Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: characterization and application

In this work we report the advantages provided by two photon excitation (2PE) implemented in a selective plane illumination microscopy (SPIM) when imaging thick scattering samples. In particular, a detailed analysis of the effects induced on the real light sheet excitation intensity distribution is performed. The comparison between single-photon and two-photon excitation profiles shows the reduction of the scattering effects and sample-induced aberrations provided by 2PE-SPIM. Furthermore, uniformity of the excitation distribution and the consequent improved image contrast is shown when imaging scattering phantom samples in depth by 2PE-SPIM. These results show the advantages of 2PE-SPIM and suggest how this combination can further enhance the SPIM performance. Phantom samples have been designed with optical properties compatible with biological applications of interest

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living β-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs’ properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living β-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions

Targeting cellular calcium homeostasis to prevent cytokine-mediated beta cell death

AbstractPro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.</jats:p

Rpl13a small nucleolar RNAs regulate systemic glucose metabolism

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that form ribonucleoproteins to guide covalent modifications of ribosomal and small nuclear RNAs in the nucleus. Recent studies have also uncovered additional non-canonical roles for snoRNAs. However, the physiological contributions of these small RNAs are largely unknown. Here, we selectively deleted four snoRNAs encoded within the introns of the ribosomal protein L13a (Rpl13a) locus in a mouse model. Loss of Rpl13a snoRNAs altered mitochondrial metabolism and lowered reactive oxygen species tone, leading to increased glucose-stimulated insulin secretion from pancreatic islets and enhanced systemic glucose tolerance. Islets from mice lacking Rpl13a snoRNAs demonstrated blunted oxidative stress responses. Furthermore, these mice were protected against diabetogenic stimuli that cause oxidative stress damage to islets. Our study illuminates a previously unrecognized role for snoRNAs in metabolic regulation