Biology subjects

Looger, L. L.

Publications and source records attributed to Looger, L. L..

8 recordsLinked to original sources

bioRxiv2026

Combined Image-Based Profiling and Biochemical Analysis of GCaMP Overexpression Effects on Mammalian Cells

Protein-based fluorescent sensors are a powerful addition to the biology toolbox for their ability to be stably expressed within living organisms, tissues, cells, and subcellular compartments, with the capacity to report on the presence of specific target molecules or other analytes. At the same time, sensor components will unavoidably present opportunities for unintended interaction with endogenous cellular machinery, potentially confounding both sensor function and cell health. Interactions with host components may not be readily predictable during the sensor design process, especially when simultaneously optimizing many other sensor parameters such as fluorescence response, dynamic range, and kinetics. Characterizing effects of sensor expression on cells is currently a laborious ad hoc process; new methods to characterize the cell expression effects of sensors and their variants could dramatically improve sensor design pipelines, laying the groundwork to recognize potentially problematic expression side effects earlier in the iterative design and testing workflow. Here, we take a dual high-content imaging-based and biochemical approach to examine sensor interactions with native cell biology, focusing on the widely used GCaMP calcium sensor. We identify a morphology-based signature of the cellular effects of high sensor expression in a neuroblastoma cell line. Subsequently, we identify biochemical interactions between GCaMP and a component of the mammalian cytoskeleton and track morphological features in sensor-expressing cells that lack these structural components. Our findings present an entry point for engineering new minimally cross-reactive sensor versions given a contextual biological understanding of sensor overexpression. We anticipate that as this and related workflows are incorporated into sensor engineering pipelines, bioorthogonality can be more systematically assessed and prioritized in diverse sensor scaffolds.

cell biology↗

bioRxiv2018

High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics, and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and tracking large populations of neurons using 2-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

neuroscience↗

bioRxiv2018

A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP

Adenosine 5 triphosphate (ATP) is a universal intracellular energy source1 and an evolutionarily ancient2 extracellular signal3-5. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responded to relevant ATP concentrations (30 M to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. iATPSnFRs represent promising new reagents for imaging ATP dynamics.

physiology↗

bioRxiv2018

Kilohertz frame-rate two-photon tomography

Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 {micro}m and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

neuroscience↗

bioRxiv2018

Multiplex imaging of quantal glutamate release and presynaptic Ca2+ at multiple synapses in situ

Information processing by brain circuits depends on Ca2+-dependent, stochastic release of the excitatory neurotransmitter glutamate. Recently developed optical sensors have enabled detection of evoked and spontaneous release at common glutamatergic synapses. However, monitoring synaptic release probability, its use-dependent changes, and its underpinning presynaptic machinery in situ requires concurrent, intensity-independent readout of presynaptic Ca2+ and glutamate release. Here, we find that the red-shifted Ca2+ indicator Cal-590 shows Ca2+-sensitive fluorescence lifetime, and employ it in combination with the novel green glutamate sensor SF-iGluSnFR variant to document quantal release of glutamate together with presynaptic Ca2+ concentration, in multiple synapses in an identified neural circuit. At the level of individual presynaptic boutons, we use multi-exposure and stochastic reconstruction procedures to reveal nanoscopic co-localisation of presynaptic Ca2+ entry and glutamate release, a fundamental unknown in modern neurobiology. This approach opens a new horizon in the quest to understand release machinery of central synapses.

neuroscience↗

bioRxiv2018

A genetically encoded fluorescent sensor for in vivo imaging of GABA

Current techniques for monitoring GABA, the primary inhibitory neurotransmitter in vertebrates, cannot follow ephemeral transients in intact neural circuits. We applied the design principles used to create iGluSnFR, a fluorescent reporter of synaptic glutamate, to develop a GABA sensor using a protein derived from a previously unsequenced Pseudomonas fluorescens strain. Structure-guided mutagenesis and library screening led to a usable iGABASnFR ({Delta}F/Fmax ~ 2.5, Kd ~ 9 M, good specificity, adequate kinetics). iGABASnFR is genetically encoded, detects single action potential-evoked GABA release events in culture, and produces readily detectable fluorescence increases in vivo in mice and zebrafish. iGABASnFR enabled tracking of: (1) mitochondrial GABA content and its modulation by an anticonvulsant; (2) swimming-evoked GABAergic transmission in zebrafish cerebellum; (3) GABA release events during inter-ictal spikes and seizures in awake mice; and (4) GABAergic tone decreases during isoflurane anesthesia. iGABASnFR will permit high spatiotemporal resolution of GABA signaling in intact preparations.

neuroscience↗

bioRxiv2017

Stability, affinity and chromatic variants of the glutamate sensor iGluSnFR

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of the glutamate sensor iGluSnFR that are functionally brighter; can detect sub-micromolar to millimolar concentrations of glutamate; and have blue, green or yellow emission profiles. These variants allow in vivo imaging where original-iGluSnFR was too dim, reveal glutamate transients at individual spine heads, and permit kilohertz imaging with inexpensive, powerful fiber lasers.

neuroscience↗

bioRxiv2017

Distinct descending motor cortex pathways and their roles in movement

Activity in motor cortex predicts specific movements, seconds before they are initiated. This preparatory activity has been observed in L5 descending pyramidal tract (PT) neurons. A key question is how preparatory activity can be maintained without causing movement, and how preparatory activity is eventually converted to a motor command to trigger appropriate movements. We used single cell transcriptional profiling and axonal reconstructions to identify two types of PT neuron. Both types share projections to multiple targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex. In a delayed-response task, these neurons produced early preparatory activity that persisted until the movement. The second type projects to motor centers in the medulla and produced late preparatory activity and motor commands. These results indicate that two motor cortex output neurons are specialized for distinct roles in motor control.

neuroscience↗