@article {49406, title = {Cell-specific chemical delivery using a selective Nitroreductase-Nitroaryl pair.}, journal = {ACS Chemical Biology}, volume = {13}, year = {2018}, month = {2018 Aug 29}, pages = {1888-96}, abstract = {

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

}, issn = {1554-8937}, doi = {10.1021/acschembio.8b00524}, author = {Gruber, Todd D and Krishnamurthy, Chithra and Grimm, Jonathan B and Tadross, Michael R and Wysocki, Laura M and Gartner, Zev J and Lavis, Luke D} } @article {48413, title = {Deconstructing behavioral neuropharmacology with cellular specificity.}, journal = {Science (New York, N.Y.)}, volume = {356}, year = {2017}, month = {2017 Apr 07}, abstract = {

Behavior has molecular, cellular, and circuit determinants. However, because many proteins are broadly expressed, their acute manipulation within defined cells has been difficult. Here, we combined the speed and molecular specificity of pharmacology with the cell type specificity of genetic tools. DART (drugs acutely restricted by tethering) is a technique that rapidly localizes drugs to the surface of defined cells, without prior modification of the native target. We first developed an AMPAR antagonist DART, with validation in cultured neuronal assays, in slices of mouse dorsal striatum, and in behaving mice. In parkinsonian animals, motor deficits were causally attributed to AMPARs in indirect spiny projection neurons (iSPNs) and to excess phasic firing of tonically active interneurons (TANs). Together, iSPNs and TANs (i.e., D2 cells) drove akinesia, whereas movement execution deficits reflected the ratio of AMPARs in D2 versus D1 cells. Finally, we designed a muscarinic antagonist DART in one iteration, demonstrating applicability of the method to diverse targets.

}, issn = {1095-9203}, doi = {10.1126/science.aaj2161}, author = {Shields, Brenda C and Kahuno, Elizabeth and Kim, Charles and Apostolides, Pierre F and Brown, Jennifer and Lindo, Sarah and Mensh, Brett D and Dudman, Joshua T and Lavis, Luke D and Tadross, Michael R} } @article {47424, title = {Movement and structure of mitochondria in oligodendrocytes and their myelin sheaths.}, journal = {Glia}, volume = {64}, year = {2016}, month = {2016 May}, pages = {810-25}, abstract = {

Mitochondria play several crucial roles in the life of oligodendrocytes. During development of the myelin sheath they are essential providers of carbon skeletons and energy for lipid synthesis. During normal brain function their consumption of pyruvate will be a key determinant of how much lactate is available for oligodendrocytes to export to power axonal function. Finally, during calcium-overload induced pathology, as occurs in ischemia, mitochondria may buffer calcium or induce apoptosis. Despite their important functions, very little is known of the properties of oligodendrocyte mitochondria, and mitochondria have never been observed in the myelin sheaths. We have now used targeted expression of fluorescent mitochondrial markers to characterize the location and movement of mitochondria within oligodendrocytes. We show for the first time that mitochondria are able to enter and move within the myelin sheath. Within the myelin sheath the highest number of mitochondria was in the cytoplasmic ridges along the sheath. Mitochondria moved more slowly than in neurons and, in contrast to their behavior in neurons and astrocytes, their movement was increased rather than inhibited by glutamate activating NMDA receptors. By electron microscopy we show that myelin sheath mitochondria have a low surface area of cristae, which suggests a low ATP production. These data specify fundamental properties of the oxidative phosphorylation system in oligodendrocytes, the glial cells that enhance cognition by speeding action potential propagation and provide metabolic support to axons. GLIA 2016;64:810-825.

}, issn = {1098-1136}, doi = {10.1002/glia.22965}, author = {Rinholm, Johanne E and Vervaeke, Koen and Tadross, Michael R and Tkachuk, Ariana N and Kopek, Benjamin G and Brown, Timothy A and Bergersen, Linda H and Clayton, David A} } @article {47315, title = {Sequential ionic and conformational signaling by calcium channels drives neuronal gene expression.}, journal = {Science (New York, N.Y.)}, volume = {351}, year = {2016}, month = {2016 Feb 19}, pages = {863-7}, abstract = {

Voltage-gated CaV1.2 channels (L-type calcium channel α1C subunits) are critical mediators of transcription-dependent neural plasticity. Whether these channels signal via the influx of calcium ion (Ca2+), voltage-dependent conformational change (VΔC), or a combination of the two has thus far been equivocal. We fused CaV1.2 to a ligand-gated Ca2+-permeable channel, enabling independent control of localized Ca2+\ and VΔC signals. This revealed an unexpected dual requirement: Ca2+\ must first mobilize actin-bound Ca2+/calmodulin-dependent protein kinase II, freeing it for subsequent VΔC-mediated accumulation. Neither signal alone sufficed to activate transcription. Signal order was crucial: Efficiency peaked when Ca2+\ preceded VΔC by 10 to 20 seconds. CaV1.2 VΔC synergistically augmented signaling by\ N-methyl-D-aspartate receptors. Furthermore, VΔC mistuning correlated with autistic symptoms in Timothy syndrome. Thus, nonionic VΔC signaling is vital to the function of CaV1.2 in synaptic and neuropsychiatric processes.

}, doi = {10.1126/science.aad3647}, author = {Li, Boxing and Tadross, Michael R and Tsien, Richard W} } @article {46368, title = {Labeling of active neural circuits in vivo with designed calcium integrators.}, journal = {Science}, volume = {347}, year = {2015}, month = {2015 Feb 13}, pages = {755-60}, abstract = {

The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.

}, issn = {1095-9203}, doi = {10.1126/science.1260922}, author = {Fosque, Benjamin F and Sun, Yi and Dana, Hod and Yang, Chao-Tsung and Ohyama, Tomoko and Tadross, Michael R and Patel, Ronak and Zlatic, Marta and Kim, Douglas S and Ahrens, Misha B and Jayaraman, Vivek and Looger, Loren L and Schreiter, Eric R} }