Fluorescence lifetime imaging: Association of cortical actin with a PIP3-rich membrane compartment

We have used fluorescence lifetime imaging (FLIM) to study actin and plasma membrane dynamics in B16-F1 melanoma cells. In the absence of a FRET acceptor, significant changes in the fluorescence lifetime of GFP were induced simply by linking the fluorophore to different functional probes, including β-actin, the PH domains of PLCδ and Akt, the Ras farnesylation signal, and the neuromodulin palmitoylation signal (MEM). In contrast, the lifetime of GFP–actin was constant despite the many different local environments of G- and F-actin within the cell. Treatment with cytochalasin D but not latrunculin A significantly shortened the lifetime of GFP–β-actin in the absence of a FRET acceptor. Robust lifetime shifts were observed using either a GFP–RFP chimera or co-transfection of GFP–MEM with RFP–MEM. In contrast to previous reports we observed a photobleaching-dependent change in the lifetime of GFP which could complicate the interpretation of FRET experiments. Of the membrane probes tested only the fluorescence lifetime of GFP–Akt was influenced by the presence of mRFP–actin, suggesting that the cortical actin meshwork is associated with a PIP3-enriched compartment of the plasma membrane. These results will aid in the design of new FRET-based approaches to study cytoskeletal interactions at the molecular level.