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Proceedings Paper

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

Paper Details

Date Published: 2 February 2006
PDF: 8 pages
Proc. SPIE 6026, ICO20: Biomedical Optics, 60260U (2 February 2006); doi: 10.1117/12.667180
Show Author Affiliations
Jie Yang, Huazhong Univ. of Science and Technology (China)
Zhihong Zhang, Huazhong Univ. of Science and Technology (China)
Bifeng Liu, Huazhong Univ. of Science and Technology (China)
Qingming Luo, Huazhong Univ. of Science and Technology (China)

Published in SPIE Proceedings Vol. 6026:
ICO20: Biomedical Optics
Gert von Bally; Qingming Luo, Editor(s)

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