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Engineering NAVIGATION: Rewiring the cell to move towards new Targets

Motivation: Why is this useful?

We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.



Neutrophils (a type of white blood cell) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.



These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the "Migration Index." For receptors that appeared to activate a migration response (Migration Index > 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand.



6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index > 3.



Here, we show an example of one of these receptors (BBa_K209442: M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:





To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand.




Common characteristics of chemotaxis receptors: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.



Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets.


Summary and Outlook

We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways.

NEXT - Tuning receptor sensitivity



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