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Published on: December 29, 2017
Fluorescence lifetime imaging: association of cortical actin with a PIP3-rich membrane compartment
Ireen König1, Juliane P Schwarz, Kurt I Anderson
1Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK.
Abstract:
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.
Insights
Fluorescence lifetime imaging (FLIM) reveals that GFP probe linkage affects fluorescence lifetime, but GFP-actin lifetime remains constant. This study advances FRET-based cytoskeletal interaction research.
Area of Science:
- Cell Biology
- Biophysics
- Molecular Imaging
Background:
- Fluorescence resonance energy transfer (FRET) is crucial for studying molecular interactions.
- Understanding protein dynamics in living cells requires precise imaging techniques.
- Actin and plasma membrane interactions are vital for cellular processes.
Purpose of the Study:
- To investigate actin and plasma membrane dynamics using fluorescence lifetime imaging (FLIM).
- To evaluate the impact of different functional probes on GFP fluorescence lifetime.
- To explore FRET-based approaches for studying cytoskeletal interactions.
Main Methods:
- Utilized fluorescence lifetime imaging (FLIM) on B16-F1 melanoma cells.
- Employed various GFP-linked functional probes (beta-actin, PH domains, signaling motifs).
- Applied FRET experiments with GFP-RFP chimeras and co-transfections.
Main Results:
- GFP fluorescence lifetime varied with probe linkage but was constant for GFP-actin.
- Cytochalasin D, not latrunculin A, shortened GFP-beta-actin lifetime without a FRET acceptor.
- GFP-Akt lifetime was influenced by mRFP-actin, suggesting association with PIP3-enriched membrane compartments.
Conclusions:
- Probe-specific environmental effects on GFP lifetime must be considered in FRET experiments.
- The cortical actin meshwork associates with PIP3-enriched plasma membrane regions.
- FLIM provides valuable insights for designing advanced FRET-based cytoskeletal studies.
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