ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Growth hormone remodels the 3D-structure of the mitochondria of inflammatory macrophages and promotes metabolic reprogramming.

Introduction: Macrophages are a heterogeneous population of innate immune cells that support tissue homeostasis through their involvement in tissue development and repair, and pathogen defense. Emerging data reveal that metabolism may control macrophage polarization and function and, conversely, phenotypic polarization may drive metabolic reprogramming. Methods: Here we use biochemical analysis, correlative cryogenic fluorescence microscopy and cryo-focused ion-beam scanning electron microscopy. Results: We demonstrate that growth hormone (GH) reprograms inflammatory GM-CSF-primed monocyte-derived macrophages (GM-MØ) by functioning as a metabolic modulator. We found that exogenous treatment of GM-MØ with recombinant human GH reduced glycolysis and lactate production to levels similar to those found in anti-inflammatory M-MØ. Moreover, GH treatment of GM-MØ augmented mitochondrial volume and altered mitochondrial dynamics, including the remodeling of the inner membrane to increase the density of cristae. Conclusions: Our data demonstrate that GH likely serves a modulatory role in the metabolism of inflammatory macrophages and suggest that metabolic reprogramming of macrophages should be considered as a new target to intervene in inflammatory diseases.

Multiphoton imaging of melanoma 3D models with plasmonic nanocapsules.

We report the synthesis of plasmonic nanocapsules and the cellular responses they induce in 3D melanoma models for their perspective use as a photothermal therapeutic agent. The wall of the nanocapsules is composed of polyelectrolytes. The inner part is functionalized with discrete gold nanoislands. The cavity of the nanocapsules contains a fluorescent payload to show their ability for loading a cargo. The nanocapsules exhibit simultaneous two-photon luminescent, fluorescent properties and X-ray contrasting ability. The average fluorescence lifetime (τ) of the nanocapsules measured with FLIM (0.3 ns) is maintained regardless of the intracellular environment, thus proving their abilities for bioimaging of models such as 3D spheroids with a complex architecture. Their multimodal imaging properties are exploited for the first time to study tumorspheres cellular responses exposed to the nanocapsules. Specifically, we studied cellular uptake, toxicity, intracellular fate, generation of reactive oxygen species, and effect on the levels of hypoxia by using multi-photon and confocal laser scanning microscopy. Because of the high X-ray attenuation and atomic number of the gold nanostructure, we imaged the nanocapsule-cell interactions without processing the sample. We confirmed maintenance of the nanocapsules' geometry in the intracellular milieu with no impairment of the cellular ultrastructure. Furthermore, we observed the lack of cellular toxicity and no alteration in oxygen or reactive oxygen species levels. These results in 3D melanoma models contribute to the development of these nanocapsules for their exploitation in future applications as agents for imaging-guided photothermal therapy. STATEMENT OF SIGNIFICANCE: The novelty of the work is that our plasmonic nanocapsules are multimodal. They are responsive to X-ray and to multiphoton and single-photon excitation. This allowed us to study their interaction with 2D and 3D cellular structures and specifically to obtain information on tumor cell parameters such as hypoxia, reactive oxygen species, and toxicity. These nanocapsules will be further validated as imaging-guided photothermal probes.

Calcite as a Precursor of Hydroxyapatite in the Early Biomineralization of Differentiating Human Bone-Marrow Mesenchymal Stem Cells.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce.

Multiparametric analysis of the effectiveness of cisplatin on cutaneous squamous carcinoma cells using two different types of adjuvants.

The objective of this study was to regulate the cytotoxicity of cisplatin (cisPt) minimizing its adverse effects. For this purpose, the lowest cisPt concentration needed to obtain a significant positive response in cutaneous squamous cell carcinoma (cSCC) was explored. Two adjuvant agents as gold nanoparticles (AuNP) and chelating tricine were tested as enhancers in cisPt treatment. Effectiveness of all treatments was assessed by means of biochemical techniques, which offer quantitative data, as well as two microscopy-based techniques that provided qualitative cell imaging. The present work confirms the effectiveness of free cisplatin at very low concentrations. In order to enhance its effectiveness while the side effects were probably diminished, cisPt 3.5 µM was administered with AuNP 2.5 mM, showing an effectiveness practically equal to that observed with free cisPt. However, the second treatment investigated, based on cisPt 3.5 µM combined with tricine 50 mM, enhanced drug effectiveness, increasing the percentage of cells dying by apoptosis. This treatment was even better in terms of cell damage than free cisPt at 15 µM. Images obtained by TEM and cryo-SXT confirmed these results, since a notable number of apoptotic bodies were detected when cisPt was combined with tricine. Thus, tricine was clearly a better adjuvant for cisPt treatments.

Unambiguous Intracellular Localization and Quantification of a Potent Iridium Anticancer Compound by Correlative 3D Cryo X-Ray Imaging.

The iridium half-sandwich complex [Ir(η5 :κ1 -C5 Me4 CH2 py)(2-phenylpyridine)]PF6 is highly cytotoxic: 15-250× more potent than clinically used cisplatin in several cancer cell lines. We have developed a correlative 3D cryo X-ray imaging approach to specifically localize and quantify iridium within the whole hydrated cell at nanometer resolution. By means of cryo soft X-ray tomography (cryo-SXT), which provides the cellular ultrastructure at 50 nm resolution, and cryo hard X-ray fluorescence tomography (cryo-XRF), which provides the elemental sensitivity with a 70 nm step size, we have located the iridium anticancer agent exclusively in the mitochondria. Our methodology provides unique information on the intracellular fate of the metallodrug, without chemical fixation, labeling, or mechanical manipulation of the cells. This cryo-3D correlative imaging method can be applied to a number of biochemical processes for specific elemental localization within the native cellular landscape.

Intracellular nanoparticles mass quantification by near-edge absorption soft X-ray nanotomography.

We used soft X-ray three-dimensional imaging to quantify the mass of superparamagnetic iron oxide nanoparticles (SPION) within whole cells, by exploiting the iron oxide differential absorption contrast. Near-edge absorption soft X-ray nanotomography (NEASXT) combines whole-cell 3D structure determination at 50 nm resolution, with 3D elemental mapping and high throughput. We detected three-dimensional distribution of SPIONs within cells with 0.3 g/cm(3) sensitivity, sufficient for detecting the density corresponding to a single nanoparticle.

Cryo-soft X-ray tomography as a quantitative three-dimensional tool to model nanoparticle:cell interaction.

BACKGROUND: Recent advances in nanoparticle design have generated new possibilities for nano-biotechnology and nano-medicine. Here we used cryo-soft X-ray tomography (cryo-SXT) to collect comprehensive three-dimensional (3D) data to characterise the interaction of superparamagnetic iron oxide nanoparticles (SPION) with a breast cancer cell line. RESULTS: We incubated MCF-7 (a human breast cancer cell line) from 0 to 24 h with SPION (15 nm average diameter, coated with dimercaptosuccinic acid), a system that has been studied previously using various microscopy and bulk techniques. This system facilitates the validation and contextualization of the new 3D data acquired using the cryo-SXT-based approach. After vitrification, samples tested by correlative cryo-epifluorescent microscopy showed SPION accumulation in acidic vesicles related to the endocytic pathway. Microscopy grids bearing MCF-7 cells were then analysed by cryo-SXT to generate whole cell volume 3D maps. Cryo-SXT is an emerging technique that benefits from high X-ray penetration into the biological material to image close-to-native vitrified cells at nanometric resolution with no chemical fixation or staining agents. This unique possibility of obtaining 3D information from whole cells allows quantitative statistical analysis of SPION-containing vesicle (SCV) accumulation inside cells, including vesicle number and size, distances between vesicles, and their distance from the nucleus. CONCLUSIONS: Correlation between fluorescent microscopy, cryo-SXT and transmission electron microscopy allowed us to identify SCV and to generate 3D data for statistical analysis of SPION:cell interaction. This study supports continuous transfer of the internalized SPION from the plasma membrane to an accumulation area near the cell nucleus. Statistical analysis showed SCV increase in number and size concomitant with longer incubation times, and therefore an increase in their accumulated volume within the cell. This cumulative effect expands the accumulation area and cell organelles such as mitochondria are consequently displaced to the periphery. Our 3D cryo-SXT approach demonstrates that a comprehensive quantitative description of SPION:cell interaction is possible, which will serve as a basis for metal-based nanoparticle design and for selection of those best suited for hyperthermia treatment, drug delivery and image diagnosis in nanobiomedicine.

A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen.

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1ß, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive and memory T-cell immune responses, as well as humoral responses. This novel HIV-1 gp120-14K immunogen might be considered as an HIV vaccine candidate for broad T and B-cell immune responses.

The structure of native influenza virion ribonucleoproteins.

The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics, which constitute a major public-health issue. The segmented negative-stranded RNAs are associated with the polymerase complex and nucleoprotein (NP), forming ribonucleoproteins (RNPs), which are responsible for virus transcription and replication. We describe the structure of native RNPs derived from virions. They show a double-helical conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase complex at the other end. This structure will be relevant for unraveling the mechanisms of nuclear import of parental virus RNPs, their transcription and replication, and the encapsidation of progeny RNPs into virions.