Letters pubs.acs.org/acschemicalbiology
Visualizing Cell Proximity with Genetically Encoded Bioluminescent Reporters Krysten A. Jones,‡ David J. Li,∥ Elliot Hui,∥ Mark A. Sellmyer,⊥ and Jennifer A. Prescher*,†,‡,§ †
Department of Chemistry, ‡Department of Molecular Biology & Biochemistry, §Department of Pharmaceutical Sciences, and Department of Biomedical Engineering, University of California, Irvine, California 92697, United States ⊥ Department of Radiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States ∥
S Supporting Information *
ABSTRACT: Cell−cell interactions underlie diverse physiological processes ranging from immune function to cell migration. Dysregulated cellular crosstalk also potentiates numerous pathologies, including infections and metastases. Despite their ubiquity in organismal biology, cell−cell interactions are difficult to examine in tissues and whole animals without invasive procedures. Here, we report a strategy to noninvasively image cell proximity using engineered bioluminescent probes. These tools comprise “split” fragments of Gaussia luciferase (Gluc) fused to the leucine zipper domains of Fos and Jun. When cells secreting the fragments draw near one another, Fos and Jun drive the assembly of functional, light-emitting Gluc. Photon production thus provides a readout on the distance between two cell types. We used the split fragments to visualize cell−cell interactions over time in vitro and in macroscopic models of cell migration. Further application of these tools in live organisms will refine our understanding of cell contacts relevant to basic biology and disease.
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cells are exposed to luciferin, the photons produced can penetrate tissues and small organisms and be captured by sensitive cameras. Luciferase-expressing cells can also be serially imaged in live animals, enabling biological processes to be monitored noninvasivey over both long time and length scales.5 While ideal for global cell tracking, traditional bioluminescence imaging lacks the spatial resolution to image cell contacts.6 Interacting cells, in theory, could be visualized using unique luciferase−luciferin pairs to selectively illuminate the distinct cell types. In practice, though, these probes cannot be readily distinguished on time scales relevant to most cellular contacts (2 cm). We envisioned using Gluc complementation to monitor cellular movements in real time. Such assays would enable changes in cell proximity to be detected from media aliquots, without perturbing the cells themselves. Toward this end, we patterned cells secreting either Jun-NGluc or Fos-CGluc at different distances (3 or 30 mm) using the stencils pictured in Figure 4A. Upon adherence and stencil removal, cell migration was monitored over time via media sampling and bioluminescence imaging (Figure 4D, Supporting Information Figure 10). Media was also replaced every 24 h to prevent the D
DOI: 10.1021/cb5007773 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
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although split reporters with faster folding rates or improved photon outputs could potentially capture such interactions. Intriguingly, since Gluc is long-lived in the extracellular environment, the complemented probe should enable the direct detection of cell−cell interactions in locations that are distinct from, and perhaps far removed from, the actual site of cell−cell interaction in vivo (e.g., in the bloodstream). We are currently evaluating this possibility in mouse models and our results will be published in due course. We also envision imaging direct cell−cell contacts by anchoring the Gluc fragments to distinct cell membranes or antibody targets. Collectively, such tools will provide a unique vantage point for visualizing cell−cell communication and, as such, improve our mechanistic understanding of biology and disease.
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METHODS
Detailed methods are available in the Supporting Information.
ASSOCIATED CONTENT
S Supporting Information *
Methods and supplementary Figures 1−10. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
K.A.J., M.A.S., and J.A.P. designed experiments. K.A.J. performed experiments, K.A.J., D.J.L., E.E.H., M.A.S., and J.A.P. analyzed and interpreted data. D.J.L. and E.E.H. provided the fabricated stencils. The manuscript was written by K.A.J. and J.A.P., with input from all authors. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a fellowship from the Hellman Family Foundation (to J.A.P.), the National Science Foundation (NSF) (CAREER to J.A.P.), and Defense Advanced Research Projects Agency (DARPA) (DP13AP00044 to E.E.H.). K.A.J. was supported by an institutional Chemical and Structural Biology Training Grant predoctoral fellowship (T32-GM10856). D.J.L. was supported by a fellowship through the NSF LifeChips program (IGERT 0549479). We also thank members of the Weiss, Edinger, Martin, and Hughes laboratories for reagents and equipment. Finally, we thank members of the Prescher and Hui laboratories for helpful discussions.
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DOI: 10.1021/cb5007773 ACS Chem. Biol. XXXX, XXX, XXX−XXX
