
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.21.608970
preprint
1
Article
Characterization, enrichment, and computational modeling of cross-linked actin networks in trabecular meshwork cells
Li Haiyan
Harvey Devon H.
Dai Jiannong
Swingle Steven P.
Compton Anthony M
Sugali Chenna Kesavulu
Dhamodaran Kamesh
Yao Jing
Lin Tsai-Yu
Sulchek Todd
Kim Taeyoon
Ethier C. Ross http://orcid.org/0000-0001-6110-3052

Mao Weiming
21 8 2024
2024.08.21.608970https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.21.608970
nihpp-2024.08.21.608970.pdf
Abstract

Purpose

Cross-linked actin networks (CLANs) are prevalent in the glaucomatous trabecular meshwork (TM), yet their role in ocular hypertension remains unclear. We used a human TM cell line that spontaneously forms fluorescently-labeled CLANs (GTM3L) to explore the origin of CLANs, developed techniques to increase CLAN incidence in GMT3L cells, and computationally studied the biomechanical properties of CLAN-containing cells.

Methods

GTM3L cells were fluorescently sorted for viral copy number analysis. CLAN incidence was increased by (i) differential sorting of cells by adhesion, (ii) cell deswelling, and (iii) cell selection based on cell stiffness. GTM3L cells were also cultured on glass or soft hydrogel to determine substrate stiffness effects on CLAN incidence. Computational models were constructed to mimic and study the biomechanical properties of CLANs.

Results

All GTM3L cells had an average of 1 viral copy per cell. LifeAct-GFP expression level did not affect CLAN incidence rate, but CLAN rate was increased from ∼0.28% to ∼50% by a combination of adhesion selection, cell deswelling, and cell stiffness-based sorting. Further, GTM3L cells formed more CLANs on a stiff vs. a soft substrate. Computational modeling predicted that CLANs contribute to higher cell stiffness, including increased resistance of the nucleus to tensile stress when CLANs are physically linked to the nucleus.

Conclusions

It is possible to greatly enhance CLAN incidence in GTM3L cells. CLANs are mechanosensitive structures that affect cell biomechanical properties. Further research is needed to determine the effect of CLANs on TM biomechanics and mechanobiology as well as the etiology of CLAN formation in the TM.
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