A Magnetic Nanoprobe Technology for Detecting Molecular Interactions in Live Cells

^* To whom correspondence should be addressed. E-mail: tkkim{at}kaist.ac.kr

Modern medicine faces the challenge of developing safer and more effective therapies. However, many drugs currently in use were identified without knowledge of their molecular targets (1, 2). Bioactive natural products are an important source of drug leads, but their modes of action are usually unknown (2). Elucidation of their physiological targets is essential for understanding their therapeutic and adverse effects, thereby enabling the development of second-generation therapeutics. Moreover, the discovery of novel targets of clinically proven compounds may suggest new therapeutic applications (3). Target identification (ID) is also important in chemical biology, where high-throughput screening is used to identify small molecules with a desired phenotype (4, 5). Despite the great benefits of such a screen, this approach has been hampered by the daunting task of target ID (1, 4, 5).

Fig. 1. Proof-of-principle experiments for MAGIC. (A) Enhanced intracellular uptake and endosomal escape of MNPs by TAT-HA2. HeLa cells were incubated with MNP coated with FITC (FITC-MNP) or with FITC and TAT-HA2 (FITC-MNP-TATHA2) in the absence or presence of 100 µM chloroquine for 12 hours. Live-cell confocal images were taken to show the intracellular distribution of FITC-labeled MNPs. Scale bars, 50 µm. (B and C) Translocation of MNPs inside cells by the magnetic field. HeLa cells were incubated with FITC-MNP-TATHA2 or QD565-TATHA2 in the presence of 100 µM chloroquine for 12 hours. Scale bars in (B), 50 µm. (D and E) DEVD-coated MNPs direct the translocation of caspase-3–mRFP by the magnetic field. HeLa cells were transfected with the expression plasmids for caspase-3–mRFP or EGFP, detached, mixed together, and replated. These cells were incubated with MNPs coated with DEVD and TAT-HA2 in the presence of 100 µM chloroquine for 12 hours. In (B) and (D), live-cell confocal images were taken before and after application of a magnetic field (M.F.), with the focal plane at the cellular basal surface. Cells expressing EGFP or caspase-3–mRFP are indicated by green or red arrowheads, respectively, in (D). Scale bar in (D), 50 µm. In (C) and (E), quantitative analysis of the signals was performed with more than 100 cells. [View Larger Version of this Image (61K GIF file)]

To visualize and track the distribution of MNPs within cells, we labeled MNPs with fluorescein isothiocyanate (FITC) (8) (Fig. 1A). FITC-MNPs were not efficiently introduced into the cell cytosol. Most of the internalized FITC-MNPs appeared to be trapped within endocytic vesicles (Fig. 1A). Attachment of transducible fusogenic TAT-HA2 peptide on the MNPs markedly enhanced endocytic uptake and subsequent release from endosomes. It is currently understood that the high density of cationic residues in the protein transduction domain of human immunodeficiency virus TAT protein causes an electrostatic interaction with the negatively charged cell surface, thus enhancing the chance of endocytic internalization, whereas the N-terminal 20 amino acids of the influenza virus hemagglutinin protein HA2 destabilizes endosomal membranes, causing them to release their contents into the cytosol (9, 10). Cotreatment with chloroquine, which is known to enhance endosome disruption (11), further increased the concentration of MNPs outside endosomes (Fig. 1A).

We next addressed whether internalized MNPs could be moved inside cells by an external magnetic field (Fig. 1, B and C). HeLa cells were transduced with MNPs coated with FITC and TAT-HA2. As a control, a luminescent nanocrystal quantum dot, QD565, which does not exhibit magnetism (12), was internalized into another set of HeLa cells with the use of TAT-HA2. Specific translocation of MNPs, but not QD565, was observed inside cells after brief application of a magnetic field (Fig. 1, B and C). Furthermore, this translocation was reversible; the MNPs rapidly diffused away upon removal of the magnetic field and were redirected when it was reapplied.

Fig. 2. Molecular target ID based on MAGIC. (A and B) HeLa cells were infected with the retroviral EGFP–fusion protein expression library (fig. S2B). After incubation of these cells with MNPs coated with FK506 and TAT-HA2 in the presence of 100 µM chloroquine for 12 hours, the subcellular localization of EGFP was examined in the absence or presence of a magnetic field (M.F.). To address potential false positives, we simultaneously monitored mRFP bicistronically coexpressed with EGFP–fusion protein. Live-cell confocal images were taken with the focal plane at the cellular basal surface (bottom) or with the pinhole size increased to collect whole-cell images (whole). Arrowheads indicate positive clones. RT-PCR and sequence analysis of mRNAs from these clones identified several proteins (Table 1) including FKBP12 (A) and an unknown protein with NCBI accession number BAB15266 [GenBank] (B). Scale bars, 100 µm. [View Larger Version of this Image (56K GIF file)]

To use MAGIC in systematic target ID for a bioactive small molecule, we exploited expression cloning based on a retroviral EGFP fusion protein expression library (Fig. 2). We sought to identify the receptor(s) for an immunosuppressant, FK506 (fig. S2A), in a genome-wide screen. A normalized EGFP-tagged cDNA library was generated (8); cDNAs from multiple human tissues were fused to the 5' or 3' end of the gene encoding EGFP, and mRFP was coexpressed as an internal control to address potential false positives (fig. S2B). This library was stably expressed in HeLa cells by retroviral transduction. After introduction of FK506-coated MNPs into these cells, a magnetic field was applied, and the subcellular localization of proteins expressed from cDNA-EGFP fusions was examined. Nineteen positive clones were identified that exhibited specific translocation of EGFP in the direction of the magnetic field while the subcellular localization of mRFP remained unchanged (Table 1 and Fig. 2). Reverse transcription polymerase chain reaction (RT-PCR) and BLAST analysis of mRNAs from these clones identified overlapping transcripts of several known FK506-binding proteins and proteins of unknown function (Table 1 and Fig. 2) (14–17). Specific interaction of these proteins with FK506 was verified by competition analysis (fig. S3). These interactions were also readily detected with other cell types (fig. S4).

Table 1. Protein targets of FK506 identified from expression cloning with the use of MAGIC. A total of 19 positive clones were identified from 10⁷ cells. The number of independent cDNA clones for each protein isolated from the screen is shown.

NCBI accession number Protein name Number isolated Reported binding to FK506 NP_463460 [GenBank] FKBP12 4 Yes (14–17) Q16645 FKBP12.6 2 Yes (17) P26885 FKBP13 1 Yes (17) AAA58475 [GenBank] FKBP25 2 Yes (15, 17) Q02790 FKBP52 3 Yes (16, 17) AAA86245 [GenBank] FKBP54 2 Yes (17) NP_068758 [GenBank] FKBP65 1 Yes (17) BAB15266 [GenBank] Unnamed 1 No BAB15220 [GenBank] Unnamed 1 No BAC03954 [GenBank] Unnamed 1 No BAD18781 [GenBank] Unnamed 1 No Total   19

The specificity of these interactions could be distinguished readily from background and false positive signals on the basis of reversible translocation manipulated by magnetic field at the initial screening stage (Fig. 2). Overall, the screen identified diverse targets with high efficiency and no false positives (Table 1). The additional targets discovered for FK506 might give some clues about the molecular mechanisms underlying its unexpected therapeutic actions and debilitating side effects (17).

To evaluate the feasibility of MAGIC for probing intracellular signaling processes, we used the NF-B/IB pathway (Fig. 3). Members of the NF-B family (e.g., RelA/p65) are sequestered in the cytoplasm by IB family members (e.g., IB). Various stimuli, including proinflammatory cytokines such as tumor necrosis factor– (TNF-), induce the phosphorylation of IB on two serine residues, Ser³² and Ser³⁶ (18). This phosphorylation results in recognition of IB by the F box protein ßTrCP of the SKP1/cullin/F-box ubiquitin ligase complex, leading to ubiquitin-dependent proteolysis of IB. These regulated protein-protein interactions allow NF-B proteins to translocate into the nucleus, resulting in the expression of target genes.

Fig. 3. Monitoring biological signaling processes by MAGIC. (A) Schematic of IB-mRFP, IB-ECFP-FKBP12, EYFP-RelA, and mRFP-ßTrCP. Symbols used in (B) and (E) are also explained. (B to D) Detection of signal-induced phosphorylation of IB. After transfection of HeLa cells with the expression plasmid for IB-mRFP, MNPs coated with antibody to phosphorylated Ser32 of IB, FITC, and TAT-HA2 were introduced into these cells. Magnetic field–induced translocation of FITC and mRFP signals was monitored before and after stimulation with TNF- (10 ng/ml) for 5 min in the absence or presence of prior treatment with SC-514 (1 mM). (E to G) Detection of signal-dependent association of IB with ßTrCP. After transfection of HeLa cells with the expression plasmids for IB-ECFP-FKBP12, EYFP-RelA, and mRFP-ßTrCP, MNPs coated with FK506 and TAT-HA2 were introduced into these cells. Magnetic field–induced translocation of enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), and mRFP signals was monitored before and after stimulation with TNF- (10 ng/ml) for 5 min in the absence or presence of prior treatment of SC-514 (1 mM). Scale bars, 50 µm. Shown in (D) and (G) are quantitative analyses of induced signals after stimulation with TNF- for 5 min with more than 100 cells. [View Larger Version of this Image (42K GIF file)]

Next we examined whether signal-dependent protein-protein interactions could be detected with MAGIC (Fig. 3, A, E to G). To entrap IB with MNPs inside cells, we used MNPs coated with FK506 and TAT-HA2 (TATHA2-MNP-FK506) together with IB tagged with FK506-binding protein, FKBP12 (IB-ECFP-FKBP12; Fig. 3, A, E to G). After expression of IB-ECFP-FKBP12, EYFP-RelA, and mRFP-ßTrCP in HeLa cells, these cells were transduced with TATHA2-MNP-FK506. Application of a magnetic field directed the translocation of ECFP signal (Fig. 3F), reflecting IB recruitment around MNPs by FK506-FKBP12 interaction inside cells. Under these conditions, EYFP-RelA, but not mRFP-ßTrCP, was directed toward the magnetic field, indicating that interaction occurs between IB and RelA, but not between IB and ßTrCP. Upon stimulation with TNF-, a marked increase in mRFP-ßTrCP translocation was observed (Fig. 3, F and G), demonstrating signal-induced interaction between IB and ßTrCP. Notably, SC-514 blocked TNF-–induced translocation of mRFP-ßTrCP (Fig. 3, F and G). Thus, MAGIC can faithfully probe molecular interactions dynamically regulated by specific signals in live cells.

In certain disease conditions (e.g., systemic inflammation), several cellular functions, such as endocytosis, are known to be compromised (19, 20). To demonstrate MAGIC in cells impaired in endocytosis, we adopted microinjection that can introduce exogenous materials directly into cells without passing through endocytic vesicles (21) (fig. S6). The signal-induced phosphorylation and protein-protein interaction between IB and RelA were probed in HeLa cells pretreated with TNF-, which inhibited the endocytic uptake of MNPs, as described (20). MNPs coated with antibody to phosphorylated Ser³² of IB or with FK506 were microinjected into TNF-–pretreated HeLa cells expressing IB-mRFP or IB-ECFP-FKBP12/EYFP-RelA/mRFP-ßTrCP, respectively (8). Signal-induced phosphorylation and interaction of the NF-B/IBpathwaywere readily detected by MAGIC in this experimental setting (fig. S6).

MAGIC offers several advantages over target ID methods currently in use. First, it directly translates a physical molecular interaction into a clear readout signal, unlike indirect readout methods that are dependent on intermediary interactions (22), overall expression profiles (23), or complex biological phenotypes (24). Thus, intrinsic false positives/negatives or error-prone deductions about molecular target(s) of a small molecule are obviated. Second, by probing such interactions in a physiologically relevant context, misleading outcomes produced by an artificial experimental setting (22, 24–28) can be greatly diminished. Third, it is amenable to dynamic, single-cell analysis of interactions. Finally, MAGIC can be used to detect a variety of biological interactions and protein modifications within live cells in a broad range of tissues and disease states. With the great advantage of being able to detect dynamic interactions between the biomolecules within mammalian cells, this technology could be exploited in genome-wide interaction screens.

The benefits of MAGIC may be best achieved through efficient and nondisruptive introduction of MNPs into cells. Prolonged incubation of cells with TAT-HA2–conjugated MNPs may affect cellular physiology, and microinjection cannot be used for large populations of cells. Other technologies for delivering biologically active cargos into cells (10) will be helpful to complement MAGIC.

References and Notes

The editors suggest the following Related Resources on Science sites:

In Science Magazine

LETTERS
Editorial Expression of Concern

Donald Kennedy and Bruce Alberts (7 March 2008)
Science 319 (5868), 1335b. [DOI: 10.1126/science.1157223]
 |  Full Text »  |  PDF »

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:

ADVERTISEMENT