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Abstract: Upon exposure to biological environments, nanoparticles are rapidly coated with biomolecules, predominantly proteins, which alter their colloidal stability, biodistribution, and cell interactions. Despite extensive efforts to investigate the nanoparticles' fate, only a few studies use high-resolution characterization methods that allow in-depth characterization, and the existing methodologies are unable to differentiate particles internalized at the onset of incubation from those taken up toward the end of an incubation period. In this study, these limitations related to incubation disparities are overcame and precisely monitored the spatiotemporal displacement of colloidally stable protein corona-coated nanoparticles within cells. An unprecedented application of cryogenic X-ray nanotomography, combined with high-resolution, super-resolution, and correlative microscopy techniques, revealed the migration of nanoparticles to the perinuclear region while monitoring the evolution of cellular organelles in fully hydrated cells under near-native conditions, without the need for contrasting agents. Notably, this tracking indicates the progressive fusion of vesicles carrying the nanoparticles intracellularly. This strategy demonstrates the potential for uncovering the temporal aspects of nanoparticle behavior within cells and can be adaptable to a wide range of nanoparticles and cell types, offering a versatile and powerful tool to follow nanoparticles in cellular environments.

Open Access

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Abstract: Iron is a crucial element integral to various fundamental biological molecular mechanisms, including magnetosome biogenesis in magnetotactic bacteria (MTB). Magnetosomes are formed through the internalization and biomineralization of iron into magnetite crystals. However, the interconnected mechanisms by which MTB uptake and regulate intracellular iron for magnetosome biomineralization remain poorly understood, particularly at the single-cell level. To gain insights we employed a holistic multiscale approach, i.e., from elemental iron species to bacterial populations, to elucidate the interplay between iron uptake dynamics and magnetosome formation in Magnetospirillum gryphiswaldense MSR-1 under near-native conditions. We combined a correlative microscopy approach integrating light and X-ray tomography with analytical techniques, such as flow cytometry and inductively coupled plasma spectroscopy, to evaluate the effects of iron and oxygen availability on cellular growth, magnetosome biogenesis, and intracellular iron pool in MSR-1. Our results revealed that increased iron availability under microaerobic conditions significantly promoted the formation of longer magnetosome chains and increased intracellular iron uptake, with a saturation point at 300 μM iron citrate. Beyond this threshold, additional iron did not further extend the magnetosome chain length or increase total intracellular iron levels. Moreover, our work reveals (i) a direct correlation between the labile Fe2+ pool size and magnetosome content, with higher intracellular iron concentrations correlating with increased magnetosome production, and (ii) the existence of an intracellular iron pool, distinct from magnetite, persisting during all stages of biomineralization. This study offers insights into iron dynamics in magnetosome biomineralization at a single-cell level, potentially enhancing the industrial biomanufacturing of magnetosomes.

Open Access

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Abstract: iruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Open Access

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Abstract: Non-junctional connexin43 (Cx43) plasma membrane hemichannels have been implicated in several inflammatory diseases, particularly playing a role in ATP release that triggers activation of the inflammasome. Therapies targeting the blocking of the hemichannels to prevent the pathological release or uptake of ions and signalling molecules through its pores are of therapeutic interest. To date, there is no close-to-native, high-definition documentation of the impact of Cx43 hemichannel-mediated inflammation on cellular ultrastructure, neither is there a robust account of the ultrastructural changes that occur following treatment with selective Cx43 hemichannel blockers such as Xentry-Gap19 (XG19). A combination of same-sample correlative high-resolution three-dimensional fluorescence microscopy and soft X-ray tomography at cryogenic temperatures, enabled in the identification of novel 3D molecular interactions within the cellular milieu when comparing behaviour in healthy states and during the early onset or late stages under inflammatory conditions. Notably, our findings suggest that XG19 blockage of connexin hemichannels under pro-inflammatory conditions may be crucial in preventing the direct degradation of connexosomes by lysosomes, without affecting connexin protein translation and trafficking. We also delineated fine and gross cellular phenotypes, characteristic of inflammatory insult or road-to-recovery from inflammation, where XG19 could indirectly prevent and reverse inflammatory cytokine-induced mitochondrial swelling and cellular hypertrophy through its action on Cx43 hemichannels. Our findings suggest that XG19 might have prophylactic and therapeutic effects on the inflammatory response, in line with functional studies.

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Abstract: Aphthovirus is a genus of the family Picornaviridae which includes Foot-and- mouth-disease virus (FMDV) and Equine rhinitis A virus (ERAV). FMDV is a highly contagious pathogen infecting cloven-hoofed animals and is hence economically important. FMDV replication takes place in the cytoplasm and induces massive rearrangement of the host cell membranes to facilitate virus replication. Rearranged membranes form structures providing the site of viral genome replication known as the replication organelle (RO). The understanding of the RO, viral proteins and site of virus assembly is not well established. This project applies various microscopy approaches to investigate details of aphthovirus replication in cells. FMDV 3A protein is known to play a key role in viral replication machinery. We generated recombinant viruses of FMDV with various tags fused to this protein, subsequently allowing 3A to be detected in confocal microscopy. We developed a split-GFP system to study the dynamics of 3A protein in vitro. We showed that 3A signals appeared contiguous to the Golgi membrane signals suggesting that it potentially serve as a main source of membrane associated with viral replication. This approach was taken with the aim of facilitating the development of a correlative light electron microscopy (CLEM) system to unravel the localisation of virus proteins and their link to RO in cells. ERAV was used as a surrogate model to study FMDV replication in a lower containment laboratory using cryo-electron vi tomography (cryo-ET). Virus particles were observed associated with membrane structures with single membrane vesicles being more predominant than double membrane vesicles in infected cells. By sub-tomogram averaging, we reconstructed 3-dimensional (3D) models of intracellular ERAV full and empty particles which were compared with structures obtained for the virus purified from tissue culture and crystallized. Additional density was identified in the ERAV empty particles potentially corresponding to RNA contact sites inside the capsid.