A5088434377- Photoreceptor and optogenetics research
- Neural dynamics and brain function
- Neuroscience and Neural Engineering
- Molecular Communication and Nanonetworks
- Neuroscience and Neuropharmacology Research
- Bacterial biofilms and quorum sensing
- Bacterial Genetics and Biotechnology
- Lipid Membrane Structure and Behavior
- Plant and Biological Electrophysiology Studies
- Retinal Development and Disorders
- Advanced battery technologies research
- RNA and protein synthesis mechanisms
- Mechanical and Optical Resonators
- bioluminescence and chemiluminescence research
- Protein Structure and Dynamics
- Microbial Inactivation Methods
- Adipose Tissue and Metabolism
- stochastic dynamics and bifurcation
- Cardiomyopathy and Myosin Studies
- Origins and Evolution of Life
- ATP Synthase and ATPases Research
- Nicotinic Acetylcholine Receptors Study
- SARS-CoV-2 detection and testing
- Spectroscopy Techniques in Biomedical and Chemical Research
- Photochromic and Fluorescence Chemistry
University of Colorado Boulder
2017-2023
Harvard University
2011-2018
Q-State Biosciences (United States)
2015-2016
Harvard University Press
2011-2013
Boston University
2006-2009
Ambedkar University Delhi
2006
Ames Research Center
2002
Bacteria have many voltage- and ligand-gated ion channels, population-level measurements indicate that membrane potential is important for bacterial survival. However, it has not been possible to probe voltage dynamics in an intact bacterium. Here we developed a method reveal electrical spiking Escherichia coli. To potential, engineered voltage-sensitive fluorescent protein based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) E. coli...
Significance Bacteria cope with changing external environments and must therefore sense respond to their local conditions. In addition sensing numerous chemical cues, in this paper we show that Escherichia coli can the mechanical environment through voltage-induced calcium flux. This mechanism is similar sensory neurons vertebrates, suggesting an ancient process of voltage-regulated mechanotransduction. Mechanical contact drives pathogenicity several species bacteria now propose a potential...
Aminoglycosides are broad-spectrum antibiotics whose mechanism of action is under debate. It widely accepted that membrane voltage potentiates aminoglycoside activity, which ascribed to voltage-dependent drug uptake. In this paper, we measured the response Escherichia coli treated with aminoglycosides and discovered bactericidal arises not from downstream effects uptake, but rather directly dysregulated potential. absence voltage, taken into cells exert bacteriostatic by inhibiting...
Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression an apolipoprotein a in the presence lipids, leading to self-assembly protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as biotechnology platform for solubilizing characterizing proteins. However, current approaches are limited because they require extensive efforts express, purify, solubilize prior insertion into NLPs. By simple addition...
The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of function. Natural developmental processes, as well many drugs pathologies change waveform, propagation, or variability (between cells over time) these parameters. Here we apply a genetically encoded dual-function calcium voltage reporter (CaViar) to study development zebrafish heart in vivo between 1.5 4 days post fertilization (dpf). We developed high-sensitivity spinning disk confocal...
Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput patch-clamp characterization. We introduce line non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 generate spontaneous electrical action potentials. These enable rapid, electrode-free screening speed sensitivity sensitive dyes or proteins on standard fluorescence microscope. screened small library mutants archaerhodopsin 3 (Arch)...
Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near site protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements...
Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge functional characterization by manual patch-clamp electrophysiology. Here, we describe optimized all-optical electrophysiology, "Optopatch," pipeline high-throughput human iPSC-derived neuronal cultures. We demonstrate method in motor neuron (iPSC-MN) model amyotrophic lateral sclerosis (ALS). In comparison iPSC-MNs with...
We developed a technique, "flash memory", to record photochemical imprint of the activity state--firing or not firing--of neuron at user-selected moment in time. The key element is an engineered microbial rhodopsin protein with three states. Two nonfluorescent states, D1 and D2, exist voltage-dependent equilibrium. A stable fluorescent state, F, reached by conversion from D2. When exposed light wavelength λ(write), population transfers D2 rate determined ⇌ F maintains membrane voltage which...
Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across disciplines, but it time-intensive and resource-consuming. Here we describe geometric (GVA) that replicates CFU measurements over 6 orders of magnitude while reducing 10-fold time consumables required. GVA computes sample's cell count on basis distribution embedded colonies growing inside pipette tip. compatible with Gram-positive...
We demonstrate the functionalization of single-walled carbon nanotubes (SWNTs) using a glow discharge for generating atomic or molecular radicals. A 30-s exposure to cold plasma H2 results in near-saturation coverage SWNT with hydrogen. Functionalization SWNTs hydrogen is confirmed by an infrared band at 2924 cm−1, characteristic C–H stretching mode. corresponding decrease ultraviolet absorption also observed, which due loss some conjugated C–C π bonds covered SWNTs.
Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) Halobacterium salinarum, AR3 readily incorporates into plasma membrane both E. coli mammalian cells. Here, we near-IR resonance Raman confocal microscopy to study effects pH potential on retinal chromophore structure. Measurements...
Proteorhodopsins are an extensive family of photoactive membrane proteins found in proteobacteria distributed throughout the world's oceans which often classified as green- or blue-absorbing (GPR and BPR, respectively) on basis their visible absorption maxima. GPR BPR have significantly different properties including photocycle lifetimes wavelength dependence pH. Previous studies revealed that these correlated with a single residue, Leu105 Gln105 although molecular for has not yet been...
Abstract Addition of glucose to starved Saccharomyces cerevisiae initiates collective NADH dynamics termed glycolytic oscillations. Numerous questions remain about the extent which single cells can oscillate, if oscillations occur in natural conditions, and potential physiological consequences In this paper, we report sustained without need for cyanide. Glucose addition immobilized induced pH that could be imaged with fluorescent sensors. A population had were heterogeneous frequency, start...
Anabaena sensory rhodopsin (ASR) is a novel microbial recently discovered in the freshwater cyanobacterium sp. PCC7120. This protein most likely functions as photosensory receptor do related haloarchaeal rhodopsins. However, unlike archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with soluble cytoplasmic analogous transducers of animal vertebrate In this study, infrared spectroscopy was used examine molecular mechanism photoactivation...
Abstract Microbial rhodopsins are an important class of light‐activated transmembrane proteins whose function is typically studied on bulk samples. Herein, we apply photochromic fluorescence resonance energy transfer to investigate the dynamics these with sensitivity approaching single‐molecule limit. The brightness a covalently linked organic fluorophore modulated by changes in absorption spectrum endogenous retinal chromophore that occur as molecule undergoes photocycle. We photocycles...
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly tissue repair humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, electrophysiology similar that embryonic cardiomyocytes. We grew patterned islands different sizes shapes, measured the effect island geometry on action potential waveform calcium dynamics using optical recordings voltage from 970 sizes. larger...
Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested occur in rhodopsin-like where retinal photoisomerization may impulsively drive structural changes nearby groups. Here, we test this possibility investigating the earliest occurring proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with ∼200 fs time resolution combined...
Proteorhodopsins are a recently discovered class of microbial rhodopsins, ubiquitous in marine bacteria. Over 4000 variants have thus far been discovered, distributed throughout the oceans world. Most fall into one two major groups, green- or blue-absorbing proteorhodopsin (GPR and BPR, respectively), on basis both visible absorption maxima (530 versus 490 nm) photocycle kinetics (∼20 ∼200 ms). For well-studied pair, these differences appear to be largely determined by identity single...
We examine the structural changes during primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of light-induced BPR spectrum recorded at 80 K to that green-absorbing (GPR) reveals there are several differences and GPR photoreactions despite similar structure retinal chromophore all-trans → 13-cis isomerization. Strong bands near 1700 cm−1 assigned previously change hydrogen...
Calcium plays numerous critical roles in signaling and homeostasis eukaryotic cells. Far less is known about calcium bacteria than cells, few genes controlling influx efflux have been identified. Previous work Escherichia coli showed that was induced by voltage depolarization, which enhanced mechanical stimulation, suggested a role bacterial mechanosensation. To identify proteins pathways affecting handling bacteria, we designed live-cell screen to monitor dynamics single cells across...