ReviewCell Biology

Toward total synthesis of cell function: Reconstituting cell dynamics with synthetic biology

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Science Signaling  09 Feb 2016:
Vol. 9, Issue 414, pp. re1
DOI: 10.1126/scisignal.aac4779

Figures

  • Fig. 1 Outline of the major themes.

    Normal cells under physiological conditions undergo proliferation and differentiation. Synthetic biology techniques enable the generation of synthetic bypasses, which enable the “basal” cell to achieve a proliferative or differentiated state without going through normal physiological pathways.

  • Fig. 2 Synthetic reconstitution of phagocytosis.

    To bypass the physiological mechanisms that mediate the phagocytosis of an apoptotic cell (left), a host cell is engineered (right) to express a plasma membrane–anchored FRB domain and a Golgi-localized fusion protein containing the PS-binding C2 domain and an FKBP domain. Upon addition of a dimerizing agent, the fusion protein is translocated to the plasma membrane, exposing the C2 domain on the cell surface so that it can bind to PS on the surface of the target apoptotic cell. Simultaneous activation of Rac1 signaling by the expression of a mutant protein in the host cell leads to actin reorganization and engulfment of the apoptotic cell. RTK, receptor tyrosine kinase; GTP, guanosine 5′-triphosphate; GDP, guanosine 5′-diphosphate; DOCK180, dedicator of cytokinesis; ELMO, engulfment and cell motility; FAK, focal adhesion kinase; Grb-2, growth factor receptor–bound protein-2; DG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Crk, CT10 regulator of kinase; Gas6, growth arrest specific 6.

  • Fig. 3 Synthetic reconstitution of directed cell migration.

    (A) Schematic of cellular chemotaxis toward native ligands through transmembrane receptors shows the activation of multiple downstream pathways. (B) Reconstitution of directed cell migration in HL-60 cells to a synthetic ligand, clozapine N-oxide (CNO), is achieved through the binding of a Gαi-coupled RASSL and activation of endogenous downstream signaling. CNO is released through a micropipette (orange) and attracts transfected HL-60 cells. Small circles in orange indicate individual CNO molecules binding to the receptors. (C) Reconstitution of directed cell migration in HeLa cells by graded activation of native Rac is achieved through a combination of CID and microfluidics. A rapamycin gradient (orange) is applied from top to bottom of the chamber, which induces the graded dimerization of Rac-GEF with a membrane-anchoring construct. Small circles in orange indicate individual rapamycin molecules entering into cells to actuate Rac. (D) Reconstitution of directed cell migration in mouse embryonic fibroblasts (MEFs) in vitro is achieved with 458-nm light to locally activate a synthetic, light-responsive Rac. A MEF is locally illuminated with 458-nm light (blue circle), which induces the formation of protrusions.

  • Fig. 4 PI(4)P depletion and PI(4,5)P2 synthesis have distinct effects on actin phenotypes.

    Actin comets can be generated by the activation of RhoA-ROCK signaling and of Arf6 without effecting upstream signaling events. (A) Actin comet formation by the PIP2 synthesis system. The increased local abundance of PI(4)P 5-kinase by rapamycin-based CID stimulates the PI(4,5)P2 synthesis” system, which increases PI(4,5)P2 abundance and reduces the amount of PI(4)P. PI(4,5)P2 synthesis promotes robust elongation of actin comet tails by providing a platform for the assembly of the endocytosis machinery. (B) Synthetic reconstitution of the actin comet formation system. In this system, endocytosis is induced by Arf6 activation by recruiting the Arf6-GEF Sec7 to the plasma membrane. In addition, activation of RhoA-ROCK signaling is achieved by ectopic expression of a constitutively active mutant of RhoA [RhoA(Q63L)] or a dominant-negative mutant of Rac1 [Rac1(T17N)]. (C) Images of the actin cytoskeleton (visualized by the actin-binding protein Evl; right) indicate synthetically generated actin comets (arrowheads). Confocal fluorescence microscopy images are of the actin cytoskeleton of cells expressing inactive “Mock” protein (mCherry), RhoA(Q63L) (middle), or Rac1(T17N) (bottom) before (left) and after (right) the rapid activation of Arf6. After Arf6 activation, robust membrane ruffling, but not actin comet formation, was observed (top). Whereas membrane ruffling was blocked by the expression of either of the GTPase mutants, actin comets were observed in cells in which RhoA-ROCK signaling and endocytosis were activated (middle and bottom). The figure is reproduced from Ueno et al. (36).

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