FRET biosensor allows spatio-temporal observation of shear stress-induced polar RhoGDIα activation

Abstract

Rho GDP-dissociation inhibitor α (RhoGDIα) is a known negative regulator of the Rho family that shuts off GDP/GTP cycling and cytoplasm/membrane translocation to regulate cell migration. However, to our knowledge, no reports are available that focus on how the RhoGDIα-Rho GTPases complex is activated by laminar flow through exploring the activation of RhoGDIα itself. Here, we constructed a new biosensor using fluorescence resonance energy transfer (FRET) technology to measure the spatio-temporal activation of RhoGDIα in its binding with Rho GTPases in living HeLa cells. Using this biosensor, we find that the dissociation of the RhoGDIα-Rho GTPases complex is increased by shear stress, and its dissociation rate varies with subcellular location. Moreover, this process is mediated by membrane fluidity, cytoskeleton and Src activity, which indicates that the regulation of RhoGDIα activation under shear stress application represents a relatively separate pathway from the shear stress-induced Rho pathway.

A high-resolution real-time quantification of astrocyte cytokine secretion under shear stress for investigating hydrocephalus shunt failure

“…In a microfluidic device, physiological shear stress can be created by fluid flow induction at a predetermined rate, in the presence of TNF-α/IL-1β to determine the influence of shear stress on specific astrocyte phenotypes. In this study, four microfluidic channels with different widths were designed to provide a multiplex culture platform to measure four different shear stress stimulations on astrocytes 31 , 32 (Fig. 2a ).…”

Structural and signaling role of lipids in plasma membrane repair

“…The nanoclusters appear to form specifically at the boundary of ordered raft domains and disordered domains where signaling lipids such as PIP 3 and PIP 2 are found. Shear stress on the plasma membrane also results in the dissociation of the negative regulator RhoGDI and its binding partner Rho (Shao et al, 2018). This dissociation has the effect of allowing Rho GTPase translocation to the membrane where it can interact with signaling lipids.…”

Heat Shock Protein 27 Phosphorylation Regulates Tumor Cell Migration under Shear Stress

“…Images were divided into 50 parts equally along the direction of shear stress as explained in Supplementary Figure S2. In combination to time sequence of photographing, the fluorescence intensity of each part at the previous moment of stimulation was used as a reference to normalize fluorescence intensity, after which a three-dimensional graph of time, space, and fluorescence intensity was drawn and a color bar given at the right side of the images that mentions the range of the FRET ratio [32,33].…”