Using Fluorescence Resonance Energy Transfer-Based Biosensors to Probe Rho GTPase Activation During Phagocytosis

Abstract

The p21-family members of Rho GTPases are important for the control of actin cytoskeleton dynamics, and are critical regulators of phagocytosis. The three-dimensional structure of phagosomes and the highly compartmentalized nature of the signaling mechanisms during phagocytosis require high-resolution imaging using ratiometric biosensors to decipher Rho GTPase activities regulating phagosome formation and function. Here we describe methods for the expression and ratiometric imaging of FRET-based Rho GTPase biosensors in macrophages during phagocytosis. As an example, we show Cdc42 activity at the phagosome over Z-serial planes. In addition, we demonstrate the usage of a new, fast, and user-friendly deconvolution package that delivers significant improvements in the attainable details of Rho GTPase activity in phagosome structures.

A spatial model of YAP/TAZ signaling reveals how stiffness, dimensionality, and shape contribute to emergent outcomes

“…6, compare A to B and A to C). Indeed, Rho biosensor experiments show that localization and activity is not biased to the basal surface of cells on 2D substrates (65)(66)(67)(68), suggesting that our 2.xD model geometry is a more relevant representation of 2D culture.…”

Characterization of Genetically Encoded FRET Biosensors for Rho-Family GTPases

“…We perform this type of analysis routinely by quantifying the rate of protrusion and other cell motility parameters. There are various methods previously described to achieve these imaging conditions and expression levels of the biosensor, including the stable expression of biosensors using viral transduction and inducible expression strategies [27, 37, 39, 40].…”

“…For macrophage cell line RAW/LR5, we directly plate them onto cleaned coverslips. Transfection procedure for RAW/LR5 cells using the Fugene HD reagent was previously described in detail [39].…”

Optogenetics: Rho GTPases Activated by Light in Living Macrophages

“…RAW/LR5 cell line can be readily made to express FRET biosensors for Rho GTPases [15,22], including those that fluoresce in near infrared (NIR) [23]. This is advantageous because NIR FRET biosensor can be used together with other optogenetic tools that require blue/green light for photo-uncaging [23].…”

“…This is advantageous because NIR FRET biosensor can be used together with other optogenetic tools that require blue/green light for photo-uncaging [23]. The description of biosensors based on CFP-YFP FRET and NIR FRET systems is beyond the scope of this work, but RAW/LR5 cells stably expressing the tet-OFF tTA are available [15]; thus, inducible NIR-FRET biosensor cell line can be readily produced following previously published [22] protocols.…”