Hypotonicity Promote Gap Closure By Lamellipodial Protrusion

Objectives: By using a gap closure model of epithelial cells and time-lapse microscope,we compared the dynamics of gap closure under normal tonicity and hypotonic medium.To explore the mechanism underlying rapid gap closure promoted by hypotonicity,we analyzed the lamellipodia and actomyosin ring formation,respectively.Methods: Using a gap closure model of epithelial cells,we found that hypotonic condition could facilitate gap closure.Moreover,instead of actomyosin ring,enhanced migration leading by lamellipodia primarily contributed to the rapid gap closure in hypotonic condition.Results: We tracked the perimeter of the gap every 5 minutes.In the hypotonic condition,the average velocity of complete closure was 3.93 ±1.04 ?m/min(n = 5,mean ± s.d.).As a control,the velocity was 1.76 ±0.18 ?m/min(n = 6,mean ± s.d.)in the isotonic condition.Also,wo tracked the lamellipodial area of the gap every 5minutes.In the hypotonic condition,the average area was 1137.46±70.26?m2(n = 5,mean ± s.d.).As a control,the area was 60.44±21.87?m2(n = 6,mean ± s.d.).In hypotonic condition,no actin clustering at the gap interface was observed.Peak of F-actin intensity is more defined(higher and narrower)in normal condition,compared with hypotonic condition.Conclusion: Hypotonic condition could facilitate gap closure.Moreover,instead of actomyosin ring,enhanced migration leading by lamellipodia primarily contributed to the rapid gap closure in hypotonic condition.Without actin cable,gap closure is more quickly in hypotonic condition which indicates cell crawling appears to be the dominant mechanism of gap closure.