Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors.
Eukaryotic cells mobilize the actin cytoskeleton to generate a remarkable diversity of morphological behaviours, including motility, phagocytosis and cytokinesis. Much of this diversity is mediated by guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases-the master regulators of the actin cytoskeleton. There are over 80 Rho GEFs in the human genome (compared to only 22 genes for the Rho GTPases themselves), and the evolution of new and diverse GEFs is thought to provide a mechanism for linking the core cytoskeletal machinery to a wide range of new control inputs. Here we test this hypothesis and ask if we can systematically reprogramme cellular morphology by engineering synthetic GEF proteins. We focused on Dbl family Rho GEFs, which have a highly modular structure common to many signalling proteins: they contain a catalytic Dbl homology (DH) domain linked to diverse regulatory domains, many of which autoinhibit GEF activity. Here we show that by recombining catalytic GEF domains with new regulatory modules, we can generate synthetic GEFs that are activated by non-native inputs. We have used these synthetic GEFs to reprogramme cellular behaviour in diverse ways. The GEFs can be used to link specific cytoskeletal responses to normally unrelated upstream signalling pathways. In addition, multiple synthetic GEFs can be linked as components in series to form an artificial cascade with improved signal processing behaviour. These results show the high degree of evolutionary plasticity of this important family of modular signalling proteins, and indicate that it may be possible to use synthetic biology approaches to manipulate the complex spatio-temporal control of cell morphology.
Yeh
Brian J
BJ
Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, San Francisco, California 94158-2517, USA.
Rutigliano
Robert J
RJ
Deb
Anrica
A
Bar-Sagi
Dafna
D
Lim
Wendell A
WA
D000818:Animals; D002460:Cell Line; D048430:Cell Shape / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D011554:Pseudopodia / Q000502:physiology; D051381:Rats; D015398:Signal Transduction; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D017868:Cyclic AMP-Dependent Protein Kinases; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins