PhD Scientific Days 2026

Poster Session 1.A - Molecular Medicine

Next-generation Green and Red LTB4 Biosensors for Intravital Microscopy

Name of the presenter

Vámosi, Boldizsár

Institute/workplace of the presenter

Semmelweis University, Department of Phisiology

Authors

Boldizsár Vámosi¹, Benoit Roux¹, Klaudia Vágó-Kiss¹, Anna Török¹, Fabian Dehne¹, Szimonetta Tamás¹, Balázs Enyedi¹
1: Semmelweis University, Department of Phisiology

Text of the abstract

Introduction:
Leukotriene B4 (LTB4) is a potent lipid mediator that plays a key role in directing neutrophil migration during the early phases of inflammation and tissue repair. Despite its importance, fundamental aspects of LTB4 synthesis and its spatiotemporal release from neutrophils in living tissues remain poorly understood, largely due to the lack of tools for direct, real-time detection. To address this limitation, we recently introduced a genetically encoded fluorescent biosensor for LTB4. Although this sensor enables visualization of LTB4 gradients both in vitro and in vivo, improvements in sensitivity, dynamic range and spectral diversity would substantially expand its applicability.
Aims:
Our goal is to engineer next-generation LTB4 biosensors optimized for intravital microscopy and to apply them to investigate neutrophil behavior with an emphasis on swarming.
Methods:
GEM-LTB4-1.0 was created by inserting a circularly permuted EGFP, connected by short linker sequences, into LTB4R1. To generate improved variants, we constructed large libraries of sensor variants by modifying the linker regions, the receptor, and the fluorescent protein. Optimization was performed in multiple iterative rounds. In parallel, red-shifted variants were generated by substituting cpEGFP with a cpRFP. Individual variants were screened at single-variant resolution, and those exhibiting the strongest LTB4-induced fluorescence responses were isolated and sequenced.
Results:
GEM-LTB4-1.0 displayed a maximal fluorescence increase of 103% under saturating stimulation. Multiple-round optimization resulted in variants with markedly higher baseline fluorescence and multiple-fold greater fluorescence response, together yielding a substantially improved signal-to-noise ratio. Additionally, we described a red-shifted sensor variant, also showing improved kinetics compared to GEM-LTB4-1.0. These novel sensors allowed us to visualize live release of LTB4 from fMLF-stimulated neutrophils.
Conclusions:
Using a high-throughput, iterative optimization pipeline, we have developed both a red-shifted LTB4 biosensor and substantially enhanced green variants. We are currently working on investigating LTB4 release dynamics in the context of neutrophil swarming using these new biosensors.
Funding:
NRDIO K 147351, ADVANCED 152292, 2025-1.2.1-HURIZONT-2025-00025 and SE250+ Excellence PhD grants