Biology subjects

Stringari, C.

Publications and source records attributed to Stringari, C..

4 recordsLinked to original sources

bioRxiv2025

In vivo autofluorescence lifetime imaging of the Drosophila brain captures metabolic shifts associated with memory formation

Neuronal energy regulation is increasingly recognized as a critical factor underlying brain functions and their pathological alterations, yet the metabolic dynamics that accompany cognitive processes remain poorly understood. As a label-free and minimally invasive technique, fluorescence lifetime imaging (FLIM) of coenzymes NADH and NADPH (collectively referred to as NAD(P)H) offers the possibility to resolve cellular metabolic profiles with high spatial precision. However, NAD(P)H FLIMs capacity to detect subtle variations in neuronal metabolism has not been demonstrated. In this study, we applied NAD(P)H FLIM to map the metabolic profiles of Drosophila neurons in vivo across multiple scales, focusing on the primary centers for associative memory: the mushroom bodies (MBs). At a broad scale, we obtained an overview of the metabolic signatures of the main brain tissue and identified a marked difference between neuropil and cortex areas. At a finer scale, our findings revealed notable heterogeneity in the basal metabolic profiles of distinct MB neuron subtypes. Measurements performed after associative olfactory learning also uncovered a low-magnitude subtype-specific metabolic shift associated with memory formation, suggesting the utility of NAD(P)H FLIM in detecting physiology-driven changes linked to brain function. These results establish a promising framework for studying the spatial heterogeneities and the dynamics of cerebral energy metabolism in vivo.

neuroscience↗

bioRxiv2023

FLUTE: a Python GUI for interactive phasor analysis of FLIM data

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The phasor approach to FLIM data is a fit-free analysis and is increasingly used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available thus limiting the widespread use of phasor analysis in biomedical research. Here we present (F)luorescence (L)ifetime (U)l(t)imate (E)xplorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of FLIM analysis, such as calibrating the FLIM data, performing interactive exploration of the phasor plot with cursors, displaying the phasor plot and the FLIM images with different lifetime contrasts and calculating the relative concentration of molecular species. The final edited datasets after applying the desired filters and thresholds can be exported for further user specific analysis. FLUTE was tested using several FLIM datasets including autofluorescence of Zebrafish embryos, cells in vitro and intact live tissues. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets and accelerates FLIM analysis for non-specialized labs. Impact statementThis work introduces the first open-source graphical user interface (GUI) for phasor analysis of Fluorescence Lifetime Microscopy (FLIM) data. Phasor analysis is increasingly used for FLIM data analysis in biomedical research as it reduces the complexity of the analysis and provides a powerful visualization of the data content and optimization of data handling with respect to multiexponential fitting. However, the development of quantitative FLIM applications in the life sciences has been until now hampered by the lack of an open source and user-friendly graphical user interface. Here we introduce FLUTE that expands some possibilities of phasor FLIM image processing, accelerates the whole FLIM analysis and simplifies the visualization and the analysis of FLIM data, thus making phasor analysis possible for a broader base of researchers. FLUTE will be of interest to researchers with interests ranging from physics to biology and will facilitate research in several biomedical fields.

bioinformatics↗

bioRxiv2023

Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes

T cell activation induces a metabolic switch generating energy required for proliferation, survival, and fueling their functions. Thus, it is essential to monitor metabolism associated to subcellular functional and structural changes. We used non-invasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NADH co-enzyme during T lymphocyte activation. 2P-FLIM measurements of the protein-bound and free NADH ratios provides a readout of the redox state (NAD+/ NADH) of the cells, and thus of their OXPHOS and glycolysis rates. Using this method, we followed the dynamics of fraction of bound NADH (fb NADH) in live single cells. Comparing fb NADH between resting and activated T cells, we show that T cell activation induces a rapid switch toward glycolysis. The switch takes only 10 minutes and remains stable for at least one hour. Three-dimensional (3D) analysis revealed that the intracellular distribution of fb NADH is symmetrically distributed in resting cells, whereas increases at the contact zone in activated cells. Finally, we show that fb NADH negatively correlates with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study shows that 2P-FLIM measurement of fb NADH is well suited to follow a fast metabolic switch in 3D, in single T lymphocytes with subcellular resolution.

immunology↗

bioRxiv2021

Coordination between metabolic transitions and gene expression by NAD+ availability during adipogenic differentiation in human cells.

Adipocytes are the main cell type in adipose tissue, a critical regulator of metabolism, highly specialized in storing energy as fat. Adipocytes differentiate from multipotent mesenchymal stromal cells through adipogenesis, a tightly controlled differentiation process involving closely interplay between metabolic transitions and sequential programs of gene expression. However, the specific gears driving this interplay remain largely obscure. Additionally, the metabolite nicotinamide adenine dinucleotide (NAD+) is becoming increasingly recognized as a regulator of lipid metabolism, being postulated as promising therapeutic target for dyslipidemia and obesity. Here, we explored the effect of manipulating NAD+ bioavailability during adipogenic differentiation from human mesenchymal stem cells. We found a previously unappreciated strong repressive role for NAD+ on adipocyte commitment, while a functional NAD+-dependent deacetylase SIRT1 appeared crucial for terminal differentiation of pre-adipocytes. Remarkably, repressing the NAD+ biosynthetic salvage pathway during adipogenesis promoted the adipogenic transcriptional program, suggesting that SIRT1 activity during adipogenesis is independent from the NAD+ salvage pathway, while two photon microscopy and extracellular flux analyses suggest that its activation relies on the metabolic switch. Interestingly, SIRT1-directed control of subcellular compartmentalization of redox metabolism during adipogenesis was evidenced by two-photon fluorescence lifetime microscopy. Significance StatementAdipocyte differentiation occurs from mesenchymal stem cells through the adipogenic process, involving sequential activation of both transcriptional and metabolic programs in a tightly coordinated manner. However, how transcriptional and metabolic transitions reciprocally interact during adipogenic differentiation remains largely obscure. Here we describe that the metabolite NAD+ is suppresses adipogenesis trough rewiring transcription, while a functional NAD+-dependent deacetylase SIRT1 is essential for terminal differentiation of pre-adipocytes. Using two-photon fluorescence lifetime microscopy, we created a metabolic map of NADH and lipid content simultaneously in live cells and described a new role for SIRT1 in the control of compartmentalization of redox metabolism during adipogenesis. These findings advance our understanding to improve therapeutical approaches targeting the NAD+-SIRT1 axis as treatment for obesity and dyslipemia.

cell biology↗