Recycling and Composing at the Appalachian Center for Craft

In summarizing our project, the 2017-2018 ESS Capstone students would like to revamp the existing recycling bins in the Appalachian Center for Craft campus along with introducing composting to the campus. After evaluating the existing recycling bins on the campus, it was decided to offer a new system of color coded bins that the Office of Sustainability could easily provide. This would replace the old bins and offer more of them to residents and other areas of the campus to provide ease of access and more incentive to recycle. Composting was introduced to the Craft Center for their on-site restaurant, The Blue Water Grille, to begin recycling their waste for future use of soil on their own grounds. This would educate residents and staff on composting and provide the campus the opportunity to be more environmentally friendly. Both projects would provide the campus with environmental benefits for almost no cost due to the Office of Sustainability providing all recycling and composting bins

MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling

Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.National Institutes of Health (U.S.) (GM0077465)National Institutes of Health (U.S.) (DK080261

MICU2 is paralogous to MICU1 and localizes to mitochondria.

<p>A. MICU1, MICU2 and MICU3 share a common ancestor and are present in multiple vertebrate species. B. RNA expression analysis of MICU1, MICU2, MICU3 and MCU across 21 mouse tissues. For each tissue, the dots represent individual replicate measures and the bars represent mean values. C. MICU2 has two evolutionarily conserved EF hands. D. Representative confocal images of HeLa cells cotransfected with MICU2-GFP and Mito-HcRed1.</p

MICU1 and MICU2 can be silenced <i>in vivo</i> in mouse liver using siRNA technology.

<p>A. <i>In vitro</i> dose-response curves of selected duplexes targeting MICU1 and MICU2. B. Relative expression of MICU1 and MICU2 mRNA after 6 weekly injections normalized to siLUC mice. C. Representative oxygen consumption traces measured in isolated mitochondria from siLUC (top) and siMICU1+2 (bottom) mice. Arrows denote addition of mitochondria, glutamate and malate (G/M), ADP and uncoupler (carbonyl cyanide m-chlorophenylhydrazone, CCCP). Respiratory control ratios (RCR) and ADP∶O ratios (P∶O) were calculated from experiments performed on three separate mice per group. D. Representative mitochondrial membrane potential traces measured in isolated mitochondria from siLUC (top) and siMICU1+2 (bottom) mice using tetramethyl rhodamine methyl ester (TMRM). E. Respiratory control ratios (RCR) and ADP∶O ratios (P∶O) were comparable among all treatment groups.</p

MICU1 and MICU2 stabilize each other's expression and interact with MCU.

<p>A. Whole cell lysates from HEK293T cells stably expressing a control shRNA (shGFP and shLACZ) or a shRNA targeting MICU1 (shMICU1_a and shMICU1_b) or MICU2 (shMICU2_a) were analyzed using qPCR and western blot. The relative mRNA is reported using β-actin as an endogenous control and normalized to shGFP for each target. Whole cell lysates were blotted with anti-MICU1, anti-MICU2 and control anti-ATP5A. B. Whole cell lysates from HEK293T cells stably expressing FLAG-GFP or FLAG-MICU1 were lysed and blotted with anti-MICU2, anti-FLAG and control anti-ATP5A. C–D. Mitochondria isolated from HEK293T cells stably expressing MCU-FLAG (C) or FLAG-MICU1 (D) were solubilized with 0.2% DDM and subjected to anti-FLAG immunoprecipitation. Immunoprecipitates and lysate were blotted with anti-FLAG, anti-MICU1, anti-MICU2 and control anti-ATP5B and anti-SDHB.</p