Morphological Observation of the Cashmere Goat Fetal Fibroblasts after mTOR Kinase Inhibition with Combination of Fluorescent Dyes and Confocal Cell Imaging

Abstract

The mammalian target of rapamycin（mTOR）is a kind of Ser/Thr kinase in mammalian cells. It can recruit and integrate input signals from nutrients, growth factors, energy and environmental stress to regulate cell growth and proliferation via different cellular processes. This study uses the fetal fibroblasts of Inner Mongolia Cashmere goat (Capra hircas) to prove that the mTOR plays a critical role in formation of the cell cytoskeleton structure. The mTOR kinase activity was inhibited in Inner Mongolia Cashmere goat fetal fibroblasts (GFb) after treatment with CCI-779 (temsirolimus), an mTOR specific inhibitor for 48 h. The results showed that GFb cells were sensitive to CCI-779. GFb cells morphology and its cytoskeleton structure changed under confocal laser scanning microscopy stained with the Fluorescent phalloidin (50µg/ml, Phalloidin-FITC5282) which combines with F-actin. In summary, mTOR signaling pathway was proved to be functional in GFb cells and acts as a key regulator to form cell structure. The Morphological results indicated that perhaps the synthesis of microfilament or organization of cytoskeleton was disrupted in GFb cells when mTOR was inhibited.