Biology subjects

Miskolci, V.

Publications and source records attributed to Miskolci, V..

7 recordsLinked to original sources

bioRxiv2026

A genetically encoded RhoG FRET biosensor reveals spatially compartmentalized RhoG-Rac1 signaling during cell protrusion

RhoG is a member of the Rho-family of small GTPases, and is closely related to the canonical Rac1 GTPase, implicated in membrane trafficking, dorsal ruffling, macropinocytosis, and cell protrusion, but its activity has been difficult to visualize directly in living cells with high spatial and temporal resolution. Here, we developed and validated a genetically encoded, single-chain Forster resonance energy transfer (FRET) biosensor for RhoG based on a C-terminal full-length RhoG and an intramolecular RhoG-binding domain derived from ELMO1. The biosensor showed a robust dynamic range when comparing constitutively active and inactive RhoG mutants, responded appropriately to regulation by RhoGDI, GAPs, and GEFs, and detected growth factor-stimulated RhoG activation in live cells. Imaging in mouse embryonic fibroblasts revealed dynamic RhoG activation at leading-edge protrusions, dorsal ruffles, and forming pinocytic and macropinocytic structures. To define the signaling relationship between RhoG and its closely related family member Rac1, we combined the RhoG biosensor with a near-infrared Rac1 FRET biosensor for simultaneous live-cell imaging. Morphodynamic mapping showed that both RhoG and Rac1 activities were positively coupled to edge protrusion, with strongest correlations near the leading-edge, but their direct coupling varied with distance from the edge, indicating partial spatial decoupling within protrusive regions. Inhibition of Src-family kinases altered RhoG dynamics, strongly suppressed Rac1 coupling to protrusion, and inverted the normal positive correlation between RhoG and Rac1 activities. Signaling microdomain analysis further showed that Src inhibition selectively prolonged Rac1 microdomain lifetimes without significantly affecting RhoG domains. Together, these results establish a new biosensor for direct visualization of RhoG activity and reveal that RhoG and Rac1 are coordinated but spatially and temporally distinct components of protrusion-associated signaling networks, with Src-family kinases playing a central role in maintaining their normal coupling.

cell biology↗

bioRxiv2026

Fibroblast response to burn injury in larval zebrafish mirrors developmental maturation and is inhibited by infiltrating neutrophils

Fibroblasts mediate tissue repair after damage, but aberrant fibroblast behavior in response to injury can result in impaired wound healing. Severe burn injury often results in tissue scarring, but the underlying mechanisms by which fibroblasts respond to burn injury and the role of inflammation in fibrosis are not well understood. Here we developed fluorescent reporters of collagen expressing mesenchymal cells enabling real time imaging of fibroblasts during homeostatic development and in response to burn injury using larval zebrafish. We find that fibroblasts derived from the mesenchyme respond to burn injury by engaging in a maturation process, characterized by the expression of vimentin, which is reminiscent of larval development. In burned tissue, fibroblast maturation is perturbed by prolonged neutrophil infiltration, resulting in disorganized extracellular matrix (ECM) and delayed ECM remodeling, which can be rescued by neutrophil depletion. This work adds to our understanding of fibroblast development in zebrafish and shows that collagen expressing mesenchymal cells regulate ECM remodeling in coordination with immune cells during burn wound healing. Summary StatementVimentin-positive fibroblasts are required for normal wound healing in larval zebrafish, but the presence of inflammatory neutrophils after burn injury impairs fibroblast maturation and collagen remodeling.

Developmental Biology↗

bioRxiv2024

Real time imaging reveals a role for macrophage protrusive motility in melanoma invasion

Macrophages are primary cells of the innate immune system that mediate tumor progression. However, the motile behavior of macrophages and interactions with tumor cells are not well understood. Here, we exploit the optical transparency of larval zebrafish and perform real time imaging of macrophage-melanoma interactions. We found that macrophages are highly motile in the tumor microenvironment. Macrophages extend dynamic projections between tumor cells that precedes invasive melanoma migration. Modulating macrophage motility with a dominant inhibitory mutation in Rac2 inhibits recruitment to the tumor and impairs tumor invasion. However, a hyperactivating mutation in Rac2 does not affect macrophage recruitment but limits macrophage projections into the melanoma mass and reduces invasive melanoma cell migration. Taken together, these findings reveal a role for Rac2-mediated macrophage protrusive motility in melanoma invasion.

cell biology↗

bioRxiv2024

Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems

Neutrophils, the most abundant leukocytes in human peripheral circulation, are crucial for the innate immune response. They are typically quiescent but rapidly activate in response to infection and inflammation, performing diverse functions such as oxidative burst, phagocytosis, and NETosis, which require significant metabolic adaptation. Deeper insights into such metabolic changes will help identify regulation of neutrophil functions in health and diseases. Due to their short lifespan and associated technical challenges, the metabolic processes of neutrophils are not completely understood. This study uses optical metabolic imaging (OMI), which entails optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic coenzymes NAD(P)H and FAD to assess the metabolic state of single neutrophils. Primary human neutrophils were imaged in vitro under a variety of activation conditions and metabolic pathway inhibitors, while metabolic and functional changes were confirmed with mass spectrometry, oxidative burst, and NETosis measurements. Our findings show that neutrophils undergo rapid metabolic remodeling to a reduced redox state indicated by changes in NAD(P)H lifetime and optical redox ratio, with a shift to an oxidized redox state during activation. Additionally, single cell OMI analysis reveals a heterogeneous metabolic response across neutrophils and human donors to live pathogen infection (Pseudomonas aeruginosa and Toxoplasma gondii). Finally, consistent OMI changes with activation were confirmed between in vitro human and in vivo zebrafish larvae neutrophils. This study demonstrates the potential of OMI as a versatile tool for studying neutrophil metabolism and underscores its use across different biological systems, offering insights into neutrophil metabolic activity and function at a single cell level.

immunology↗

bioRxiv2023

Light sheet autofluorescence lifetime imaging with a single photon avalanche diode array

Single photon avalanche diode (SPAD) array sensors can increase the imaging speed for fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser scanning to widefield geometries. While a SPAD camera in epi-fluorescence geometry enables widefield FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus features masks weak cell autofluorescence and biases lifetime measurements. Here, we address this problem by integrating the SPAD camera in a light sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling fast label-free FLIM of single-cell NAD(P)H autofluorescence. The feasibility of this NAD(P)H light sheet FLIM system was confirmed with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times, and in vivo NAD(P)H light sheet FLIM was demonstrated with live neutrophil imaging in a zebrafish tail wound, also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light sheet geometries, indicating a 30X to 6X frame rate advantage for the light sheet compared to the laser scanning geometry. This light sheet system provides faster frame rates for 3D NAD(P)H FLIM for live cell imaging applications such as monitoring single cell metabolism and immune cell migration throughout an entire living organism.

bioengineering↗

bioRxiv2020

In vivo fluorescence lifetime imaging captures metabolic changes in macrophages during wound responses in zebrafish

The effector functions of macrophages across the spectrum of activation states in vitro are linked to profound metabolic rewiring. However, the metabolism of macrophages remains poorly characterized in vivo. To assess changes in the intracellular metabolism of macrophages in their native inflammatory microenvironment, we employed two-photon fluorescence lifetime imaging microscopy (FLIM) of metabolic coenzymes NAD(P)H and FAD. We found that pro-inflammatory activation of macrophages in vivo was associated with a decrease in the optical redox ratio [NAD(P)H/(NAD(P)H+FAD)] relative to a pro-resolving population during both infected and sterile inflammation. FLIM also resolved temporal changes in the optical redox ratio and lifetime variables of NAD(P)H in macrophages over the course of sterile inflammation. Collectively, we show that non-invasive and label-free imaging of autofluorescent metabolic coenzymes is sensitive to dynamic changes in macrophage activation in interstitial tissues. This imaging-based approach has broad applications in immunometabolism by probing in real time the temporal and spatial metabolic regulation of immune cell function in a live organism. SignificanceMetabolic regulation of macrophage effector functions has recently emerged as a key concept in immune cell biology. Studies rely on in vitro and ex vivo approaches to study macrophage metabolism, however the high plasticity of these cells suggest that removal from their native microenvironment may induce changes in their intracellular metabolism. Here, we show that fluorescence lifetime imaging microscopy of metabolic coenzymes captures dynamic changes in the metabolic activity of macrophages while maintaining them in their endogenous microenvironment. This approach also resolves variations on a single-cell level, in contrast to bulk measurements provided by traditional biochemical assays, making it a potentially valuable tool in the field of immunometabolism.

immunology↗

bioRxiv2018

High cholesterol diet modulates macrophage polarization and liver inflammation during early hepatocellular carcinoma progression in zebrafish

Diabetes and obesity have been associated with nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) and increased incidence of hepatocellular carcinoma (HCC). Here we use optically transparent zebrafish to visualize liver inflammation and disease progression in a NAFLD/NASH-HCC model. We combined a high-cholesterol diet (HCD) with a transgenic zebrafish HCC model induced by hepatocyte-specific activated {beta}-catenin and found that diet induced an increase in liver size and enhanced angiogenesis and neutrophil infiltration in the liver. Although macrophage number was not affected by diet, HCD induced changes in macrophage morphology and polarization with an increase in liver associated TNF-positive macrophages. Treatment with metformin altered macrophage polarization and reduced liver size in NAFLD/NASH-associated HCC larvae. Moreover, ablation of macrophages limited progression in NAFLD/NASH-associated HCC larvae but not in HCC alone. These findings suggest that HCD alters macrophage polarization and exacerbates the liver inflammatory microenvironment and cancer progression in a zebrafish model of NAFLD/NASH-associated HCC.

cancer biology↗