Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases

Abstract

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.

G-Trap Assay I: Small GTPases as sensitive immune response biomarkers for active bacteremia

“…GTP binding assays were performed using a GTPase effector trap flow cytometry assay (G-Trap). [36, 37] This protocol has been fully described in a methods chapter. [37] A 48-well plate is seeded with 20,000 Vero E6 or TIME cells in 100 μL of culture medium per well, resulting in about 50,000 cells the next day.…”

“…[36, 37] This protocol has been fully described in a methods chapter. [37] A 48-well plate is seeded with 20,000 Vero E6 or TIME cells in 100 μL of culture medium per well, resulting in about 50,000 cells the next day. First, the culture medium is removed and replaced with 100 μL of serum-free medium overnight.…”

“…[35][36][37] We devised an assay called G-Trap [37] capable of quantitatively measuring activated GTP-bound Rho family GTPases due to host immune-inflammatory mediators and demonstrating value for early recognition of patient immune response to infection. [37] For initial proof-of-principle, we used lipopolysaccharide (LPS), a bacterial endotoxin, plasma from a patient with a culture-confirmed infection, and Fms-like tyrosine kinase-3 ligand (Flt-3L), a hematopoietic growth factor, [38,39] to stimulate GTP binding to Rho GTPases in phagocytes and endothelial cells via a common pathway [40] (Fig. 2B-D).…”

G-Trap Assay II: Characterization of blood Leukocyte Functionality differentiates immune activation and immune suppression in bacteremia patient samples

“…45 µl of cleared lysate was then analyzed for GTPase activity with G-Trap beads, functionalized with PAK-1 effector beads. 56 We used the CBC with diff. data to determine how much Rac1•GTP was produced by single cells.…”