Dissection of a QTL Hotspot on Mouse Distal Chromosome 1 that Modulates Neurobehavioral Phenotypes and Gene Expression

A remarkably diverse set of traits maps to a region on mouse distal chromosome 1 (Chr 1) that corresponds to human Chr 1q21–q23. This region is highly enriched in quantitative trait loci (QTLs) that control neural and behavioral phenotypes, including motor behavior, escape latency, emotionality, seizure susceptibility (Szs1), and responses to ethanol, caffeine, pentobarbital, and haloperidol. This region also controls the expression of a remarkably large number of genes, including genes that are associated with some of the classical traits that map to distal Chr 1 (e.g., seizure susceptibility). Here, we ask whether this QTL-rich region on Chr 1 (Qrr1) consists of a single master locus or a mixture of linked, but functionally unrelated, QTLs. To answer this question and to evaluate candidate genes, we generated and analyzed several gene expression, haplotype, and sequence datasets. We exploited six complementary mouse crosses, and combed through 18 expression datasets to determine class membership of genes modulated by Qrr1. Qrr1 can be broadly divided into a proximal part (Qrr1p) and a distal part (Qrr1d), each associated with the expression of distinct subsets of genes. Qrr1d controls RNA metabolism and protein synthesis, including the expression of ∼20 aminoacyl-tRNA synthetases. Qrr1d contains a tRNA cluster, and this is a functionally pertinent candidate for the tRNA synthetases. Rgs7 and Fmn2 are other strong candidates in Qrr1d. FMN2 protein has pronounced expression in neurons, including in the dendrites, and deletion of Fmn2 had a strong effect on the expression of few genes modulated by Qrr1d. Our analysis revealed a highly complex gene expression regulatory interval in Qrr1, composed of multiple loci modulating the expression of functionally cognate sets of genes

Involvement of the Cytokine MIF in the Snail Host Immune Response to the Parasite Schistosoma mansoni

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions

Modelling Vesicular Release at Hippocampal Synapses

We study local calcium dynamics leading to a vesicle fusion in a stochastic, and spatially explicit, biophysical model of the CA3-CA1 presynaptic bouton. The kinetic model for vesicle release has two calcium sensors, a sensor for fast synchronous release that lasts a few tens of milliseconds and a separate sensor for slow asynchronous release that lasts a few hundred milliseconds. A wide range of data can be accounted for consistently only when a refractory period lasting a few milliseconds between releases is included. The inclusion of a second sensor for asynchronous release with a slow unbinding site, and thereby a long memory, affects short-term plasticity by facilitating release. Our simulations also reveal a third time scale of vesicle release that is correlated with the stimulus and is distinct from the fast and the slow releases. In these detailed Monte Carlo simulations all three time scales of vesicle release are insensitive to the spatial details of the synaptic ultrastructure. Furthermore, our simulations allow us to identify features of synaptic transmission that are universal and those that are modulated by structure

Quintuple labeling in the electron microscope with genetically encoded enhanced horseradish peroxidase.

Genetic encoded multilabeling is essential for modern cell biology. In fluorescence microscopy this need has been satisfied by the development of numerous color-variants of the green fluorescent protein. In electron microscopy, however, true genetic encoded multilabeling is currently not possible. Here, we introduce combinatorial cell organelle type-specific labeling as a strategy for multilabeling. First, we created a reliable and high sensitive label by evolving the catalytic activity of horseradish peroxidase (HRP). We then built fusion proteins that targeted our new enhanced HRP (eHRP) to three cell organelles whose labeling pattern did not overlap with each other. The labeling of the endoplasmic reticulum, synaptic vesicles and the plasma membrane consequently allowed for triple labeling in the EM. The combinatorial expression of the three organelle-specific constructs increased the number of clearly distinguishable labels to seven. This strategy of multilabeling for EM closes a significant gap in our tool set and has a broad application range in cell biology

High resolution fluorescent image of eHRP labeled neurons in culture.

<p>eHRP was targeted to the endoplasmic reticulum and visualized with Amplex Red.</p

Neuronal cells expressing a fusion protein of eHRP and synaptotagmin 1 (eHRPsyt1).

<p>(a) Numerous vesicles (arrows) of various sizes are HRP-positive in a young cultured hippocampal neuron. At some locations eHRPsyt1 can be found at the plasma membrane (arrowhead). Bar = 200 nm. (b) An electron micrograph of an eHRPsyt1-positive presynaptic bouton. Note the numerous synaptic vesicles distributed throughout the synaptic vesicle cluster. Some labeled synaptic vesicles are also docked at the active zone (to the left). Bar = 200 nm.</p

Live imaging correlated with light-electron microscopy.

<p>Cultured hippocampal neurons were transfected with eGFP (a) and ER-targeted eHRP (b). The arrowheads point to the HRP-labeled dendrite. (c) Electron micrograph of the same HRP-labeled dendrite as in (b). The ultrastructure is optimally preserved. The arrows point to the labeled ER. (d) Unlabeled (bottom) and labeled (top) dendrites when imaged in the EM. Note the high signal-to-noise ratio of the HRP-positive ER (arrows) compared to the ER in the unlabeled dendrite (d). Both dendrites are innervated by presynaptic boutons (s). Bars in (c) and (d) = 500 nm.</p

Quintuple labeling of neuroblastoma cells in a single sample.

<p>Each unique label and/or combination of labeling corresponds to a unique color. The three single labelings are in red, green and blue. The double labelings are in yellow, turquoise, and magenta. The triple labeling is in white. (a) An unlabeled cell. The horizontal arrowheads point at the endoplasmic reticulum, the vertical arrowheads at small vesicles with an electron-lucent lumen. The arrows demarcate the plasma membrane. (b) A cell expressing ER-targeted eHRP (arrowheads). (c) Expression of eHRPsyt1. Note the numerous small eHRP-positive vesicles in the cytosol (e.g. arrowheads). (d) Single expression of eHRP targeted to the plasma membrane. The arrows point at the labeled surface of the cell. (e) Double expression ER-targeted eHRP and eHRPsyt1. The horizontal arrowheads point to the labeled ER and the vertical arrowheads to vesicle labeling. (f) Double labeling with eHRP targeted to the plasma membranes and eHRPsyt1. The arrows point at the labeled cell surface and the arrowheads to labeled vesicles. (g) Double labeling of ER-targeted eHRP and eHRP targeted to the plasma membrane. Again, the arrows indicate the labeled plasma membrane and the horizontal arrowheads the labeled ER. (h) Triple labeling with all three fusion proteins. Horizontal arrows mark the labeled ER, vertical arrows labeled vesicles, and arrows point at the labeled cell surface. Bars = 500 nm in (a), 200 nm in (b), 1 μm in (c), 200 nm in (d), 500 nm in (e), 200 nm in (f), 200 nm in (g), 1 μm in (h).</p