Karginov, A. V. | Search bioRxiv

bioRxiv2022

Parallel Activation of Src and Hif1α Increases Localized Glycolytic ATP Generation for Re-assembly of Endothelial Adherens Junctions

Endothelial adherens junctions (AJs) are critical for the regulation of vascular barrier integrity and undergo dis-assembly during inflammatory injury, thus causing vascular leakiness. AJ re-assembly is thus necessary for restoration of the endothelial barrier following the initial injury. Here we examine the metabolic underpinnings that drive restoration of vascular integrity. In response to inflammatory stimuli, the glycolysis regulatory enzyme PFKFB3 is activated, resulting in a rapid and sustained increase of intracellular glycolytic ATP, especially in the proximity of AJs at the plasma membrane. We engineered a novel chemo-genetic construct (RapT) which allowed for precise temporal control of PFKFB3 recruitment to the plasma membrane. Activation of RapT by rapamycin during the barrier restoration phase increased regional ATP and accelerated AJ re-assembly. Mechanistically, we observed that PFKFB3 is activated through two modes. Src-mediated post-translational phosphorylation rapidly increases PFKFB3 activity. Using another chemo-genetic approach to temporally control Src activity, we found that Src activates PFKFB3 by binding to and phosphorylating it at residues Y175, Y334, and Y363. Tyrosine-phospho-deficient mutants of PFKFB3 at these residues block the glycolytic activation upon inflammatory stimuli. In parallel, elevated reactive oxygen species generated during inflammatory stimulation create pockets of regional hypoxia and allow for increased Hif1-mediated transcription of PFKFB3, leading to sustained glycolytic activation. Moreover, inhibition of PFKFB3 delays AJ reassembly and restoration of vascular integrity both in vitro and in vivo. In conclusion, we show that while inflammatory activation acutely compromises the endothelial barrier, inflammatory signaling also concomitantly generates a metabolic milieu in anticipation of the subsequent re-assembly of AJs and restoration of the vascular barrier.

cell biology↗

bioRxiv2021

FRET and LRET Biosensors for Cell-based Imaging and Screening of Rac1 Activation

Rac1 is a key regulator of several cell signaling pathways and dysregulated Rac1 activation has been implicated in cancer. Genetically encoded Forster resonance energy transfer (FRET) biosensors with enhanced dynamic range enabled live cell fluorescence imaging of Rac1 activity and a cell lysate-based assay of Rac1 inhibition in 96-well plates. We prepared HEK293 cell lines that stably expressed polypeptides with a general domain sequence (N- to C-terminus) of 1) FRET acceptor; 2) Rac/Cdc42 binding domain of human p21 protein kinase A (residues 68-150); 3) a linker domain; 4) FRET donor; and 5) full-length Rac1. Activated Rac1 binds to the protein kinase A domain, bringing donors and acceptors close together to increase FRET. We evaluated the effects on FRET signal dynamic range of alpha helical linkers comprised of alternating repeats of roughly four glutamate and four arginine or lysine residues. So-called ER/K linkers had limited effects on conventional FRET biosensors that incorporated the fluorescent protein (FP) pairs mCerulean/YPet, or mTFP1(cp227)/mVenus(cp229). Fluorometric measurements of cells that co-expresssed biosensors with positive (TIAM1) or negative (RhoGDI) Rac1 regulators revealed significant dynamic range enhancement in only one FP construct (mCerulean/YPet with 20 nm ER/K linker) relative to an analogous structure that incorporated an unstructured linker. We transfected this construct into a cell line that stably expressed a rapamycin-inducible c-Src analog (RapR-Src) and observed activation of Rac1 at protruding edges following rapamycin stimulation. In cells that expressed lanthanide-based FRET (LRET biosensors) that incorporated a luminescent terbium complex donor and GFP fluorescent acceptor, time-gated luminescence (TGL) measurements showed substantial gains in dynamic range that increased with linker length (up to 1200%). We robustly detected small molecule Rac1 inhibition following lysis of LRET biosensor-expressing cells grown directly in 96-well plates. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.

biochemistry↗