Real-time detecting gelatinases activity in living cells by FRET imaging

Jie Yang; Zhihong Zhang; Bifeng Liu; Qingming Luo

doi:10.1117/12.667180

2 February 2006 Real-time detecting gelatinases activity in living cells by FRET imaging

Jie Yang, Zhihong Zhang, Bifeng Liu, Qingming Luo

Proceedings Volume 6026, ICO20: Biomedical Optics; 60260U (2006) https://doi.org/10.1117/12.667180
Event: ICO20:Optical Devices and Instruments, 2005, Changchun, China

Abstract

Degradation of the extracellular matrix by Matrix metalloproteinases (MMPs) not only enhances tumor invasion, but also affects tumor cell behaviour and leads to cancer progression. To monitor gelatinases (contain MMP2 and MMP9) activity in living cells, we constructed a vector that encoded a gelatinases recognition site (GRS) between citrine (mutation of EYFP Q69M) in N terminal and ECFP in C terminal. Because Gelatinases are secretory proteins and act outside of cell, an expressing vector displayed the fusion protein on cellular surface was used for this FRET gene probe. On expression of YFP-GRS-ECFP in MCF-7 cells that expressed no gelatinases, we were able to observe the efficient transfer of energy from excited ECFP to YFP within the YFP-GRS-ECFP molecule. However, the fusion protein YFP-GRS-ECFP was expressed in MDA-MB 453s cell line with high secretory gelatinases, so YFP-GRS-ECFP was cleaved by gelatinases, no such transfer of energy was detected and fluorescence signal disappeared in YFP channel since YFP protein was cut down. Moreover, Doxycycline, a MMP inhibitor, could make FRET signal increase and fluorescence signal appeared in YFP channel. Thus, the FRET probe YFP-GRS-ECFP can sensitively and reliably monitor gelatinases activation in living cells and can be used for screening MMP inhibitors.

Citation Download Citation

Jie Yang, Zhihong Zhang, Bifeng Liu, and Qingming Luo "Real-time detecting gelatinases activity in living cells by FRET imaging", Proc. SPIE 6026, ICO20: Biomedical Optics, 60260U (2 February 2006); https://doi.org/10.1117/12.667180

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KEYWORDS

Fluorescence resonance energy transfer

Luminescence

Proteins

Cancer

Tumors

Signal detection

Fluorescence spectroscopy

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