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 beta-actin, the PH domains of PLCdelta 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-beta-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.
König
Ireen
I
Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK.
Schwarz
Juliane P
JP
Anderson
Kurt I
KI
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008856:Microscopy, Fluorescence; D018129:Phosphatidylinositol Phosphates / Q000032:analysis
D018129:Phosphatidylinositol Phosphates; D049452:Green Fluorescent Proteins