TIRF microscopic investigation of the insulin granules and the actin cytoskeleton in primary beta cells and measurement of the cellular energy metabolism with PercevalHR

Bisherige Untersuchungen von Insulin-Granula wurden meist mit dem Fokus auf fusionierende Granula durchgeführt, indem diese manuell gezählt wurden. Das Aktin-Zytoskelett soll bezüglich der Fusion der Granula eine Doppelrolle einnehmen, sowohl als Barriere als auch als Transportstruktur bei Stimulation. Da die Dynamik des Aktins und der Granula energieabhängig sind, ist es außerdem interessant die dynamische ATP/ADP-Ratio in MIN6- und primären Beta-Zellen zu betrachten. In dieser Arbeit wurde das Verhalten der Insulin-Granula in primären Beta-Zellen aus NMRI-Mäusen mittels TIRF-Mikroskopie betrachtet und Anwender-unabhängig ausgewertet. Außerdem wurden Struktur und Lokalisation des Aktin-Zytoskeletts in Abhängigkeit zu den Insulin-Granula untersucht. Die dynamische Änderung der ATP/ADP-Ratio wurde mit dem PercevalHR-Label bestimmt. Die Insulin-Granula in den Beta-Zellen wiesen eine konstante Bewegung vom Zellinneren zur Plasmamembran und zurück ins Zellinnere auf. Auf diese Weise wurde jedes Granulum im Durchschnitt innerhalb von 25 Sekunden 13,3-mal an der Plasmamembran ausgetauscht. Es war überraschend, dass mit einem 30 mM Glucose-Stimulus keine erhöhten Exozytoseraten in den Beta-Zellen beobachtet werden konnten. Allerdings konnte eine sehr hohe Aktin-Dichte an der gesamten Kontaktfläche zum umströmenden Medium beobachtet werden. Parallele Untersuchung der Granula und des Aktin-Zytoskeletts zeigten eine gegenseitig ausschließende Lokalisation beider zellulärer Strukturen, die in vielen Zellen mit steigender Glucose-Konzentration zunahm. Die Messungen der ATP/ADP-Ratio zeigten beim Wechsel von 0 mM Glucose auf 30 mM Glucose, sowohl in MIN6-Zellen als auch in primären Beta-Zellen eine deutliche Steigerung. Es konnte gezeigt werden, dass einzelne Beta-Zelle kein ideales Modell ist um die Exozytose von Insulin-Granula zu untersuchen, da diese nicht mit insulinotropen Stimuli wie 30 mM Glucose gesteigert werden konnte. Ein möglicher Grund dafür könnte das Aktin-Zytoskelett sein, das in Einzelzellen sehr dichte Strukturen an der Zellmembran ausbildet. Diese dichten Aktin-Strukturen könnten die Insulin-Granula von der Plasmamembran abschirmen und die Exozytose verhindern. Es wäre daher von Vorteil die Exozytose von Insulin-Granula an ganzen Inseln untersuchen zu können. Die dynamische ATP/ADP-Ratio ließ sich mit PercevalHR in MIN6-Zellen und in primären Beta-Zellen bestimmen. Der Verlauf der ATP/ADP-Ratio in primären Beta-Zellen besser reproduzierbar.So far, most of the investigations on Insulin granules focused on granule fusion by manually counting the fusion events. Considering the granule fusion the actin cytoskeleton is believed to act in a dual manner, on one site as a barrier against uncontrolled exocytosis and on the other site as a transport structure in stimulated exocytosis. Since the dynamics of actin and granules are energy-dependent, dynamic measurements of the ATP/ADP-ratio in beta cells are of high interest for these investigations. All measurements were performed in single beta cells. The number and the mobility of insulin granules and the structure and dynamic of the actin cytoskeleton were analysed with TIRF microscopy. The analysis of the insulin granules was performed with an observer-independent program. The dynamic change of the ATP/ADP ratio was measured with the PercevalHR label. In primary beta cells, the insulin granules showed a continuous movement from the centre of cell to the plasma membrane and back into the cell interior. The mean exchange rate of every granule at the plasma membrane was 13.3 times in 25 seconds. Surprisingly stimulation with 30 mM Glucose did not affect the rate of exocytotic events in beta cells. Actually, the density of the cortical actin network was very high, where the beta cells had direct contact to the surrounding medium. Simultaneous investigations of the actin cytoskeleton and the insulin granules revealed a mutually exclusive localisation of actin and granules, which increased in most beta cells with glucose stimulation. The measurements with the indicator PercevalHR showed a strong increase in ATP/ADP-Ratio in both MIN6 cells and primary beta cells, when the glucose concentration was raised from 0 mM to 30 mM. Since the number of exocytotic events did not increase with a 30 mM glucose stimulus primary beta cells are no ideal model system for the investigation of granule exocytosis. The dense structures of the cortical actin network in single beta cells might be an explanation for this behaviour. These dense cortical actin structures might prevent the granules from reaching the plasma membrane and finally from exocytosis. Whole islets would be a favourable model for the measurement of granule exocytosis. The dynamic change of ATP/ADP ratio was successfully measured with PercevalHR in both MIN6 cells and primary beta cells. In these measurements, the results with primary beta cells were more reproducible compared to MIN6 cells

A Parallel Perifusion Slide From Glass for the Functional and Morphological Analysis of Pancreatic Islets.

An islet-on-chip system in the form of a completely transparent microscope slide optically accessible from both sides was developed. It is made from laser-structured borosilicate glass and enables the parallel perifusion of five microchannels, each containing one islet precisely immobilized in a pyramidal well. The islets can be in inserted via separate loading windows above each pyramidal well. This design enables a gentle, fast and targeted insertion of the islets and a reliable retention in the well while at the same time permitting a sufficiently fast exchange of the media. In addition to the measurement of the hormone content in the fractionated efflux, parallel live cell imaging of the islet is possible. By programmable movement of the microscopic stage imaging of five wells can be performed. The current chip design ensures sufficient time resolution to characterize typical parameters of stimulus-secretion coupling. This was demonstrated by measuring the reaction of the islets to stimulation by glucose and potassium depolarization. After the perifusion experiment islets can be removed for further analysis. The live-dead assay of the removed islets confirmed that the process of insertion and removal was not detrimental to islet structure and viability. In conclusion, the present islet-on-chip design permits the practical implementation of parallel perifusion experiments on a single and easy to load glass slide. For each immobilized islet the correlation between secretion, signal transduction and morphology is possible. The slide concept allows the scale-up to even higher degrees of parallelization

The forebrain synaptic transcriptome is organized by clocks but its proteome is driven by sleep

Neurons have adapted mechanisms to traffic RNA and protein into distant dendritic and axonal arbors. Taking a biochemical approach, we reveal that forebrain synaptic transcript accumulation shows overwhelmingly daily rhythms, with two-thirds of synaptic transcripts showing time-of-day-dependent abundance independent of oscillations in the soma. These transcripts formed two sharp temporal and functional clusters, with transcripts preceding dawn related to metabolism and translation and those anticipating dusk related to synaptic transmission. Characterization of the synaptic proteome around the clock demonstrates the functional relevance of temporal gating for synaptic processes and energy homeostasis. Unexpectedly, sleep deprivation completely abolished proteome but not transcript oscillations. Altogether, the emerging picture is one of a circadian anticipation of messenger RNA needs in the synapse followed by translation as demanded by sleep-wake cycles

Development of a Sandwich ELISA to Measure Exposure to Occupational Cow Hair Allergens

Background: Cow hair and dander are important inducers of occupational allergies in cattle-exposed farmers. To estimate allergen exposure in farming environments, a sensitive enzyme immunoassay was developed to measure cow hair allergens. Methods: A sandwich ELISA was developed using polyclonal rabbit antibodies against a mixture of hair extracts from different cattle breeds. To assess the specificity of the assay, extracts from other mammalian epithelia, mites, molds and grains were tested. To validate the new assay, cow hair allergens were measured in passive airborne dust samples from the stables and homes of farmers. Dust was collected with electrostatic dust fall collectors (EDCs). Results: The sandwich ELISA was found to be very sensitive (detection limit: 0.1 ng/ml) and highly reproducible, demonstrating intra-and interassay coefficients of variation of 4 and 10%, respectively. The assay showed no reactivity with mites, molds and grains, but some cross-reactivity with other mammalian epithelia, with the strongest reaction with goat. Using EDCs for dust sampling, high concentrations of bovine allergens were measured in cow stables (4,760-559,400 mu g/m(2)). In addition, bovine allergens were detected in all areas of cattle farmer dwellings. A large variation was found between individual samples (0.3-900 mu g/m(2)) and significantly higher values were discovered in changing rooms. Conclusion: The ELISA developed for the detection of cow hair proteins is a useful tool for allergen quantification in occupational and home environments. Based on its low detection limit, this test is sensitive enough to detect allergens in passive airborne dust. Copyright (C) 2011 S. Karger AG, Base

Regulation of B cell homeostasis and activation by the tumor suppressor gene CYLD

B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-κB (NF-κB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLDex7/8 mice), which is a deubiquitinating enzyme that is integral to NF-κB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occurs in this strain. The B lymphocytes themselves exhibit prolonged survival and manifest a variety of signaling disarrangements that do not occur in mice with a complete deletion of CYLD. Although both the full-length and the mutant CYLD are able to interact with Bcl-3, a predominant nuclear accumulation of Bcl-3 occurs in the CYLD mutant B cells. More dramatic, however, is the accumulation of the NF-κB proteins p100 and RelB in CYLDex7/8 B cells, which, presumably in combination with nuclear Bcl-3, results in increased levels of Bcl-2 expression. These findings suggest that CYLD can both positively and negatively regulate signal transduction and homeostasis of B cells in vivo, depending on the expression of CYLD splice variants

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer.

Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM -/- patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations

Genome-wide interaction analysis of menopausal hormone therapy use and breast cancer risk among 62,370 women

Use of menopausal hormone therapy (MHT) is associated with increased risk for breast cancer. However, the relevant mechanisms and its interaction with genetic variants are not fully understood. We conducted a genome-wide interaction analysis between MHT use and genetic variants for breast cancer risk in 27,585 cases and 34,785 controls from 26 observational studies. All women were post-menopausal and of European ancestry. Multivariable logistic regression models were used to test for multiplicative interactions between genetic variants and current MHT use. We considered interaction p-values < 5 × 10–8 as genome-wide significant, and p-values < 1 × 10–5 as suggestive. Linkage disequilibrium (LD)-based clumping was performed to identify independent candidate variants. None of the 9.7 million genetic variants tested for interactions with MHT use reached genome-wide significance. Only 213 variants, representing 18 independent loci, had p-values < 1 × 105. The strongest evidence was found for rs4674019 (p-value = 2.27 × 10–7), which showed genome-wide significant interaction (p-value = 3.8 × 10–8) with current MHT use when analysis was restricted to population-based studies only. Limiting the analyses to combined estrogen–progesterone MHT use only or to estrogen receptor (ER) positive cases did not identify any genome-wide significant evidence of interactions. In this large genome-wide SNP-MHT interaction study of breast cancer, we found no strong support for common genetic variants modifying the effect of MHT on breast cancer risk. These results suggest that common genetic variation has limited impact on the observed MHT–breast cancer risk association

PHIP - a novel candidate breast cancer susceptibility locus on 6q14.1

Most non-BRCA1/2 breast cancer families have no identified genetic cause. We used linkage and haplotype analyses in familial and sporadic breast cancer cases to identify a susceptibility locus on chromosome 6q. Two independent genome-wide linkage analysis studies suggested a 3 Mb locus on chromosome 6q and two unrelated Swedish families with a LOD > 2 together seemed to share a haplotype in 6q14.1. We hypothesized that this region harbored a rare high-risk founder allele contributing to breast cancer in these two families. Sequencing of DNA and RNA from the two families did not detect any pathogenic mutations. Finally, 29 SNPs in the region were analyzed in 44,214 cases and 43,532 controls from BCAC, and the original haplotypes in the two families were suggested as low-risk alleles for European and Swedish women specifically. There was also some support for one additional independent moderate-risk allele in Swedish familial samples. The results were consistent with our previous findings in familial breast cancer and supported a breast cancer susceptibility locus at 6q14.1 around the PHIP gene.Peer reviewe