Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, and for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future.

Here we generate FRET biosensors for guanine exchange factors (GEFs) by inserting a fluorescent protein pair in a structural “hinge” common to many GEFs. Fluorescent biosensors can map the activation of signaling molecules in space and time, but it has not been possible to quantify how different activation events affect one another or contribute to a specific cell behavior. By imaging the GEF biosensors in the same cells as red-shifted biosensors of Rho GTPases, we can apply partial correlation analysis to parse out the extent to which each GEF contributes to the activation of a specific GTPase in regulating cell movement. Through analysis of spontaneous cell protrusion events we identify when and where the GEF Asef regulates the GTPases Cdc42 and Rac1 to control cell edge dynamics. This approach exemplifies a powerful means to elucidate the real-time connectivity of signal transduction networks.

Lysophophatidic acid (LPA), a biologically active phospholipid that is ubiquitously present in tissues and organs, provokes cellular responses such as proliferation, apoptosis, differentiation and migration via activation of G-protein coupled receptors. These receptors activate a broad range of intracellular signaling cascades to mediate these responses. Using microfluidic chambers that generate and maintain stable gradients, we observed that chemotaxis of fibroblasts to LPA has higher directional fidelity than chemotaxis provoked by the receptor tyrosine kinase (RTK) ligand platelet-derived growth factor (PDGF). Unlike fast moving amoeboid cells, mesenchymal cells such as fibroblasts do not require PI3K for chemotaxis to a GPCR ligand. In addition, the Arp2/3 complex is not required for fibroblast GPCR-based chemotaxis in either 2D or 3D environments. Our data indicate that combinatorial regulation of myosin II involving global activation by RhoA/ROCK and local inhibition of myosin II at the leading edge by PKC results in highly efficient chemotaxis of fibroblasts to LPA. Based on these observations, we develop a simple mathematical model to explain how dual regulation of myosin II is responsible for enhanced chemotaxis in LPA gradients relative to PDGF. Using pharmacological approaches, we test predictions of this model and modulate the fidelity of LPA and PDGF chemotaxis.