Biology subjects

Shaner, N. C.

Publications and source records attributed to Shaner, N. C..

10 recordsLinked to original sources

bioRxiv2025

Control of Synaptic Communication through Molecularly Engineered Bioluminescence Light Emission and Sensing

Synapses are sites of intercellular communication between neurons and from neurons to target organs, and of signal integration that underly physiological and behavioral responses. We have developed a modular platform, Interluminescence (Int), for experimental control of synaptic transmission: bioluminescent light, generated by a luciferase oxidizing a luciferin, from a presynaptic neuron is used to activate transsynaptic optogenetic ion channels in the postsynaptic neuron. Two strategies can modulate postsynaptic neurons in the presence of luciferin. In the Act-Int approach, a luciferase is genetically expressed in synaptic vesicles and released during depolarization-induced presynaptic vesicle fusion and exocytosis. In the Persist-Int approach, a luciferase is tethered to the presynaptic membrane where it can support sustained transsynaptic signaling. Both strategies can activate postsynaptic neurons with comparable efficacy under the conditions tested. By design, the modularity of the platform permits the use of luciferases and opsins ranging in brightness and light sensitivity, with the luciferase targeted to different subcellular regions of the presynaptic neuron, and the postsynaptic opsin being excitatory or inhibitory. Our results demonstrate the utility and versatility of Interluminescence to mediate synapse-specific transmission that is either activity-dependent or activity-independent.

neuroscience↗

bioRxiv2023

Towards a Brighter Constellation: Multi-Organ Neuroimaging of Neural and Vascular Dynamics in the Spinal Cord and Brain

SignificancePain is comprised of a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. AimHere, we aimed to develop and validate new tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations were targeted to developing novel imaging hardware that addresses the many challenges of multi-site imaging. The second key set of innovations were targeted to enabling bioluminescent imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity and decreased resolution due to scattering of excitation signals. ApproachWe designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one-or two-photon microscopy or optogenetic stimulation). We also tested the viability for bioluminescent imaging, and developed a novel modified miniscope optimized for these signals (BLmini). ResultsHere, we describe novel universal implants for acute and chronic simultaneous brain-spinal cord imaging and optical stimulation. We further describe successful imaging of bioluminescent signals in both foci, and a new miniscope, the BLmini, which has reduced weight, cost and form-factor relative to standard wearable miniscopes. ConclusionsThe combination of 3D printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a new coalition of methods for understanding spinal cord-brain interactions. This work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior.

bioengineering↗

bioRxiv2023

Engineering luminopsins with improved coupling efficiencies

SignificanceLuminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. AimWe investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. ApproachWe quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons. ResultsLuciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. ConclusionsWhole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Forster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

molecular biology↗

bioRxiv2023

A Bioluminescent Activity Dependent (BLADe) Platform for Converting Neuronal Activity to Photoreceptor Activation

Genetically encoded sensors and actuators have advanced the ability to observe and manipulate cellular activity, yet few non-invasive strategies enable cells to directly couple their intracellular states to user-defined outputs. We promote a bioluminescent activity-dependent (BLADe) platform that facilitates programmable feedback through genetically encoded light generation. Using calcium (Ca{superscript 2}) flux as a model, we engineered a Ca{superscript 2}-dependent luciferase that functions as an activity-gated light source capable of photoactivating light-sensing actuators. As an initial demonstration of the versatility of this platform we present two separate use cases in neurons. In the first application, the presence of luciferin triggers Ca{superscript 2} dependent local illumination that provides activity dependent gene expression by activating a light-sensitive transcription factor. In the second application, neuronal activity-driven Ca{superscript 2} fluctuations via locally generated bioluminescence control neural dynamics through opsin activation in single cells, populations and intact tissue. BLADe can be expanded to couple any signal that bioluminescent enzymes can be engineered to detect with the wide variety of photosensing actuators. This modular strategy of coupling an activity dependent light emitter to a light sensing actuator offers, in principle, a generalizable framework for state dependent cell-autonomous control across biological systems.

neuroscience↗

bioRxiv2023

CaBLAM! A high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase

Measuring ongoing cellular activity is essential to understanding the dynamic functions of biological organisms. The most popular current approach is imaging fluorescence-based genetically encoded Ca2+ indicators (GECIs). While fluorescent probes are useful in many contexts, bioluminescence-based GECIs--probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein--have several distinct advantages. Because bioluminescent (BL) GECIs do not use the bright extrinsic excitation light required for fluorescence, BL GECIs do not photobleach, do not suffer from nonspecific autofluorescent background, and do not cause phototoxicity. Further, BL GECIs can be applied in contexts where directly shining photons on an imaging target is not possible. Despite these advantages, the use of BL GECIs has to date been limited by their small changes in bioluminescence intensity, high baseline signal at resting Ca2+ concentrations, and suboptimal Ca2+ affinities. Here, we describe a new BL GECI, CaBLAM (Ca2+ BioLuminescence Activity Monitor), that displays much higher dynamic range than previous BL GECIs and has a Ca2+ affinity suitable for capturing physiological changes in cytosolic Ca2+ concentration. With these improvements, CaBLAM captures single-cell and subcellular resolution activity at high frame rates in cultured neurons and in vivo, and allows multi-hour recordings in mice and behaving zebrafish. This new advance provides a robust alternative to traditional fluorescent GECIs that can enable or enhance imaging across many experimental conditions.

neuroscience↗

bioRxiv2023

A FRET-based excitatory luminopsin with improved efficacy of bioluminescence-driven activation

SignificanceBioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). AimTo further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence- driven) LMO activation. ApproachWe developed novel luciferases optimized for Forster resonance energy transfer (FRET) when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). ResultsA new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard), and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. ConclusionsWe report a robust new option for achieving multiple modes of control in a single actuator, and a promising engineering strategy for continued improvement of BL-OG.

neuroscience↗

bioRxiv2021

Single-Domain SARS-CoV-2 S1 and RBD Antibodies Isolated from Immunized Llama Effectively Bind Targets of the Wuhan, UK, and South African Strains in vitro

The spreading of SARS-CoV-2 variants has become a major challenge of the current fight against the pandemic. Of particular concerns are the strains that have arisen from the United Kingdom (UK) and South Africa. The UK variant spreads rapidly and is projected to overtake the original strain in the US as early as in March 2021, while the South African variant appears to evade some effects of the current vaccines. Potential false-negative diagnosis using currently available antigen kits that may not recognize these variants could cause another wave of community infection. Therefore, it is imperative that antibodies used in the detection kits are validated for binding against these variants. Here we report that the nanoantibodies (nAbs in our terminology, also referred to as VHH fragments, single domain antibodies, nanobodies) that we have developed for rapid antigen detection test bind the receptor binding domain (RBD) of the S1 protein from the original COVID-SARS-2 virus as well as those from the UK and South African variants. This finding validates our antibodies used in our assay for the detection of these major variant strains.

immunology↗

bioRxiv2019

Aequorea victoria’s secrets

Using mRNA-Seq and de novo transcriptome assembly, we identified, cloned and characterized nine previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from avGFP. Among these FPs are the brightest GFP homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including two that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

biochemistry↗

bioRxiv2018

Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling

Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here we develop a method to rapidly deplete tubulin glutamlyation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

cell biology↗

bioRxiv2017

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

Genetically encoded Forster Resonance Energy Transfer (FRET) based biosensors report on changes in biochemical states in single living cells. The performance of biosensors depends on their brightness and dynamic range, which are dependent on the characteristics of the fluorescent proteins that are employed. Cyan fluorescent protein (CFP) is frequently combined with yellow fluorescent protein (YFP) as FRET pair in biosensors. However, current YFPs are prone to photobleaching and pH changes. In addition, more efficient acceptors may yield biosensors that have higher contrast. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Forster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mChery and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. The sensor with mScarlet-I as acceptor and mTurquoise2 as donor shows a higher dynamic range in ratiometric FRET imaging experiments and less variability than with mCherry as acceptor, due to the high quantum yield and efficient maturation of mScarlet-I. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.

cell biology↗