Luciferase fragment complementation imaging in preclinical cancer studies

Abstract

The luciferase fragment complementation assay (LFCA) enables molecular events to be non-invasively imaged in live cells in vitro and in vivo in a comparatively cheap and safe manner. It is a development of previous enzyme complementation assays in which reporter genes are split into two, individually enzymatically inactive, fragments that are able to complement one another upon interaction. This complementation can be used to externally visualize cellular activities. In recent years, the number of studies which have used LFCAs to probe questions relevant to cancer have increased, and this review summarizes the most significant and interesting of these. In particular, it focuses on work conducted on the epidermal growth factor, nuclear and chemokine receptor families, and intracellular signaling pathways, including IP3, cAMP, Akt, cMyc, NRF2 and Rho GTPases. LFCAs which have been developed to image DNA methylation and detect RNA transcripts are also discussed.

Keywords: Cancer; Imaging; Luciferase.

Figure 1. Summary of luciferase fragment complementation strategies

The luciferase enzyme can be split into fragments (NL and CL), which, depending upon the specific site of cleavage, will or will not spontaneously complement. Fragments which do not spontaneously complement are able to complement when brought into close proximity. Such fragments can be used to image the interaction of two proteins of interest by either a simple complementation strategy (A) or by a split-intein (NI and CI) mediated splicing strategy (B). The complementation strategy (A) is reversible and enables interaction dynamics to be visualized. In addition to imaging protein-protein interactions, luciferase fragment complementation can also be used to image the presence of a specific cellular factor by adjacent binding (C), a conformational change in a peptide/protein (D) or protein/peptide cleavage (E). In this strategy, the luciferase fragments are fused to two self associating peptides (A and B), which are only able to bind to one another once enzymatic cleavage has occurred. Luciferase fragments which spontaneously complement have also been identified and are thought to offer the potential to image protein localization (E) and cellular macromolecule delivery (F).

Figure 2. Imaging estrogen receptor biology

(A) By imaging the interaction of estrogen receptor α (ERα) with the coactivator AIB1, activation of ERα by estrogen (E) and subsequent inhibition by the anti-estrogen ICI (I) can be non-invasively imaged in vivo. P = placebo. (B) Strategy to simultaneously image ERα/ERα and ERα/ERβ dimerization using Renilla (NR/CR) and firefly (NF/CF) luciferases, respectively. Figures have been adapted from [52] and [32], respectively.

Figure 3. Dual wavelength ratiometric sensor for imaging cAMP levels

Takeuchi and colleagues have successfully imaged cAMP levels using an engineered C terminal click beetle (CB) luciferase fragment (MC) which can complement with the N terminal fragment of CB red (NR) and green (NG) luciferases to emit red or green light, respectively. In the presence of cAMP, the intramolecular fusion protein undergoes a conformational change due to the inclusion of the cAMP binding domain of PKA (PKA-BD). Figure has been adapted from [63].

Figure 4. Imaging of DNA methylation

(A) Badran and colleagues have imaged global DNA methylation using the CpG island binding protein MBD1 (M). In high methylation conditions, luciferase fragment fused MBD1 proteins bind to adjacent CpG islands (red dots) and complementation between luciferase fragments can occur. (B) Using artificially engineered zinc finger proteins (ZF) which recognize specific DNA sequences, Huang and colleagues have imaged methylation at the L1PA2 locus. In the absence of methylation, the DNA is more accessible and the ZF proteins can bind to the DNA, leading to complementation between the intein fragments NI and CI, and splicing together of the two luciferase fragments. Figures adapted from [14] and [15], respectively.

Figure 5. Imaging of RNA transcripts

(A) Furman and colleagues have imaged the presence of specific RNA transcripts using the double stranded DNA binding proteins EC2 and Aart. Single stranded DNA probes, attached to double stranded DNA recognition sequences for EC2 and Aart, are engineered for the RNA of interest. These serve as a platform for luciferase fragment complementation. An alternative method for imaging the presence of specific RNAs has been developed by Kobatake's group. The group use the HIV-1 Rev-peptide and BIV Tat-peptide (blue polygon) which undergo a conformational change upon binding to the RNAs (depicted in red) RRE-RNA and TAR-RNA, respectively (B.i and ii). In order to detect a specific RNA of choice, the group produced split RNA probes to anneal to the RNA of interest and reform the RRE-RNA or TAR-RNA (B.iii). Once reformed, the RRE-RNA or TAR-RNA is detected by the HIV-1 Rev-peptide or BIV Tat-peptide and a change in luciferase activity is observed (B.iv). Figures adapted from [16] and [18], respectively.