Authors: Chunyan Cao, Huijing Liu & Gaolin Liang
We developed a new “smart” Eu-based probe (2) which is susceptive to furin, a protease overexpressed in cancer cells. Upon furin cleavage, 2 condenses to form oligomers and the latter self-assemble into Europium nanparticles (Eu-NPs) on site. Two-photon laser microscopy (TPLM) imaging of MDA-MB-468 cells incubated with 2 showed strong fluorescence signals from Golgi networks, suggesting 2 was under the action of furin and trapped at/near the locations of furin (i.e., Golgi networks). TPLM imaging of MDA-MB-468 cells incubated with the scrambled control of 2 (i.e., 2-Scr) at same condition only exhibits uniform, weak fluorescence signals. These results suggest that 2 could be a useful probe for TPLM imaging of furin activity in cancer cells. We describe herein a detailed protocol of cell preparation and TPLM imaging with 2.
The trans-Golgi protease furin is a protein convertase playing crucial roles in homeostasis, and in diseases ranging from anthrax and Ebola fever to Alzheimer’s disease and cancer (1). Increase of furin in tumors correlates with the increase of membrane type 1-matrix metalloproteinase (MT1-MMP), which activates extracellular pro-MMP2 to induce rapid tumor growth and metastasis (2). Therefore, noninvasive imaging of furin activity offers a valuable tool to probe tumor growth and migration in real time and directly assess the anti-cancer efficacy of drugs in vivo (3). It has been reported that the majority of human breast cancer cells overexpress furin (4).Traditional immunofluorescence staining of MDA-MB-468 cells indicates that furin is predominantly located in the trans-Golgi networks of this type of breast cancer cells (5). While there are very few methods that have been reported to image furin activity directly, Rao and coworkers developed two methods of intracellular condensation and intramolecular macrocyclization for imaging furin activity in living cells using fluorescence probes (6-7). Two-photon laser microscopy (TPLM) is a fluorescence imaging technique that allows imaging of living tissues up to a very high depth. It uses red-shifted excitation light to excite fluorescent dyes. For each excitation, two photons of the infrared light are absorbed simultaneously. TPLM can be a superior technique due to its deep tissue penetration, efficient light detection and reduced phototoxicity (8). Furin preferentially cleaves Arg-X-Lys/Arg-Arg↓X motifs, where Arg is arginine, Lys is lysine, X can be any amino acid residue and ↓indicates the cleavage site (9). Combining these two advantages above, recently we developed Acetyl-Arg-Val-Arg-Arg-Cys(StBu)-Lys(Eu-DOTA)-CBT (2) for imaging furin-controlled condensation in MDA-MB-468 cells (Fig. 1) (10). Its scrambled control, 2-Scr, was studied in parallel. In brief, 2 contains a RVRR peptide sequence for furin cleavage and cell membrane translocation, disulfided Cys for supplying the 1,2-aminothiol group for condensation with the cyano group on the benzothiazole motif, Lys conjugated with Eu-DOTA for TPLM. With the probes designed, we successfully imaged the furin-controlled intracellular condensation of 2, as well as the location and activity of furin (Fig. 2). We describe herein a detailed protocol of cell preparation and TPLM imaging with 2.
The two europium probes developed in this protocol could be used as one pair for TPLM imaging furin activities in cancer cells. Figure 1 shows the chemical structures of the two probes used in this protocol, in which 2 has a RRVR peptide substrate for furin cleavage. Following this protocol to prepare cell samples, 2 condenses to form Europium nanparticles (Eu-NPs) intracellularly resulting in strong fluorescence emission from the locations of furin (i.e., Golgi networks), as exampled in figure 2a. In contrast, since 2-Scr is not susceptive to furin, TPLM imaging of cancer cells incubated with 2-Scr will exhibit uniform, weak fluorescence signal, as illustrated in Figure 2b.
Figure 1: Chemical structures of 2 and 2-scr.
Figure 2: TPLM images (λex = 725 nm, λem = 565-636 nm) of MDA-MB-468 cells incubated with 2 (a) or 2-Scr (b) at 100 μM for 8 h and then rinsed and fixed prior to imaging. Scale bar: 20 μm.
The authors are grateful to the Center for Integrative Imaging (CII) of Hefei National Laboratory for Physical Science at the Microscale for the imaging facilities.
Controlled intracellular self-assembly of gadolinium nanoparticles as smart molecular MR contrast agents. Chun-Yan Cao, Ying-Ying Shen, Jian-Dong Wang, Li Li, and Gao-Lin Liang. Scientific Reports 3() 03/01/2013 doi:10.1038/srep01024
Chunyan Cao & Gaolin Liang, CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China
Huijing Liu, Department of Neurobiology and Biophysics, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China
Correspondence to: Gaolin Liang ([email protected])
Source: Protocol Exchange (2013) doi:10.1038/protex.2013.005. Originally published online 3 January 2013.