3D Protein Dynamics in the Cell Nucleus

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.     

Climate change impacts on Mediterranean vegetation are amplified at low altitudes

Aim

In the face of ongoing climate warming, we wanted to quantify impacts on vegetation at one of the major climatic and biogeographical boundaries of Europe, the limit between the Mediterranean and Eurosiberian biogeographical regions. We analyse temperature and moisture requirements of plants along altitudinal gradients at regional scale in the period 1980–2020 and we explore if changes coincide with observed changes in the same regions in terms of measured climatic data.

Location

Southern France.

Time period

1980–2020.

Taxa

Vascular plants.

Methods

We calculated shifts in plants’ temperature and moisture requirements for a large floristic database from south-eastern France (SIMETHIS) during the period 1980–2020 along altitudinal gradients by using ecological indicator values (EIV). Additionally, we analysed standardized weather station data from the same area and period, to investigate whether floristic changes are synchronized with climate changes.

Results

Vegetation data suggest a linear increase in temperature requirements of plant communities from 1980 to 2020 with a greater change at low altitudes. Upward shifts in temperature requirements coincided with observed climate change although warming did not show a general trend towards greater increases at low altitudes. Data on vegetation and climate suggest an upward shift of respectively 150 and 300 m for the boundary between Mediterranean and temperate belts. Moisture requirements of vegetation indicate an increase of the frequency of dry adapted species at low altitudes but an increase towards higher moisture requirements at high altitudes. Comparing vegetation responses with climate data suggests that responses are faster at low altitudes.

Main conclusions

Our analyses show that strong general changes in vegetation are underway and highlight faster responses of vegetation to warming in low altitudes compared to high altitudes and demonstrate the need for reliable data on vegetation and climate changes, especially on water balance.     

Orexins acting at native OX(1) receptor in colon cancer and neuroblastoma cells or at recombinant OX(1) receptor suppress cell growth by inducing apoptosis

Screening of 26 gut peptides for their ability to inhibit growth of human colon cancer HT29-D4 cells grown in 10% fetal calf serum identified orexin-A and orexin-B as anti-growth factors. Upon addition of either orexin (1 microM), suppression of cell growth was total after 24 h and >70% after 48 or 72 h, with an EC(50) of 5 nm peptide. Orexins did not alter proliferation but promoted apoptosis as demonstrated by morphological changes in cell shape, DNA fragmentation, chromatin condensation, cytochrome c release into cytosol, and activation of caspase-3 and caspase-7. The serpentine G protein-coupled orexin receptor OX(1)R but not OX(2)R was expressed in HT29-D4 cells and mediated orexin-induced Ca(2+) transients in HT29-D4 cells. The expression of OX(1)R and the pro-apoptotic effects of orexins were also indicated in other colon cancer cell lines including Caco-2, SW480, and LoVo but, most interestingly, not in normal colonic epithelial cells. The role of OX(1)R in mediating apoptosis was further demonstrated by transfecting Chinese hamster ovary cells with OX(1)R cDNA, which conferred the ability of orexins to promote apoptosis. A neuroblastoma cell line SK-N-MC, which expresses OX(1)R, also underwent growth suppression and apoptosis upon treatment with orexins. Promotion of apoptosis appears to be an intrinsic property of OX(1)R regardless of the cell type where it is expressed. In conclusion, orexins, acting at native or recombinant OX(1)R, are pro-apoptotic peptides. These findings add a new dimension to the biological activities of these neuropeptides, which may have important implications in health and disease, in particular colon cancer.     

Phototransformable fluorescent proteins: which one for which application?

In these last two decades , fluorescent proteins (FPs) have become highly valued imaging tools for cell biology, owing to their compatibility with living samples, their low levels of invasiveness and the possibility to specifically fuse them to a variety of proteins of interest. Remarkably, the recent development of phototransformable fluorescent proteins (PTFPs) has made it possible to conceive optical imaging experiments that were unimaginable only a few years ago. For example, it is nowadays possible to monitor intra- or intercellular trafficking, to optically individualize single cells in tissues or to observe single molecules in live cells. The tagging specificity brought by these genetically encoded highlighters leads to constant progress in the engineering of increasingly powerful, versatile and non-cytotoxic FPs. This review is focused on the recent developments of PTFPs and highlights their contribution to studies within cells, tissues and even living organisms. The aspects of single-molecule localization microscopy, intracellular tracking of photoactivated molecules, applications of PTFPs in biotechnology/optobiology and complementarities between PTFPs and other microscopy techniques are particularly discussed.     

Assessment of hepatic glucose metabolism by indirect calorimetry in combination with a non-invasive technique using naturally enriched 13C glucose in healthy children and adolescents

The metabolic fate of hepatic glucose can be best studied using invasive techniques such as tracer infusions and frequent blood sampling which have been revealed to be impractical in the pediatric age group. The aim of this study was to develop a non-invasive method based on indirect calorimetry and expired 13CO2 monitoring in order to gain insight into the mechanisms leading to impaired glucose tolerance in children and teenagers. As a first step, net glucose oxidation (NGO) and energy expenditure (EE) were measured in 47 subjects (range 7.5-17.3 years) of whom 18 were prepubertal (P1), 11 in early puberty (P2-P3) and 18 in late puberty (P4-P5) after 3-hourly loads of 180 mg/kg of oral maize glucose containing naturally enriched 13C. Isotope analysis allowed to calculate exogenous and endogenous glucose oxidation (EXGO, ENGO) and, hence, to derive TGS and NGS, that is glycogen turnover. NGO and EE decreased significantly with pubertal progression, reflecting higher metabolism at younger ages, whereas EXGO remained constant. TGS did not change significantly whereas NGS showed a significant negative correlation with pubertal progression: this can be explained by the fact that glycogenolysis exceeded glycogen synthesis in this experimental setting. This non-invasive method appears to be a promising tool to study the fate of hepatic glucose and therefore glycogen turnover in children at risk of developing glucose intolerance and/or type 2 diabetes.