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In JoVE (1)
Other Publications (2)
Articles by Christopher Pohlmeyer in JoVE
JoVE Bioengineering
Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
1Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University, 2Graduate School of Pharmaceutical Sciences, University of Tokyo, 3Biomedical Engineering, Johns Hopkins University
A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.
Other articles by Christopher Pohlmeyer on PubMed
A Photocleavable Rapamycin Conjugate for Spatiotemporal Control of Small GTPase Activity
We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.
Triggering Actin Comets Versus Membrane Ruffles: Distinctive Effects of Phosphoinositides on Actin Reorganization
A limited set of phosphoinositide membrane lipids regulate diverse cellular functions including proliferation, differentiation, and migration. We developed two techniques based on rapamycin-induced protein dimerization to rapidly change the concentration of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. First, using a membrane-recruitable form of PI(4)P 5-kinase, we increased PI(4,5)P(2) synthesis from phosphatidylinositol 4-phosphate [PI(4)P] and found that COS-7, HeLa, and human embryonic kidney 293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P(2) without consuming PI(4)P induced membrane ruffles. These distinct phenotypes were mediated by dynamin-mediated vesicular trafficking and mutually inhibitory crosstalk between the small guanosine triphosphatases Rac and RhoA. Our results indicate that the effect of PI(4,5)P(2) on actin reorganization depends on the abundance of other phosphoinositides, such as PI(4)P. Thus, combinatorial regulation of phosphoinositide concentrations may contribute to the diversity of phosphoinositide functions.
