Optical Tools To Study the Isoform-Specific Roles of Small GTPases in Immune Cells

Abstract

Despite the 92% homology of the hematopoietic cell-specific Rac2 to the canonical isoform Rac1, these isoforms have been shown to play non-redundant roles in immune cells. To study isoform-specific dynamics of Rac in live cells we developed a genetically-encoded, single-chain FRET-based biosensor for Rac2. We also made significant improvements to our existing single-chain Rac1 biosensor. We optimized the biosensor constructs for facile expression in hematopoietic cells and performed functional validations in murine macrophage sublines of RAW264.7 cells. Rac2, along with Rac1and Cdc42, have been implicated in the formation of actin-rich protrusions by macrophages, but their individual activation dynamics have not been previously characterized. We found that both Rac1 and Rac2 had similar activation kinetics yet they had very distinct spatial distributions in response to the exogenous stimulus, fMLP. Active Rac1 was mainly localized to the cell periphery, while active Rac2 was distributed throughout the cell with an apparent higher concentration in the perinuclear region. We also performed an extensive morphodynamic analysis of Rac1, Rac2 and Cdc42 activities during the extension of random protrusions. We found that Rac2 appears to play a leading role in the generation of random protrusions, as we observed an initial strong activation of Rac2 in regions distal from the leading edge, followed by the activation of Rac1, a second burst of Rac2 and then Cdc42 immediately behind the leading edge. Overall, isoform-specific biosensors that have been optimized for expression should be valuable for interrogating the coordination of Rho family GTPase activities in living cells.

Platelet-derived growth factor receptor-α and Ras-related C3 botulinum toxin substrate-1 regulate mechano-responsiveness of lung fibroblasts

“…27795), mVenus N1 (catalog no. 27793; Addgene, Cambridge, MA); modification V2 Rac1 WT FRET (Förster resonance energy transfer) biosensor; mCerulean donor, and mVenus acceptor (Louis Hodgson, Albert Einstein University, New York, NY) (38). The biosensor was cloned into the pacAd5CMVmcspA shuttle vector and incorporated into Adenovirus 5 (University of Iowa Viral Vector Core, Iowa City, IA).…”

“…Others have shown that Rac1 mediates mechanosensitive responses (43). When GTP, rather than GDP, is bound to the Rac1-FRET biosensor, it undergoes a conformational change, increasing the proximity between the mCerulean donor and mVenus acceptor, thereby enabling FRET (38).…”

Kalirin/Trio Rho GDP/GTP exchange factors regulate proinsulin and insulin secretion

“…For studies probing the effects of GEF1 domain inhibition, these cell permeant inhibitors were added either at the start of the chase period (pulse chase) or during a 3-h preincubation before the start of immunofluorescence-labeling experiments. Plasmid: The Rac1 FRET biosensor to monitor Rac1 localization and activity has been described in detail (Moshfegh et al 2014, Miskolci et al 2016 and consisted of monomeric Cerulean (mCerulean) fluorescent protein, two tandem p21-binding domains (PBD; amino-acid residues 70-149) from Pak1, separated by a linker, then followed by monomeric Venus (mVenus) fluorescent protein and a full-length region of Rac1. For ratiometric imaging, 3 × 10 5 cells were transiently transfected with the Rac1 FRET biosensor using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions.…”

“…Whole cell average Rac1 activity was determined by thresholding the cell area in the FRET/donor ratio image using Metamorph Software. To measure the 'edge over core' ratio of Rac1 activities, we performed unbiased edge erosion measurements of ratio intensities as described previously (Miskolci et al 2016). We measured the average FRET/donor intensities within a region defined as distance of ten pixels (2.2 µm) from the edge and compared this to the average FRET/donor intensity from the rest of the cell body.…”

“…To determine that cell-surface association also represented Rac1 activation, we transiently expressed a genetically encoded FRET-based Rac1 biosensor whose localization and activity have been described in detail (Moshfegh et al 2014, Miskolci et al 2016. ITX3 was used to assess the role of Kalirin/Trio in the relocalization and activity of Rac1 in response to high glucose.…”