Autophagy is increased in cryptorchid testis resulting in abnormal spermatozoa.

Autophagy is involved in spermatogenesis by regulating germ cell maturation. This catabolic process increases with hyperthermic conditions to prevent the accumulation of damaged organelles. Cryptorchidism is associated with impairment of germ cell maturation revealed by the presence of immature forms of sperm cells in ejaculates. The aim of the present study was to evaluate the status of autophagy in sperm cells from cryptorchid patients. Semen samples of cryptorchid patients and normozoospermic controls were analyzed by immunocytochemistry and electron microscopy. Autophagy proteins, autophagy-related protein 9 (ATG9) and microtubule-associated protein, 1A/1B-light chain 3 (LC3) were localized by immunocytochemistry on the acrosome and on the equatorial segment of sperm cells. LC3 was also detected in the midpiece of cryptorchid sperm tail. Autophagy substrate p62 protein was present in the acrosome and in the postequatorial segment of sperm in control samples, but not in the cryptorchid ones. Transmission electron microscopy revealed double-membrane-limited autophagosomes in postequatorial part of spermatozoa head and midpiece in cryptorchid samples. Partly degraded mitochondria were frequently discerned in autophagic vacuoles. In conclusion, autophagy is increased in sperm cells from patients with cryptorchid history comparatively to control. Our work provides insights into the role of autophagy in the maturation and survival of human male gametes in pathological conditions. Thus, regulating autophagy could represent a potential way to improve sperm quality in cryptorchid men.

Aurora kinase A localises to mitochondria to control organelle dynamics and energy production.

Many epithelial cancers show cell cycle dysfunction tightly correlated with the overexpression of the serine/threonine kinase Aurora A (AURKA). Its role in mitotic progression has been extensively characterised, and evidence for new AURKA functions emerges. Here, we reveal that AURKA is located and imported in mitochondria in several human cancer cell lines. Mitochondrial AURKA impacts on two organelle functions: mitochondrial dynamics and energy production. When AURKA is expressed at endogenous levels during interphase, it induces mitochondrial fragmentation independently from RALA. Conversely, AURKA enhances mitochondrial fusion and ATP production when it is over-expressed. We demonstrate that AURKA directly regulates mitochondrial functions and that AURKA over-expression promotes metabolic reprogramming by increasing mitochondrial interconnectivity. Our work paves the way to anti-cancer therapeutics based on the simultaneous targeting of mitochondrial functions and AURKA inhibition.

A targeted 3D EM and correlative microscopy method using SEM array tomography.

Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric array tomography for asymmetric samples and model organisms (Caenorhabditis elegans, Drosophila melanogaster, Danio rerio). We modified a diamond knife to simplify serial section array acquisition with minimal artifacts. After array acquisition, the arrays were transferred to a glass coverslip or silicon wafer support. Using light microscopy, the arrays were screened rapidly for initial recognition of global anatomical features (organs or body traits). Then, using SEM, an in-depth study of the cells and/or organs of interest was performed. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.

The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival.

Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na(+)/H(+) exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration).

Hypermutator Salmonella Heidelberg induces an early cell death in epithelial cells.

We have previously described that a strain of Salmonella Heidelberg with a hypermutator phenotype, B182, adhered strongly to HeLa cells. In this work, we showed that this hypermutator Salmonella strain invaded HeLa epithelial cells and induced cytoskeleton alteration. Those changes lead to HeLa cell death which was characteristic of apoptosis. For the first time, we showed that this hypermutator strain induced apoptosis associated with the activation of caspases 2, 9 and 3. Complementation of B182 strain showed a decrease in cells death induction. In the presence of other Salmonella Heidelberg with a normomutator phenotype, such as WT and SL486, cell death and caspase 3 were undetectable. These results suggested that early apoptosis and caspase 3 activation were specific to B182. Besides, B182 induced LDH release and caspase 3 activation in CaCo-2 and HCT116 cells. Heat-treated B182 and diffusible products failed to induce this phenotype. Epithelial cells treatment with cytochalasin D caused the inhibition of B182 internalisation and caspase 3 activation. These results showed that this cell death required active S. Heidelberg B182 protein synthesis and bacterial internalisation. However sipB and sopB, usually involved in apoptosis induced by Salmonella were not overexpressed in B182, contrary to fimA and fliC. Comparative genome analysis showed numerous mutations as in rpoS which would be more investigated. The role of the hypermutator phenotype might be suspected to be implicated in these specific features. This result expands our knowledge about strong mutators frequently found in bacterial organisms isolated from clinical specimens.

Toxicity, genotoxicity and proinflammatory effects of amorphous nanosilica in the human intestinal Caco-2 cell line.

Silica (SiO2) in its nanosized form is now used in food applications although the potential risks for human health need to be evaluated in further detail. In the current study, the uptake of 15 and 55nm colloidal SiO2 NPs in the human intestinal Caco-2 cell line was investigated by transmission electron microscopy. The ability of these NPs to induce cytotoxicity (XTT viability test), genotoxicity (γH2Ax and micronucleus assay), apoptosis (caspase 3), oxidative stress (oxidation of 2,7-dichlorodihydrofluorescein diacetate probe) and proinflammatory effects (interleukin IL-8 secretion) was evaluated. Quartz DQ12 was used as particle control. XTT and cytokinesis-block micronucleus assays revealed size- and concentration-dependent effects on cell death and chromosome damage following exposure to SiO2 nanoparticles, concomitantly with generation of reactive oxygen species (ROS), SiO2-15nm particles being the most potent. In the same way, an increased IL-8 secretion was only observed with SiO2-15nm at the highest tested dose (32µg/ml). TEM images showed that both NPs were localized within the cytoplasm but did not enter the nucleus. SiO2-15nm, and to a lower extent SiO2-55nm, exerted toxic effects in Caco-2 cells. The observed genotoxic effects of these NPs are likely to be mediated through oxidative stress rather than a direct interaction with the DNA. Altogether, our results indicate that exposure to SiO2 NPs may induce potential adverse effects on the intestinal epithelium in vivo.

Modeling of supramolecular centrosymmetry effect on sarcomeric SHG intensity pattern of skeletal muscles.

A theoretical far-field second harmonic generation (SHG) imaging radiation pattern is calculated for muscular myosin taking into account both Gouy effect and light diffraction under high focusing excitation. Theoretical analysis, in agreement with experimental results obtained on healthy Xenopus muscles, shows that the increase on intensity at the middle of the sarcomeric SHG intensity pattern is generated by an off-axis constructive interference related to the specific antipolar distribution of myosin molecules within the sarcomere. The best fit of the experimental sarcomeric SHG intensity pattern was obtained with an estimated size of antiparallel, intrathick filaments' packing-width of 115 ± 25 nm localized at the M-band. During proteolysis, experimental sarcomeric SHG intensity pattern exhibits decrease on intensity at the center of the sarcomere. An effective intra- and interthick filaments centrosymmetry of 320 ± 25 nm, in agreement with ultrastructural disorganization observed at the electron microscopy level, was necessary to fit the experimental sarcomeric SHG intensity pattern. Our results show that sarcomeric SHG intensity pattern is very sensitive to misalignment of thick filaments and highlights the potential usefulness of SHG microscopy to diagnose proteolysis-induced muscular disorders.