PhD Scientific Days 2025

Poster Session II. - B: Molecular Medicine

A Genetically Encoded Fluorescent Biosensor to Investigate fMLF Release in Tissue-Damage Responses

Name of the presenter

Al-Sheraji Nada Mohamed

Institute/workplace of the presenter

Semmelweis University Department of Physiology

Authors

Nada Mohamed Al-Sheraji^1,2, Szimonetta Xénia Tamás^1,2, Diána Patrícia Kaszás^1,2, Attila Puskás¹, Fabian Dehne^1,2, Benoit Roux^1,2, Petra Dunkel³, Klaudia Vágó-Kiss^1,2, Balázs Enyedi^1,2

1: Department of Physiology, Semmelweis University, Budapest, Hungary
2: HCEMM-SE Inflammatory Signaling Research Group, Department of Physiology, Semmelweis University, Budapest, Hungary
3: Department of Organic Chemistry, Semmelweis University, Budapest, Hungary

Text of the abstract

Formyl peptides, such as fMLF (formyl-Met-Leu-Phe), act as both pathogen- and damage-associated molecular patterns (PAMPs/DAMPs), guiding innate immune cells to sites of infection and tissue damage via formyl peptide receptors (FPRs). Despite their potency as chemoattractants, the dynamics of fMLF release during infections, tissue damage, or cell death remain poorly understood.

To detect fMLF release, we developed a genetically encoded fluorescent biosensor, GEM-fMLF, by inserting circularly permuted EGFP (cpEGFP) into the third intracellular loop of FPR1. Screening over 100,000 variants differing in cpEGFP insertion site and linker sequences yielded sensors with precise membrane localization, high affinity (detection threshold ~5 nM), and a robust fluorescence shift upon fMLF binding. After isolating and sequencing the best GEM-fMLF 1.0 variants, we created second generation of GEM-fMLF sensor libraries by randomizing amino acid sequences in the linker regions. These modifications led to substantial diversity in sensor performance, with some variants exhibiting up to 1200% increase in fluorescence signal upon fMLF stimulation. Furthermore, to enable multiplex imaging with green fluorescent dyes or sensors, we have developed libraries of red fMLF sensor variants by inserting a circularly permuted RFP into FPR1 with random linker length and sequence.

To specifically examine eukaryotic fMLF release, we employed miniSOG2, a photosensitizer protein targeted to the plasma membrane or outer mitochondrial membrane, to induce localized cell or mitochondrial lysis by localized illumination with blue light.

Additionally, we synthetized caged fMLF and used localized 385 nm illumination to release fMLF during live imaging experiments with human neutrophils, FPR1-expressing HEK293A cells and our GEM-fMLF biosensor. Using GEM-fMLF, we could visualize and quantify the amounts of local fMLF release and assess how these gradients trigger waves of calcium signaling and the migration of human neutrophils towards the localized sources of fMLF.

In summary, GEM-fMLF provides a sensitive and specific tool to monitor fMLF dynamics during infection, eukaryotic cell rupture and fMLF uncaging. Future applications in zebrafish models will further elucidate the role of fMLF in sterile injury-induced inflammation and immune regulation.

Supported by SE 250 Excellence PhD Scholarship