Volume 30 Issue 5 - May 6, 2016 PDF
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Mechanosensitive store-operated calcium entry regulates the formation of cell polarity
Yi-Wei Huang1, Shu-Jing Chang2, Hans I-Chen Harn3, Hui-Ting Huang4, Hsi-Hui Lin1, Meng-Ru Shen3,5,*, Ming-Jer Tang1,3, and Wen-Tai Chiu2,3,6
1 Department of Physiology, National Cheng Kung University, Tainan, Taiwan
2 Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan
3 Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan
4 Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan
5 Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan
6 Medical Device Innovation Center, National Cheng Kung University, Tainan, Taiwan
 
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The formation of cell polarity is essential for directional migration, which plays an important role in the physiological and pathological processes of an organism. For example, neuronal cells migration during corticogenesis, leukocytes extravasation at inflammatory site, angiogenesis, wound repair and cancer metastasis are typical cases of directional migration. Previous studies have indicated that calcium (Ca2+) is concentrated at the rear end of polarized cells. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx in non-excitable cells, and the major molecular components in the regulation of SOCE are the ER Ca2+ sensor stromal interaction molecule 1 (STIM1) and two plasma membrane Ca2+ channels transient receptor potential channel 1 (TRPC1) and Orai1. To examine the critical role of SOCE, we employed human bone osteosarcoma U2OS cells, which exhibit distinct morphological polarity during directional migration. Our analyses showed that Ca2+ was concentrated at the rear end of cells and that extracellular Ca2+ influx was important for cell polarization. Inhibition of SOCE using specific inhibitors disrupted the formation of cell polarity in a dose-dependent manner. Moreover, the channelosomal components caveolin-1, TRPC1 and Orai1 were concentrated at the rear end of polarized cells. Knockdown of TRPC1 or a TRPC inhibitor, but not knockdown of Orai1, reduced cell polarization. Furthermore, disruption of lipid rafts or overexpression of caveolin-1 contributed to the downregulation of cell polarity. On the other hand, we also found that cell polarity, SOCE activity and cell stiffness were markedly decreased by low substrate rigidity, which may be caused by the disorganization of actin filaments and microtubules that occurs while regulating the activity of the mechanosensitive TRPC1 channel.
Ca2+ regulates the formation of cell polarity. Ca2+ is concentrated at the rear end of polarized cells. Fluo-4/AM, a chemical Ca2+ fluorescent probe, was used to evaluate the cytosolic Ca2+ levels in polarized and non-polarized U2OS cells. Cells were treated with 2μM Fluo-4/AM for 30 minutes. The fluorescence images were taken using a confocal microscope under excitation by a 488 nm laser. Fluo-4/AM emission is presented in green fluorescence (upper panel) and pseudocolor (lower panel) images.
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