Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the context of tissue regeneration, tumor development, and therapeutic responses. Particularly, the calvarial bone marrow is a highly dynamic tissue, where the hematopoietic fate is interconnected with the surrounding microenvironment, with specialized vessels responding to normal and pathologic hematopoiesis. Traditional imaging of fixed tissues offers static information, often limiting a comprehensive understanding of these processes. The integration of transgenic animals expressing cell-specific markers, live cell tracers, advancements in imaging equipment, and the use of specialized chambers has elevated intravital microscopy to a pivotal tool for gaining insights into dynamic biological phenomena. One application of intravital imaging is the investigation of tumor vessel behavior and therapeutic effects. A newly designed 3D-printed titanium head fixation implant can be stably connected to the mouse skull and is suitable for longitudinal imaging during multiple sessions. The proposed protocol allows for the spatial and temporal examination of vascular dynamics in the calvarial bone marrow, including visualization and quantification of vascular heterogeneity, interaction with stromal and hematopoietic cells, and measurement of vascular functional parameters. Additionally, the technique enables the visualization of established vascular beds and the monitoring of therapeutic effects, stem cell mobilization, and the localization of chemotherapeutic compounds over time using two-photon microscopy. Overall, this intravital longitudinal imaging protocol provides a comprehensive platform for investigating both tumor vessel behavior and hematopoietic cell dynamics, offering valuable insights into the intricate processes governing these biological phenomena.

Neural tissues of the central nervous system are among the softest and most fragile in the human body, protected from mechanical perturbation by the skull and the spine. In contrast, the enteric nervous system is embedded in a compliant, contractile tissue and subject to chronic, high-magnitude mechanical stress. Do neurons and glia of the enteric nervous system display specific mechanical properties to withstand these forces? Using nano-indentation combined with immunohistochemistry and second harmonic generation imaging of collagen, we discovered that enteric ganglia in adult mice are an order of magnitude more resistant to deformation than brain tissue. We found that glia-rich regions in ganglia have a similar stiffness to neuron-rich regions and to the surrounding smooth muscle: ∼3 kPa at a 3 μm indentation depth and ∼7 kPa at an 8 μm depth. Differences in the adhesion strength of the different tissue layers to the glass indenter were scarce. The collagen shell surrounding ganglia and inter-ganglionic fibers may play a key role in strengthening the enteric nervous system to resist the manifold mechanical challenges it faces.

The viscosity that ensures the controlled diffusion of biomolecules in cells is a crucial biophysical parameter. Consequently, fluorescent probes capable of reporting viscosity variations are valuable tools in bioimaging. In this field, red-shifted probes are essential, as the widely used and gold standard probe remains green-emitting molecular rotors based on BODIPY. Here, we demonstrate that pyrrolyl squaraines, red-emissive fluorophores, exhibit high sensitivity over a wide viscosity range from 30 to 4890 mPa·s. Upon alkylation of the pyrrole moieties, the probes improve their sensitivity to viscosity through an enhanced twisted intramolecular charge transfer phenomenon. We utilized this scaffold to develop a plasma membrane probe, pSQ-PM, that efficiently stains the plasma membrane in a fluorogenic manner. Using fluorescence lifetime imaging, pSQ-PM enabled efficient sensing of viscosity variations in the plasma membrane under various conditions and in different cell lines (HeLa, U2OS, and NIH/3T3). Moreover, upon incubation, pSQ-PM stained the membrane of intracellular vesicles and suggested that the lysosomal membranes displayed enhanced fluidity.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.

While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of β1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.

Alveolar macrophages (AM) are crucial to COPD pathogenesis. Evidence suggests they have defective internalisation mechanisms which could drive disease exacerbations where human rhinovirus (HRV) is isolated. We hypothesised HRV infection of macrophages could impair their internalisation functions. AM or monocyte derived macrophages (MDM) were challenged with HRV16 and then with various triggers and internalisation assessed. Changes to the actin cytoskeleton following HRV16 infection and following particle internalisation were assessed by microscopy. AM or MDM challenged with HRV16 demonstrated deficient internalisation towards multiple triggers. HRV16 exaggerated deficient internalisation in COPD AM. HRV16 altered the actin cytoskeleton in macrophages driving persistence of actin around internalised particles. Screening for actin regulators affected by HRV16 identified Arpin, a negative Arp2/3 regulator to be significantly decreased after infection. Re-expression of Arpin after infection restored internalisation defects. Further, Arpin depletion impaired internalisation in a similar manner as HRV16 with internalisation stalling at the same point with significantly reduced bacterial uptake. These results confirm that HRV16 targets Arpin in macrophages to impair particle internalisation. Our results demonstrate HRV16 impairs various macrophage internalisation pathways by targeting Arpin. Hence, HRV16 can hijack macrophage functions and an important phagocytic regulator possibly explaining why COPD patients show secondary bacterial infection post virus infection with increased bacterial numbers.

Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n = 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p < 0.001) and mature endothelial cells (p < 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p = 0.03), capillary density (CD31) (p = 0.01) and myofiber regeneration (p = 0.04) was observed. Also, a significant necrosis (p = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.

Human rhinovirus is a causative agent of severe exacerbations of chronic obstructive pulmonary disease (COPD). COPD is characterised by an increased number of alveolar macrophages with diminished phagocytic functions, but how rhinovirus infection affects macrophage function is still unknown. Here, we describe that human rhinovirus 16 impairs bacterial uptake and receptor-mediated phagocytosis in macrophages. The stalled phagocytic cups contain accumulated F-actin. Interestingly, we find that human rhinovirus 16 downregulates the expression of Arpin, a negative regulator of the Arp2/3 complex. Importantly, re-expression of the protein rescues defective internalisation in human rhinovirus 16-treated cells, demonstrating that Arpin is a key factor targeted to impair phagocytosis. We further show that Arpin is required for efficient uptake of multiple targets, for F-actin cup formation and for successful phagosome completion in macrophages. Interestingly, Arpin is recruited to sites of membrane extension and phagosome closure. Thus, we identify Arpin as a central actin regulator during phagocytosis that it is targeted by human rhinovirus 16, allowing the virus to perturb bacterial internalisation and phagocytosis in macrophages.

The organization and dynamics of chromatin within the interphase nucleus as chromosome territories (CTs) and the relationship with transcriptional regulation are not fully understood. We studied a natural example of chromosomal disorganization: aneuploidy due to trisomies 13, 18 and 21. We hypothesized that the presence of an extra copy of one chromosome alters the CT distribution, which perturbs transcriptional activity. We used 3D-FISH to study the position of the chromosomes of interest (18 and 21) in cultured amniocytes and chorionic villus cells from pregnancies with a normal or aneuploid karyotype. We studied the volumes of nuclei and CTs in both conditions and performed a compared transcriptome analysis. We did not observe any differences between euploid and aneuploid cells in terms of the radial and relative CT positions, suggesting that the same rules govern nuclear organization in cases of trisomy. We observed lower volumes for CTs 18 and 21. Overall genome expression profiles highlighted changes in the expression of a subset of genes in trisomic chromosomes, while the majority of transcriptional changes concerned genes located on euploid chromosomes. Our results suggest that a dosage imbalance of the genes on trisomic chromosomes is associated with a disturbance of overall genomic expression.

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

Dans le noyau interphasique, la chromatine est organisée en territoires chromosomiques (TC) [1]. Le lien entre cette organisation et la régulation de l’expression du génome reste méconnu. Afin de préciser cette organisation, nous avons étudié un modèle de désorganisation : les aneuploïdies en supposant que la présence d’un chromosome surnuméraire pouvait entraîner une perturbation de la distribution des TC, elle-même conduisant à une modification de l’activité transcriptionnelle. Étude de la position des TC 18 et 21 par FISH 3D dans les noyaux interphasiques de fœtus aneuploïdes et témoins. Comparaison des volumes des noyaux et des TC. Parallèlement, une analyse transcriptomique comparative pangénomique des cellules a été réalisée afin d’objectiver des dérégulations d’expression. Nous n’avons pas observé de différence de positions des TC 18 et 21 entre les fœtus atteints et les témoins. Si les volumes nucléaires n’étaient pas modifiés, ceux des TC l’étaient. Nous avons mis en évidence des modifications d’expression d’une partie des gènes portés par les chromosomes d’intérêt ; mais également de gènes sur les autres chromosomes. En cas d’aneuploïdie, l’organisation des TC aneuploïdes dans le noyau semble globalement respectée. Il existerait cependant une diminution du volume de ces TC sans modification du volume des noyaux, interrogeant au détriment de quel compartiment, se fait cette compaction. L’analyse transcriptomique renforce l’idée d’une combinaison d’un effet de dosage génique et d’une rupture de l’homéostasie : aux gènes dérégulés portés par les chromosomes trisomiques, s’ajoutent des modifications d’expression du reste du génome. Ces dérégulations concernent de multiples voies impliquées dans le développement mais aussi des gènes au potentiel rôle dans l’apparition de traits phénotypiques. Il semblerait exister un lien entre le déséquilibre génique des chromosomes aneuploïdes et les dérégulations de l’ensemble du génome. La participation de la réorganisation des TC reste à affiner.

Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1 infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150(Glued), and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150(Glued), hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1 infected macrophages, leading to strong alterations in phagolysosome biogenesis.

BACKGROUND:Roberts syndrome (RBS) is a rare autosomal recessive disorder mainly characterized by growth retardation, limb defects and craniofacial anomalies. Characteristic cytogenetic findings are "railroad track" appearance of chromatids and premature centromere separation in metaphase spreads. Mutations in the ESCO2 (establishment of cohesion 1 homolog 2) gene located in 8p21.1 have been found in several families. ESCO2, a member of the cohesion establishing complex, has a role in the effective cohesion between sister chromatids. In order to analyze sister chromatids topography during interphase, we performed 3D-FISH using pericentromeric heterochromatin probes of chromosomes 1, 4, 9 and 16, on preserved nuclei from a fetus with RBS carrying compound heterozygous null mutations in the ESCO2 gene.RESULTS:Along with the first observation of an abnormal separation between sister chromatids in heterochromatic regions, we observed a statistically significant change in the intranuclear localization of pericentromeric heterochromatin of chromosome 1 in cells of the fetus compared to normal cells, demonstrating for the first time a modification in the spatial arrangement of chromosome domains during interphase.CONCLUSION:We hypothesize that the disorganization of nuclear architecture may result in multiple gene deregulations, either through disruption of DNA cis interaction -such as modification of chromatin loop formation and gene insulation - mediated by cohesin complex, or by relocation of chromosome territories. These changes may modify interactions between the chromatin and the proteins associated with the inner nuclear membrane or the pore complexes. This model offers a link between the molecular defect in cohesion and the complex phenotypic anomalies observed in RBS.

Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser(727)) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser(727)-phosphorylated STAT3 molecule (pSTAT3Ser(727)) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer(727) modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser(727), but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser(727) overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser(727) appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser(727) could be a promising new therapeutic approach.