Cell signaling nanoimaging

Oxidative signaling

Reactive Oxygen Species (or ROS), such as H₂O₂, play a major in numerous patho-physiolgical process, notably in the triggering and evolution of complex patholgoies, such as some cancers or (auto)-inflammation related diseases. Their quantitative monitoring in cells and tissues is thus essential for their deciphering and could be a major asset in the perspective of designing future treatments. However, current methods of ROS detection or imaging often lack the quantitativity and/or the dynamics to address this issue and accurately reveal signaling mechanisms. We are thus developing the use of lanthanide-based nanoparticle as nanosensors for quantiative ROS imaging at high spatial and temporal resolution. YVO₄:Eu nanoparticles are indeed luminescent through the presence of Eu³⁺ doping ions, whose redox state can be modulated optically or chemically. Through single nanoparticle imaging, the oxidative properties of their environment can thus be monitored quantitatively (Figure 1).

Figure1. Top: Principle of YVO₄:Eu nanoparticle photoreduction and reoxidation. Bottom: Photoreduction curve of single nanoparticules under resonant illumination (466 nm) and calibration sheet determining the local ROS concentration around a single nanoparticle in fonction of its photoluminescence.

The use of redox sensititve in cells allows the measurement of intracellular ROS concentration in response to various stimuli. We notably study the oxidative signaling in vascular and kidney cell to reveal the molecular mechanisms controlling their pathos-physiological behavior, including contraction, proliferation and migration. We notably identify regulation mechanisms (involving EGF, PDGF, ET-1,.. pathways) of ROS homeostasis and their organization and dynamics at sub-cellular scales in normal or pathological, vascular, kidney,... cells in (Figure 2)

Figure 2. Top left: YVO₄:Eu nanoparticles internalized in Vascular Smooth Muscle Cells (VSMCs). Top right: Quantitative ROS respsonse of VSMCs to ET-1, in presence (green line) or in the absence (blue line) of EGFR inhibition, compared to molecular models (red lines). Bottom left. Single YVO₄:Eu nanoparticles in HeLa cells submitted to a PDGF chemotactic gradient in a microchannel. Bottom right. Intracellullar ROS profile in cells submitted to a PDGF gradient

Based on the nanoparticle luminescence properties, we are furthermore developing methodologies for ROS probing at larger scales, either on-chip or in vivo (FIgure 3) in pathological models. The objective of this work is thus the monitoring of pathological, e.g. inflammation-related, transitions at the molecular scale, in complex, physiologically relevant, systems.

Figure 3. Top. Luminescence images of GdVO₄:Eu nanoparticles injected in a mouse ear before and 10 min after the triggering of a topic inflammation. Bottom. Oxidative response dynamics of single inflammated ears (plain lines) compared to an auto-amplification model, including ROS production by immune cells and their ROS dependent recruitment.

Membrane receptor nano-dynamics

The cell response to various stimuli rely on the spatial organization of the involved signaling pathways, notably the activation of membrane receptors, which may be regulated by their dynamics and organization. In order to probe these properties and their functional role, we develop the tracking of single membrane proteins through luminescent nanoparticles (YVO₄:Eu) imaging. This method provides minute-long trajectories of single receptors at high spatial and temporal resolution (typically 30 nm/20 ms) in living cells (Figure 4), whose analysis through advanced numerical methods may provide a quantitative characterization of their mobility and of the energy landscape regulating their nano-organization. Based on this approach, we notably classified the type of confinement of different receptors (EGFR, toxin receptors,...) and identified their molecular regulators (Figure 4). We furthermore investigate the effect of this membrane organization impairment in receptor activation (EGFR) and subsequent cell response, notably in the context of pathological kidney function regulation. These studies contribute at identifying quantitatively critical events at the molecular scale, which may then constitute key target in the regulation of cell and organ functions.

Top left. Nanoparticle labeling of EGFR and image of single EGFRs at the membrane of MDCK cells. Top right. Typical trajectory of a single membrane protein (EGFR). Bottom left. Measurements of the confining spring constant of the energy landscape of different receptors (EGFR, transferrin, a and e toxin) and effects of cholesterol-enriched rafts (ChlOx) or actin meshwork disruption (latB). Bottom right. Models of nano-organization for , e toxin receptor, EGFR and transferin receptors.

References

Confinement energy landscape classification reveals membrane receptor nano-organization mechanisms C. Yu, M. Richly, T. Hoang, M. El Beheiry, S. Türkcan, J-B. Masson, A. Alexandrou, C.I. Bouzigues Biophys. J., 123 ,1882 (2023)

In vivo ROS production during inflammation revealed by lanthanide-based nanoparticle imaging M. Abdesselem, N. Petri, R. Kuhner, F. Mousseau, V. Rouffiac, C. Laplace, A. Alexandrou, C.I. Bouzigues Biomed. Opt. Exp., 14 ,5392 (2023)

Fast quantitative ROS detection based on single multi-color lanthanide nanoparticle imaging Dual-color imaging of lanthanide-based single nanoparticles enables fast quantitative ROS detection and reveals endothelin-1 signaling pathway kinetics in living cells M. Abdesselem R. Ramodiharilafy, L. Devys, T. Gacoin, A. Alexandrou, C.I. Bouzigues. Nanoscale 9, 656-665 (2017)

Mapping the intracellular concentration of reactive oxygen species. Bouzigues C, Alexandrou A. Med Sci. 30,848-50 (2014)

Regulation of the ROS Response Dynamics and Organization to PDGF Motile Stimuli Revealed by Single Nanoparticle Imaging. Bouzigues CI, Nguyên TL, Ramodiharilafy R, Claeson A, Tharaux PL, Alexandrou A Chem. Biol. 21, 647 (2014)

Single Eu-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells. Casanova D*, Bouzigues C*, Nguyên TL*, Ramodiharilafy RO, Bouzhir-Sima L, Gacoin T, Boilot JP, Tharaux PL, Alexandrou A. Nat Nanotechnol. 4, 581. (2009)