Biology subjects

Barsi-Rhyne, B.

Publications and source records attributed to Barsi-Rhyne, B..

4 recordsLinked to original sources

bioRxiv2022

Discrete GPCR-triggered endocytic modes enable β-arrestins to flexibly regulate cell signaling

{beta}-arrestins are master regulators of cellular signaling that operate by desensitizing ligand-activated G protein-coupled receptors (GPCRs) at the plasma membrane and promoting their subsequent endocytosis. The endocytic activity of {beta}-arrestins is ligand-dependent, triggered by GPCR binding, and increasingly recognized to have a multitude of downstream signaling and trafficking consequences that are specifically programmed by the bound GPCR. However, only one biochemical mode for GPCR-mediated triggering of the endocytic activity is presently known- displacement of the {beta}-arrestin C-terminus (CT) to expose CCP-binding determinants that are masked in the inactive state. Here we revise this view by uncovering a second mode of GPCR-triggered endocytic activity that is independent of the {beta}-arrestin CT and, instead, requires the cytosolic base of the {beta}-arrestin C-lobe (CLB). We further show each of the discrete endocytic modes is triggered in a receptor-specific manner, with GPCRs that bind {beta}-arrestin transiently ( class A) primarily triggering the CLB-dependent mode and GPCRs that bind more stably ( class B) triggering both the CT and CLB-dependent modes in combination. Moreover, we show that each mode has opposing effects on the net signaling output of receptors- with the CLB-dependent mode promoting rapid signal desensitization and the CT-dependent mode enabling prolonged signaling. Together, these results fundamentally revise understanding of how {beta}-arrestins operate as efficient endocytic adaptors while facilitating diversity and flexibility in the control of cell signaling.

cell biology↗

bioRxiv2021

Membrane phosphoinositides stabilize GPCR-arrestin complexes and offer temporal control of complex assembly and dynamics

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized some GPCRs may complex with arrestin, while others interact transiently; this difference affects receptor signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in arrestin recruitment and GPCR-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one requiring PIP-binding for arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes by promoting a receptor core-engaged state of the complex. As allosteric modulators of GPCR-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. The dependance on membrane PIPs provides a mechanism for arrestin release from transiently associated GPCRs, allowing their rapid recycling, while explaining how stably associated GPCRs are able to engage G proteins at endosomes.

biochemistry↗

bioRxiv2020

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

biochemistry↗

bioRxiv2018

Robust and sensitive GFP-based cGMP sensor for real time imaging in intact Caenorhabditis elegans

cGMP is a ubiquitous second messenger that plays a role in sensory signaling and plasticity through its regulation of ion channels and kinases. Previous studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities, including light, heat, gases, salt and odor. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other sensors and fluorescent markers. Here, we demonstrate that WincG2, a codon-optimized version of the cpEGFP-based cGMP sensor FlincG3, can be used in C. elegans to visualize rapidly changing cGMP levels in living, behaving animals using a single fluorophore. We coexpressed the sensor with the blue light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles and found that the rate of WincG2 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that WincG2 responds linearly upon NaCl concentration changes and SDS presentation in the cell bodies of the gustatory neuron ASER and the nociceptive phasmid neuron PHB, respectively. Intriguingly, WincG2 fluorescence in the ASER cell body decreased in response to a NaCl concentration downstep and either stopped decreasing or increased in response to a NaCl concentration upstep, which is opposite in sign to previously published calcium recordings. These results illustrate that WincG2 can be used to report rapidly changing cGMP levels in an intact animal and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.\n\nAuthor SummarycGMP is a second messenger that plays an important role in sensory signaling and neural plasticity. Previous genetic and biochemical studies indirectly suggest that cGMP is spatiotemporally regulated in neurons to modulate neural activity. While a FRET-based sensor for cGMP has been used in intact Caenorhabditis elegans to examine its spatiotemporal regulation in neurobiological processes, its use has been limited due to the complicated setup required to image this type of sensor. Here, we describe a GFP-based cGMP sensor that has been codon optimized for use in C. elegans and demonstrate that it responds robustly and reliably to endogenously changing cGMP levels. We show that the sensor responds to cGMP production by coexpressing it with blue light-activated guanylyl cyclases, and we show that it responds to NaCl and sodium dodecyl sulfate when expressed in a gustatory and nociceptive neuron, respectively. We think that this sensor can be used to investigate the spatiotemporal regulation of cGMP in neurons and its relationship to neural activity.

neuroscience↗