Correlative cryo-microscopy pipelines for in situ cellular studies.

Correlative cryo-microscopy pipelines combining light and electron microscopy and tomography in cryogenic conditions (cryoCLEM) on the same sample are powerful methods for investigating the structure of specific cellular targets identified by a fluorescent tag within their unperturbed cellular environment. CryoCLEM approaches circumvent one of the inherent limitations of cryo EM, and specifically cryo electron tomography (cryoET), of identifying the imaged structures in the crowded 3D environment of cells. Whereas several cryoCLEM approaches are based on thinning the sample by cryo FIB milling, here we present detailed protocols of two alternative cryoCLEM approaches for in situ studies of adherent cells at the single-cell level without the need for such cryo-thinning.

Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking.

Tunneling nanotubes (TNTs) are thin membranous channels providing a direct cytoplasmic connection between remote cells. They are commonly observed in different cell cultures and increasing evidence supports their role in intercellular communication, and pathogen and amyloid protein transfer. However, the study of TNTs presents several pitfalls (e.

Tunneling nanotubes provide a route for SARS-CoV-2 spreading.

Neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection represent a major issue in long coronavirus disease. How SARS-CoV-2 gains access to the brain and how infection leads to neurological symptoms are not clear because the principal means of viral entry by endocytosis, the angiotensin-converting enzyme 2 receptor, are barely detectable in the brain. We report that human neuronal cells, nonpermissive to infection through the endocytic pathway, can be infected when cocultured with permissive infected epithelial cells.

Fine intercellular connections in development: TNTs, cytonemes, or intercellular bridges?

Intercellular communication is a fundamental property of multicellular organisms, necessary for their adequate responses to changing environment. Tunneling nanotubes (TNTs) represent a novel means of intercellular communication being a long cell-to-cell conduit. TNTs are actively formed under a broad range of stresses and are also proposed to exist under physiological conditions.

HMGA1 negatively regulates NUMB expression at transcriptional and post transcriptional level in glioblastoma stem cells.

Glioblastoma (GBM) is a lethal, fast-growing brain cancer, affecting 2-3 per 100,000 adults per year. It arises from multipotent neural stem cells which have reduced their ability to divide asymmetrically and hence divide symmetrically, generating increasing number of cancer stem cells, fostering tumor growth. We have previously demonstrated that the architectural transcription factor HMGA1 is highly expressed in brain tumor stem cells (BTSCs) and that its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency in serial passages, suggesting a shift from symmetric to asymmetric division.

Effect of tolytoxin on tunneling nanotube formation and function.

Tunneling nanotubes (TNTs) are actin-containing membrane protrusions that play an essential role in long-range intercellular communication. They are involved in development of various diseases by allowing transfer of pathogens or protein aggregates as well as organelles such as mitochondria. Increase in TNT formation has been linked to many pathological conditions.

Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells.

The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution.

Cell Biology of Prion Protein.

Cellular prion protein (PrP) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The precise function of PrP remains elusive but may depend upon its cellular localization. PrP misfolds to a pathogenic isoform PrP, the causative agent of neurodegenerative prion diseases.

Regulation of sub-compartmental targeting and folding properties of the Prion-like protein Shadoo.

Shadoo (Sho), a member of prion protein family, has been shown to prevent embryonic lethality in Prnp mice and to be reduced in the brains of rodents with terminal prion diseases. Sho can also affect PrP structural dynamics and can increase the prion conversion into its misfolded isoform (PrP), which is amyloidogenic and strictly related to expression, intracellular localization and association of PrP to lipid rafts. We reasoned that if Sho possesses a natural tendency to convert to amyloid-like forms in vitro, it should be able to exhibit "prion-like" properties, such as PK-resistance and aggregation state, also in live cells.

HMGA1 silencing reduces stemness and temozolomide resistance in glioblastoma stem cells.

Objective: Glioblastoma multiforme (GBM) develops from a small subpopulation of stem-like cells, which are endowed with the ability to self-renew, proliferate and give rise to progeny of multiple neuroepithelial lineages. These cells are resistant to conventional chemo- and radiotherapy and are hence also responsible for tumor recurrence. HMGA1 overexpression has been shown to correlate with proliferation, invasion, and angiogenesis of GBMs and to affect self-renewal of cancer stem cells from colon cancer.

The 37/67 kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67 kDa LR cell surface localization and interaction with the cellular prion protein.

The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrP(C)) and to be required for pathological PrP (PrP(Sc)) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrP(C) interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrP(C) in the presence of specific LR inhibitor compounds has not been investigated yet.

The end of mitosis from a phosphatase perspective.

Transition through mitosis, the cell division cycle phase deputed to segregate replicated chromosomes, requires a wave of protein phosphorylation. While in the past decades a wealth of information has been gathered on the major kinase activities responsible for the onset of mitosis, only recently has a picture emerged of how their effects are reversed by protein phosphatases at the end of mitosis. Here, we summarized some recent data on the relevance for protein phosphatases in the reversal of mitotic phosphorylation required to complete mitosis in vertebrate cells.

Visual lateralization in wild striped dolphins (Stenella coeruleoalba) in response to stimuli with different degrees of familiarity.

Background: Apart from findings on both functional and motor asymmetries in captive aquatic mammals, only few studies have focused on lateralized behaviour of these species in the wild.

Methodology/principal Findings: In this study we focused on lateralized visual behaviour by presenting wild striped dolphins with objects of different degrees of familiarity (fish, ball, toy). Surveys were conducted in the Gulf of Taranto, the northern Ionian Sea portion delimited by the Italian regions of Calabria, Basilicata and Apulia.

Nrf3-Pla2g7 interaction plays an essential role in smooth muscle differentiation from stem cells.

Objective: Phospholipase A2, group 7 (Pla2g7) is an important mediator in cardiovascular development and diseases because of its divergent physiological and pathological functions in inflammation and oxidative stress. However, little is known about the functional role of Pla2g7 in smooth muscle cell (SMC) differentiation from stem cells.

Methods And Results: In the present study, embryonic stem cells were cultivated on collagen IV-coated plates to allow SMC differentiation.

Volumetric assessment of cerebral asymmetries in dogs.

In the present study we quantified volumetric brain asymmetries from computed tomography (CT) scans in 12 healthy dogs, using a semi-automated technique for assessing in vivo structure asymmetry. Volumetric assessment of asymmetrical cerebral lateral ventricle (ALV) was also investigated. Our results showed that seven dogs exhibited a right hemisphere significantly greater than the left, two dogs had a left-greater-than-right hemisphere asymmetry, and finally two dogs displayed no significant brain volumetric asymmetry.

Catecholamine plasma levels following immune stimulation with rabies vaccine in dogs selected for their paw preferences.

Epinephrine and norepinephrine plasma levels were assessed in dogs in relation to paw preference following an immune challenge with rabies vaccine. The results showed that both catecholamines increased after the vaccine administration, confirming the main role of the sympathetic nervous system in the modulation activity between the brain and the immune system. Moreover, ambidextrous dogs showed a significantly higher increase of epinephrine levels 8 days after immunization with respect to right- and left-pawed dogs, suggesting that the biological activity of this molecule could be key for a different immune response with regard to laterality.

Crucial role of nrf3 in smooth muscle cell differentiation from stem cells.

Rationale: Nuclear factor erythroid 2-related factor (Nrf)3, a member of the cap 'N' collar family of transcription factors that bind to the DNA-antioxidant responsive elements, is involved in reactive oxygen species balancing and in muscle precursor migration during early embryo development.

Objective: To investigate the functional role of Nrf3 in smooth muscle cell (SMC) differentiation in vitro and in vivo.

Methods And Results: Nrf3 was upregulated significantly following 1 to 8 days of SMC differentiation.

Dogs turn left to emotional stimuli.

During feeding behaviour, dogs were suddenly presented with 2D stimuli depicting the silhouette of a dog, a cat or a snake simultaneously into the left and right visual hemifields. A bias to turn the head towards the left rather than the right side was observed with the cat and snake stimulus but not with the dog stimulus. Latencies to react following stimulus presentation were lower for left than for right head turning, whereas times needed to resume feeding behaviour were higher after left rather than after right head turning.

Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow.

Background: Histone deacetylase 3 (HDAC3) is known to play a crucial role in the differentiation of endothelial progenitors. The role of HDAC3 in mature endothelial cells, however, is not well understood. Here, we investigated the function of HDAC3 in preserving endothelial integrity in areas of disturbed blood flow, ie, bifurcation areas prone to atherosclerosis development.

Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow.

X-box binding protein 1 (XBP1) is a key signal transducer in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas of atherosclerotic lesions in the arteries of ApoE(-/-) mice, which was related to the severity of lesion development.

Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis.