PhD Scientific Days 2025

Poster Session II. - J: Theoretical and Translational Medicine

Investigating the role of fMLF in tissue-damage responses and developing a novel genetically encoded fluorescent fMLF biosensor

Name of the presenter

Tamás Szimonetta Xénia

Institute/workplace of the presenter

Department of Physiology

Authors

Szimonetta Tamás¹, Nada Mohamed Al-Sheraji¹, Diána Patrícia Kaszás¹, Anna Török¹, Fabian Dehne¹, Klaudia Vágó-Kiss¹, Balázs Enyedi¹

1: Department of Physiology

Text of the abstract

Bacteria-derived fMLF (N-formyl-methionine-leucine-phenylalanine) is a well-known chemoattractant interacting with formyl peptide receptors (FPR) on leukocytes. While fMLF is also released from eukaryotic cell mitochondria during cell death or tissue damage, the specific cell types and death mechanisms involved in fMLF gradient formation remain unclear.

Illuminating plasma or mitochondrial membrane-targeted, optogenetically activatable miniSOG2 with 435 nm light induces rapid membrane damage and cell death.
To visualize fMLF gradients, we developed two approaches tested on HEK293A cells. FMLF-activated FPR-1, in conjunction with G16, significantly increased intracellular calcium levels, detectable by jGCaMP8m. Despite the FPR-1 receptor's high sensitivity, other cell death-related molecules, such as ATP, can influence calcium levels.
To specifically detect fMLF release, we engineered an FPR1-based genetically encoded fluorescent biosensor. A circularly permuted green fluorescent protein was inserted into the FPR1's third intracellular loop using C- and N-terminal linkers. FMLF binding induced a conformational change, altering biosensor fluorescence intensity.

By integrating miniSOG2 into plasma and mitochondrial membranes, we developed a tool potentially capable of inducing fMLF release from a single cell, leading to calcium level fluctuations in surrounding HEK293 cells and human neutrophils. To visualize fMLF, we created two systems: the FPR1-jGCaMP8m system for effectively detecting point source diffusion and a genetically encoded fluorescent biosensor exhibiting a 50% intensity increase in response to fMLF.

By further developing our sensor prototypes and integrating both them and the miniSOG2 system into a live zebrafish model, we can gain more knowledge about fMLF gradients formed during tissue damage and understand more about how sterile tissue injury influences neutrophil migration.

This research was supported by a grant of the New National Excellence Program of the Ministry for Culture and Innovation of Hungary from the source of the National Research, Development and Innovation Fund.