Fluorescence Lifetime Imaging Application to Probe GTPase Activation in Macrophage Cell Line, Using the Time-Domain FastFLIM Modality

The p21 Rho family of small GTPases, RhoA, Rac1, and Cdc42, play vital roles in regulating actin dynamics and cell motility. These GTPases alternate between active (GTP-bound) and inactive (GDP-bound) states to modulate downstream signaling pathways that control cellular behavior. Monitoring their activation dynamics is essential for understanding cell morphodynamics and physiology, particularly in hematopoietic cells like monocytes and macrophages that are highly motile. Fluorescence resonance energy transfer (FRET)-based biosensors enable real-time visualization of Rho GTPase activities, but conventional ratiometric approaches can be limiting due to nonlinearity, making data interpretation challenging. Fluorescence lifetime imaging microscopy (FLIM) offers a quantitative alternative by directly measuring the change in donor fluorophore lifetime during FRET, circumventing acceptor imaging and ratiometric limitations. However, traditional FLIM methods can be technically challenging due to high photon demands and complex equipment. We discuss an alternative method of FLIM imaging using a time-domain FastFLIM system that supports rapid, sub-second imaging with reduced photon requirements, enabling visualization and quantification of FRET in a macrophage cell line. We demonstrate the utility of FastFLIM in RAW264.7/LR5 macrophages expressing a single-chain Rac GTPase FRET biosensor, showing Rac1 activation in response to mCSF1 (murine colony-stimulating factor 1) stimulation. This approach provides quantitative FRET data on GTPase dynamics, and we discuss herein practical guidance for researchers employing FastFLIM to study cell signaling.