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Schreiter Lab / Publications
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39 Publications

Showing 1-10 of 39 results
08/01/17 | Genetically encoded biosensors.
Marvin JS, Looger LL, Lee RT, Schreiter ER
USPTO. 2017 Aug 01;B2:

The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

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06/12/17 | Neural signatures of dynamic stimulus selection in Drosophila.
Sun Y, Nern A, Franconville R, Dana H, Schreiter ER, Looger LL, Svoboda K, Kim DS, Hermundstad AM, Jayaraman V
Nature Neuroscience. 2017 Jun 12;20(8):1104-13. doi: 10.1038/nn.4581

Many animals orient using visual cues, but how a single cue is selected from among many is poorly understood. Here we show that Drosophila ring neurons—central brain neurons implicated in navigation—display visual stimulus selection. Using in vivo two-color two-photon imaging with genetically encoded calcium indicators, we demonstrate that individual ring neurons inherit simple-cell-like receptive fields from their upstream partners. Stimuli in the contralateral visual field suppressed responses to ipsilateral stimuli in both populations. Suppression strength depended on when and where the contralateral stimulus was presented, an effect stronger in ring neurons than in their upstream inputs. This history-dependent effect on the temporal structure of visual responses, which was well modeled by a simple biphasic filter, may determine how visual references are selected for the fly's internal compass. Our approach highlights how two-color calcium imaging can help identify and localize the origins of sensory transformations across synaptically connected neural populations.

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03/01/17 | All-optical functional synaptic connectivity mapping in acute brain slices using CaMPARI.
Zolnik TA, Sha F, Johenning F, Schreiter ER, Looger LL, Larkum ME, Sachdev RN
The Journal of Physiology. 2017 Mar 01;595(5):1465-77. doi: 10.1113/JP273116

The calcium-modulated photoactivatable ratiometric integrator CaMPARI (Fosque et al., 2015) facilitates the study of neural circuits by permanently marking cells active during user-specified temporal windows. Permanent marking enables measurement of signals from large swathes of tissue and easy correlation of activity with other structural or functional labels. One potential application of CaMPARI is labeling neurons postsynaptic to specific populations targeted for optogenetic stimulation, giving rise to all-optical functional connectivity mapping. Here, we characterized the response of CaMPARI to several common types of neuronal calcium signals in mouse acute cortical brain slices. Our experiments show that CaMPARI is effectively converted by both action potentials and sub-threshold synaptic inputs, and that conversion level is correlated to synaptic strength. Importantly, we found that conversion rate can be tuned: it is linearly related to light intensity. At low photoconversion light levels CaMPARI offers a wide dynamic range due to slower conversion rate; at high light levels conversion is more rapid and more sensitive to activity. Finally, we employed CaMPARI and optogenetics for functional circuit mapping in ex vivo acute brain slices, which preserve in vivo-like connectivity of axon terminals. With a single light source, we stimulated channelrhodopsin-2-expressing long-range posteromedial (POm) thalamic axon terminals in cortex and induced CaMPARI conversion in recipient cortical neurons. We found that POm stimulation triggers robust photoconversion of layer 5 cortical neurons and weaker conversion of layer 2/3 neurons. Thus, CaMPARI enables network-wide, tunable, all-optical functional circuit mapping that captures supra- and sub-threshold depolarization. This article is protected by copyright. All rights reserved.

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01/30/17 | Axonal Endoplasmic Reticulum Ca(2+) Content Controls Release Probability in CNS Nerve Terminals.
de Juan-Sanz J, Holt GT, Schreiter ER, de Juan F, Kim DS, Ryan TA
Neuron. 2017 Jan 30;93(4):867-81. doi: 10.1016/j.neuron.2017.01.010

Although the endoplasmic reticulum (ER) extends throughout axons and axonal ER dysfunction is implicated in numerous neurological diseases, its role at nerve terminals is poorly understood. We developed novel genetically encoded ER-targeted low-affinity Ca(2+) indicators optimized for examining axonal ER Ca(2+). Our experiments revealed that presynaptic function is tightly controlled by ER Ca(2+) content. We found that neuronal activity drives net Ca(2+) uptake into presynaptic ER although this activity does not contribute significantly to shaping cytosolic Ca(2+) except during prolonged repetitive firing. In contrast, we found that axonal ER acts as an actuator of plasma membrane (PM) function: [Ca(2+)]ER controls STIM1 activation in presynaptic terminals, which results in the local modulation of presynaptic function, impacting activity-driven Ca(2+) entry and release probability. These experiments reveal a critical role of presynaptic ER in the control of neurotransmitter release and will help frame future investigations into the molecular basis of ER-driven neuronal disease states.

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05/24/16 | Design and synthesis of a calcium-sensitive photocage.
Heckman LM, Grimm JB, Schreiter ER, Kim C, Verdecia MA, Shields BC, Lavis LD
Angewandte Chemie (International ed. in English). 2016 May 24:. doi: 10.1002/anie.201602941

Photolabile protecting groups (or "photocages") enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input-output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca(2+) -sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca(2+) ]. Caging a fluorophore with this ion-sensitive moiety yields an "ion integrator" that permanently marks cells undergoing high Ca(2+) flux during an illumination-defined time period. Our general design concept demonstrates a new class of light-sensitive material for cellular imaging, sensing, and targeted molecular delivery.

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03/24/16 | Sensitive red protein calcium indicators for imaging neural activity.
Dana H, Mohar B, Sun Y, Narayan S, Gordus A, Hasseman JP, Tsegaye G, Holt GT, Hu A, Walpita D, Patel R, Macklin JJ, Bargmann CI, Ahrens MB, Schreiter ER, Jayaraman V, Looger LL, Svoboda K, Kim DS
eLife. 2016 Mar 24;5:. doi: 10.7554/eLife.12727

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

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11/01/15 | Structural basis for the antipolymer activity of Hb ζ22βsζ2βs2 trapped in a tense conformation.
Safo MK, Ko T, Schreiter ER, Russell JE
Journal of Molecular Structure. 2015 Nov;1099:99-107. doi: 10.1016/j.molstruc.2015.06.047

The phenotypical severity of sickle cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb S, Hb α2β2s) to contain embryonic ζ globin in place of adult α-globin subunits (Hb ζ2β2s). Crystallographical analyses of liganded Hb ζζ2β2s, though, demonstrate a tense (T-state) quaternary structure that paradoxically predicts its participation in--rather than its exclusion from--pathological deoxyHb S polymers. We resolved this structure-function conundrum by examining the effects of α → ζ exchange on the characteristics of specific amino acids that mediate sickle polymer assembly. Superposition analyses of the βs subunits of T-state deoxyHb α2β2s and T-state CO-liganded Hb ζ2β2s reveal significant displacements of both mutant βsVal6 and conserved β-chain contact residues, predicting weakening of corresponding polymer-stabilizing interactions. Similar comparisons of the α- and ζ-globin subunits implicate four amino acids that are either repositioned or undergo non-conservative substitution, abrogating critical polymer contacts. CO-Hb ζ2βs2 additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological βsVal6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ2β2s suggests that it does not co-assemble with deoxyHb S in vivo  . In sum, the antipolymer activities of Hb ζ2β2s appear to arise from both repositioning and replacement of specific α- and βs-chain residues, favoring an alternate T-state solution structure that is excluded from pathological deoxyHb S polymers. These data account for the antipolymer activity of Hb ζ2β2s, and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.

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10/09/15 | A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.
Henderson MJ, Baldwin HA, Werley CA, Boccardo S, Whitaker LR, Yan X, Holt GT, Schreiter ER, Looger LL, Cohen AE, Kim DS, Harvey BK
PloS one. 2015 Oct 09;10(10):e0139273. doi: 10.1371/journal.pone.0139273

Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

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09/18/15 | Green-to-red photoconversion of GCaMP.
Ai M, Mills H, Kanai M, Lai J, Deng J, Schreiter E, Looger L, Neubert T, Suh G
PLoS One. 2015 Sep 18;10(9):e0138127. doi: 10.1371/journal.pone.0138127

Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs-including GCaMP3, GCaMP5 and GCaMP6-can be converted from green to red following exposure to blue-green light (450-500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.

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02/13/15 | Labeling of active neural circuits in vivo with designed calcium integrators.
Fosque BF, Sun Y, Dana H, Yang C, Ohyama T, Tadross MR, Patel R, Zlatic M, Kim DS, Ahrens MB, Jayaraman V, Looger LL, Schreiter ER
Science. 2015 Feb 13;347(6223):755-60. doi: 10.1126/science.1260922

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.

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