The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity.

Imaging neuromodulation with synthetic probes is an emerging technology for studying neurotransmission. However, most synthetic probes are developed through conjugation of fluorescent signal transducers to preexisting recognition moieties such as antibodies or receptors. We introduce a generic platform to evolve synthetic molecular recognition on the surface of near-infrared fluorescent single-wall carbon nanotube (SWCNT) signal transducers. We demonstrate evolution of molecular recognition toward neuromodulator serotonin generated from large libraries of ~6.9 × 1010 unique ssDNA sequences conjugated to SWCNTs. This probe is reversible and produces a ~200% fluorescence enhancement upon exposure to serotonin with a Kd = 6.3 μM, and shows selective responsivity over serotonin analogs, metabolites, and receptor-targeting drugs. Furthermore, this probe remains responsive and reversible upon repeat exposure to exogenous serotonin in the extracellular space of acute brain slices. Our results suggest that evolution of nanosensors could be generically implemented to develop other neuromodulator probes with synthetic molecular recognition.

All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (Vmem). Mounting evidence suggests a relationship between resting Vmem and the physiology of non-excitable cells with implications in diverse areas, including cancer, cellular differentiation, and body patterning. A lack of non-invasive methods to record absolute Vmem limits our understanding of this fundamental signal. To address this need, we developed a fluorescence lifetime-based approach (VF-FLIM) to visualize and optically quantify Vmem with single-cell resolution in mammalian cell culture. Using VF-FLIM, we report Vmem distributions over thousands of cells, a 100-fold improvement relative to electrophysiological approaches. In human carcinoma cells, we visualize the voltage response to growth factor stimulation, stably recording a 10–15 mV hyperpolarization over minutes. Using pharmacological inhibitors, we identify the source of the hyperpolarization as the Ca2+-activated K+ channel KCa3.1. The ability to optically quantify absolute Vmem with cellular resolution will allow a re-examination of its signaling roles.

Here, we report that treatment of syngeneic mouse tumors transduced to overexpress human epidermal growth factor receptor-2 (HER2) with the anti-human HER2 antibody trastuzumab upregulated the level of programmed death-ligand 1 (PD-L1), an important negative regulator of T-cell response, in a transgenic mouse model immune-tolerant to human HER2. We further found that trastuzumab alone had no detectable effect on the level of PD-L1 expression in monocultures of HER2-overexpressing human breast cancer cells but upregulated PD-L1 in the same panel of HER2-overexpressing breast cancer cells when they were co-cultured with human peripheral blood mononuclear cells, and the upregulation of PD-L1 could be blocked by an IFNγ-neutralizing antibody. Inhibition of HER2 intrinsic signaling via HER2 expression knockdown or kinase inhibition had variable and cell-context-specific effects on downregulating the PD-L1 level. Analysis of The Cancer Genome Atlas database showed no direct correlation between HER2 and PD-L1 at the messenger RNA level. Trastuzumab-mediated upregulation of PD-L1 through engagement of immune effector cells may function as a potential mechanism of trastuzumab resistance. Our data justify further investigation of the value of adding anti-PD-1 or anti-PD-L1 therapy to trastuzumab-based treatment.

Electrical potential differences across lipid bilayers play foundational roles in cellular physiology. Plasma membrane voltage is the most widely studied; however, the bilayers of organelles like mitochondria, lysosomes, nuclei, and the endoplasmic reticulum (ER) also provide opportunities for ionic compartmentalization and the generation of transmembrane potentials. Unlike plasma membranes, organellar bilayers, cloistered within the cell, remain recalcitrant to traditional approaches like patch-clamp electrophysiology. To address the challenge of monitoring changes in organelle membrane potential, we describe the design, synthesis, and application of the LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine-based Voltage Reporter) for optically monitoring membrane potential changes in the ER of living cells. We pair a tetrazine-quenched RhoVR for voltage sensing with a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting to the ER. Bright fluorescence is observed only at the coincidence of the LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of the LUnAR RhoVR and Cer-TCO is voltage-sensitive and that the LUnAR RhoVR can be targeted to an intact ER in living cells. Using the LUnAR RhoVR, we use two-color, ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release from internal stores. Finally, we use the LUnAR RhoVR to directly visualize functional coupling between the plasma–ER membranes in patch clamped cell lines, providing the first direct evidence of the sign of the ER potential response to plasma membrane potential changes. We envision that the LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology.

Voltage-sensitive fluorescent reporters can reveal fast changes in the membrane potential in neurons and cardiomyocytes. However, in many cases, illumination in the presence of the fluorescent reporters results in disruptions to the action potential shape that limits the length of recording sessions. We show here that a molecular prosthetic approach, previously limited to fluorophores, rather than indicators, can be used to substantially prolong imaging in neurons and cardiomyocytes.

Release and reuptake of neuromodulator serotonin, 5-HT, is central to mood regulation and neuropsychiatric disorders, whereby imaging serotonin is of fundamental importance to study the brain's serotonin signaling system. We introduce a reversible near-infrared nanosensor for serotonin (nIRHT), for which synthetic molecular recognition toward serotonin is systematically evolved from ssDNA-carbon nanotube constructs generated from large libraries of 6.9 × 10 10 unique ssDNA sequences. nIRHT produces a ~200% fluorescence enhancement upon exposure to serotonin with a Kd = 6.3 µM affinity.nIRHT shows selective responsivity towards serotonin over serotonin analogs, metabolites, and receptortargeting drugs, and a 5-fold increased affinity for serotonin over dopamine. Further, nIRHT can be introduced into the brain extracellular space in acute slice, and can be used to image exogenous serotonin reversibly. Our results suggest evolution of nanosensors could be generically implemented to rapidly develop other neuromodulator probes, and that these probes can image neuromodulator dynamics at spatiotemporal scales compatible with endogenous neuromodulation.Serotonin, or 5-hydroxytryptamine (5-HT) is a monoamine neuromodulator that plays diverse roles in the central nervous system and is critically implicated in the etiology and treatment of mood and psychiatric disorders 1 . 5-HT also plays critical roles in modulating memory and learning 2, 3 , and in the regulation of mood 4 , sleep 5 , and appetite 5 . As such, aberrations in 5-HT signaling are thought to underlie multiple mental health disorders including major depressive disorder 6 , bipolar disorder 7 , autism 8 , schizophrenia 9 , anxiety 10 , and addiction 11 . Classical neurotransmission occurs through rapid activation of ligand-gated ion channels in which neurotransmitter signaling is confined at the synaptic cleft and thus to singular synaptic partners 12 . However, 5-HT acts primarily as a neuromodulator and may undergo signaling through extrasynaptic diffusion, enabling few neurons to affect broader networks of neuronal activity through 5-HT volume transmission. As such, there is great interest in monitoring the spatial component of 5-HT modulation in addition to its temporal dynamics. Current methods for measuring 5-HT include tools adapted from analytical chemistry to measure 5-HT in the brain extracellular space with varying levels of selectivity and spatiotemporal resolution: fast-scan cyclic voltammetry leverages redox chemistry to enable rapid temporal measurement of 5-HT by inserting a carbon electrode into brain tissue 13 .Microdialysis is an analytical chemistry method in which fluid samples from brain tissue are subject to downstream analysis with chromatography 14 . Recently, a field-effect transistor sensor modified with a 5-HT aptamer was reported to exhibit a highly sensitive and selective electrochemical response to 5-HT in physiological conditions 15 . However, previous methods suffer from limited spatial resolution, and microdialysis sampling oc...

Membrane potential (Vmem) is a fundamental biophysical signal present in all cells. Vmem signals range in time from milliseconds to days, and they span lengths from microns to centimeters. Vmem affects many cellular processes, ranging from neurotransmitter release to cell cycle control to tissue patterning. However, existing tools are not suitable for Vmem quantification in many of these areas. In this review, we outline the diverse biology of Vmem, drafting a wish list of features for a Vmem sensing platform. We then use these guidelines to discuss electrode-based and optical platforms for interrogating Vmem. On the one hand, electrode-based strategies exhibit excellent quantification but are most effective in short-term, cellular recordings. On the other hand, optical strategies provide easier access to diverse samples but generally only detect relative changes in Vmem. By combining the respective strengths of these technologies, recent advances in optical quantification of absolute Vmem enable new inquiries into Vmem biology. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.