Scedosporium apiospermum is a saprophytic filamentous fungus involved in human infections, of which the virulence factors that contribute to pathogenesis are still poorly characterized. In particular, little is known about the specific role of dihydroxynaphtalene (DHN)-melanin, located on the external layer of the conidia cell wall. We previously identified a transcription factor, PIG1, which may be involved in DHN-melanin biosynthesis. To elucidate the role of PIG1 and DHN-melanin in S. apiospermum, a CRISPR-Cas9-mediated PIG1 deletion was carried out from two parental strains to evaluate its impact on melanin biosynthesis, conidia cell-wall assembly, and resistance to stress, including the ability to survive macrophage engulfment. ΔPIG1 mutants did not produce melanin and showed a disorganized and thinner cell wall, resulting in a lower survival rate when exposed to oxidizing conditions, or high temperature. The absence of melanin increased the exposure of antigenic patterns on the conidia surface. PIG1 regulates the melanization of S. apiospermum conidia, and is involved in the survival to environmental injuries and to the host immune response, that might participate in virulence. Moreover, a transcriptomic analysis was performed to explain the observed aberrant septate conidia morphology and found differentially expressed genes, underlining the pleiotropic function of PIG1.

Gold nanoclusters (Au NCs) are attractive luminescent nanoprobes for biomedical applications. In vivo biosensing and bioimaging requires the delivery of the Au NCs into subcellular compartments. In this view, we explore here the possible encapsulation of ultra-small-sized red and blue emitting Au NCs into liposomes of various sizes and chemical compositions. Different methods were investigated to prepare vesicles containing Au NCs in their lumen. The efficiency of the process was correlated to the structural and morphological aspect of the Au NCs’ encapsulating vesicles thanks to complementary analyses by SAXS, cryo-TEM, and confocal microscopy techniques. Cell-like-sized vesicles (GUVs) encapsulating red or blue Au NCs were successfully obtained by an innovative method using emulsion phase transfer. Furthermore, exosome-like-sized vesicles (LUVs) containing Au NCs were obtained with an encapsulation yield of 40%, as estimated from ICP-MS.

Urine cytology is non-invasive, easy to collect, with medium sensitivity and a high specificity. It is an effective way to detect high-grade bladder cancer (BC), but it is less effective on low-grade BC because the rate of equivocal results is much higher. Recently, the fluorescent properties of plasma membranes of urothelial tumor cells (UTC) found in urine cytology have been shown to be useful in improving the early detection of BC. This phenomenon is called peri-membrane fluorescence (PMF). Based on previous studies that have identified the PMF on UTCs, the main objective was to characterize this phenomenon. For this study, a software was specially created to quantify the PMF of all tested cells and different treatments performed. PMF was not found to be a morphological and discriminating feature of UTCs, all cells in shape and not from urine show PMF. We were able to highlight the crucial role of plasma membrane integrity in the maintenance of PMF. Finally, it was found that the induction of a strong cellular stress induced a decrease in PMF, mimicking what was observed in non-tumor cells collected from urine. These results suggest that PMF is found in cells able to resist this stress, such as tumor cells.

Protein kinases are key regulators of intracellular signaling in specific subcellular compartments and in micro‐ or nano‐domains. Genetically encoded biosensors based on Förster's resonance energy transfer (FRET) are powerful tools to track kinase dynamics. Yet, they are typically limited by spatial resolution. Aurora kinase A (AURKA), a multifunctional serine/threonine kinase, has recently emerged as a critical regulator of mitochondrial physiology. However, visualising AURKA activation and activity with sub‐diffraction precision remains a challenge. Here, we introduce BioSenSRRF, a versatile approach combining conventional FRET biosensors with super‐resolution radial fluctuations (SRRF) microscopy. BioSenSRRF requires no modification of existing probes, can be implemented using standard microscopy setups, and is supported by publicly available image analysis tools . With BioSenSRRF, we uncover that mitochondrial AURKA activation and activity are compartmentalized into distinct mitochondrial domains containing the ATP synthase. These subdomains depend on AURKA catalytic activity, and they can be altered using validated AURKA inhibitors. Finally, we demonstrate that the cancer‐associated polymorphism F31I enhances AURKA activation and ATP production on ATP synthase‐enriched subdomains. Altogether, BioSenSRRF provides a broadly accessible framework to enhance the spatial resolution of genetically encoded biosensors. This strategy opens new avenues for dissecting the subcellular organization of kinases and their contribution to physiology and disease.

Protein kinases are key regulators of intracellular signaling, showing activities in specific subcellular compartments and in micro- or nano-domains. Genetically encoded biosensors based on Förster’s Resonance Energy Transfer (FRET) are powerful tools to track kinase dynamics. Yet, they are typically limited by spatial resolution and remain challenging to implement with super-resolution microscopy approaches that require specific fluorophores or optical setups. Aurora kinase A (AURKA), a multifunctional serine/threonine kinase, has recently emerged as a critical regulator of mitochondrial physiology. However, visualising AURKA activation and activity in living cells with sub-diffraction precision remains a challenge. Here, we introduce BioSenSRRF, a versatile approach that combines conventional FRET biosensors with Super-Resolution Radial Fluctuations (SRRF) microscopy. BioSenSRRF requires no modification of existing probes, can be implemented using standard microscopy setups, and is supported by a publicly available ImageJ framework to streamline image processing and data analysis. Using this pipeline, we imaged AURKA activation at mitochondria and its activity in regulating mitochondrial ATP levels. We uncover that AURKA activation and activity are compartmentalised into distinct mitochondrial domains containing the mitochondrial ATP synthase. These subdomains depend on the catalytic activity of AURKA, and they can be altered using previously validated AURKA inhibitors. Finally, we demonstrate that the cancer-associated polymorphism F31I enhances AURKA activation and ATP production on ATP synthase-enriched subdomains, underscoring its pathological relevance. Altogether, BioSenSRRF provides a broadly accessible framework to enhance the spatial resolution of genetically encoded biosensors. This strategy opens new avenues for dissecting the subcellular organisation of protein complexes and their contribution to physiology and disease states.