Actin polymerization triggers gastric epithelial repair of damage
Authors: Aihara Eitaro , Matthis Andrea L. , Engevik Kristen A. , Turner Jerrold R. , Zhang Tongli , Montrose Marshall H. 
Published: June 2021

Background

Gastric epithelial repair, mediated by cell migration and dead cell exfoliation, is a rapid process. Actin dynamics, in collaboration with myosin, are essential for the cell migration during epithelial repair of damage. We previously demonstrated that actin dynamics play an important role in the repair of damage in vivo mouse stomach and in vitro gastric organoids (gastroids). To extend our current knowledge, we examined the repair of damage in gastroids generated from human actin‐GFP (HuGE) transgenic mouse stomach and investigated the involvement of actin polymerization and myosin in this process.

Methods

Gastroids were generated from gastric corpus of HuGE mice. Hoechst33342 (10 μg/ml) was added to image the nucleus. Selective high intensity illumination of single gastric epithelial cell nucleus (two‐photon; 730 nm light) caused a microscopic lesion via photodamage. Dynamic changes in actin‐GFP and damage size were quantified over time. In some cases, Lucifer yellow (20 μM) or Alexa647‐10K dextran (10 μM) was added to medium to determine cellular and/or epithelial leakage.

Results

In response to photodamage of gastroid epithelium, actin‐GFP expression increased within ~1 min in the lateral membranes neighboring the damaged cell, and stayed elevated in this position and in lamellipodia during the cellular migration inward that closes the gap at the basal pole of the dead cell. Synchronous dead cell exfoliation was observed. When no intentional damage was imposed, similar events occurred during natural cell shedding into the lumen of the gastroids. Assuming a bistable switch controlling actin polymerization, we constructed a mathematical model whose simulations recaptured the temporal order of the observed repair processes, and the model predicted actin‐mediated force led to the observed dead cell exfoliation. In undamaged gastroids, Cytochalasin D (inhibitor of actin assembly at F‐actin barded ends: 1 μM) did not alter total cellular actin‐GFP intensity, while Latrunculin A (inhibitor of G‐actin polymerization: 2 μM) reduced GFP intensity. Both inhibitors increased paracellular leakage in the gastroid over the time, and completely inhibited the damage‐induced increase of actin, as well as repair of damage. In contrast, in undamaged gastroids, Jasplakinolide (actin filament stabilizer: 1 μM) facilitated actin‐GFP accumulation in the membrane and decreased GFP in the cytosolic compartment in a time‐dependent manner. Short time exposures to Jasplakinolide or Latrunculin A were imposed such that there was not effect on basal actin‐GFP intensity. In this condition, Jasplakinolide did not affect repair of damage, but Latrunculin A completely inhibited repair. Blebbistatin (Myosin II inhibitor, 10 μM) significantly inhibited repair of damage and slowed dead cell ejection, with no effect on GFP‐actin accumulation.

Conclusion

New F‐actin polymerization or elongation is necessary to initiate gastric epithelial repair of damage. Furthermore, recruitment of myosin II supports generation of force to exfoliate the dead cell.

Support or Funding Information

NIH RO1 DK102551.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.