stratification and barrier function. Herein, we describe a complementary cell culture model to study the functions of PARD3 in greater detail. METHODS. PARD3-knockout (PARD3KO) cells were generated by CRISPR/Cas9-mutagenesis in hTCEpi, a telomerase-immortalized human CE cell line. We compared hTCEpi and PARD3KO cellular proliferation by CellTiter-Blue, crystal violet, xCELLigence Real Time Cell Analyzer, Ki67 staining and flow cytometry, extracellular matrix (ECM) affinity by adhesion assays and trypsinization time, actin polymerization by phalloidin staining, barrier function by Real-Time Cell Analyzer and FITC-dextran permeability assays, and relevant markers’ expression by quantitative RT-PCR, immunoblots, and immunofluorescence. RESULTS. Deconvolution of sequencing data, immunoblots, and immunofluorescence confirmed PARD3 ablation in PARD3KO cells, which displayed 1.44-to 1.66-fold faster proliferation with a shorter doubling time of 19.06 hours compared with 21.84 hours for hTCEpi cells. The G1-S phase regulators CCND2, CCNE, and CDK2 were upregulated, while the mitosis-promoting factor components CCNB1 and CDK1 were downregulated in PARD3KO cells, consistent with their increased stalling at S phase revealed by flow cytometry. PARD3KO cells also displayed 0.5- to 0.8-fold weaker adhesion to different components of ECM, 1.37-fold faster trypsin-mediated detachment, 1.4-fold faster gap filling, disrupted actin cytoskeleton, nuclear β-catenin, upregulated epithelial– mesenchymal transition markers ZEB1, TWIST1, SLUG, and α-smooth muscle actin, and defective barrier function coupled with decreased expression of cell junctional components. CONCLUSIONS. PARD3 regulates human CE cell proliferation, ECM-binding, migration, F-actin polymerization, and barrier function. Loss of PARD3 activity pushes CE cells toward epithelial–mesenchymal transition. Keywords: corneal epithelium, Pard3, Par3, apical-basal polarity, tight junction, actin cytoskeleton, barrier function T he corneal epithelium (CE) is a transparent, nonkera tinized stratified squamous tissue.1 Being the most anterior tissue of the eye, it serves as a barrier that protects the rest of the eye from external insults and microbial pathogens.2 The CE barrier function and structural rigidity depend on multiprotein cell junction complexes that are connected with the cytoskeleton.2 Cellular polarity plays an important role in coordinating the structure and function of cell junction components and the cytoskeleton in diverse epithelial cells, including the CE.3 Despite the known requirement of a healthy CE for sight, and the wellestablished role of cellular polarity in epithelial form and function, CE polarity remains understudied. Polarity, a defining feature of most ep

FAQs

How were PARD3-knockout human corneal epithelial cells generated?

PARD3KO cells were created by CRISPR/Cas9 mutagenesis of PARD3 in hTCEpi, a telomerase-immortalized human corneal epithelial cell line.

What changes in proliferation and cell-cycle regulators were observed after PARD3 loss?

PARD3KO cells showed faster proliferation and a shorter doubling time, with upregulation of CCND2, CCNE, and CDK2, and downregulation of CCNB1 and CDK1, consistent with S-phase stalling.

How does loss of PARD3 affect adhesion, cytoskeleton, and barrier function?

PARD3KO cells exhibited reduced adhesion to extracellular matrix components, faster trypsin-mediated detachment, disrupted actin cytoskeleton, altered junctional component expression, and defective barrier function measured by real-time assays and FITC-dextran permeability.

Sep 30, 2026

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