Biology subjects

Furness, S. G. B.

Publications and source records attributed to Furness, S. G. B..

3 recordsLinked to original sources

bioRxiv2025

A novel pH-sensitive probe to quantify autophagy on high throughput/content imaging platforms

Autophagy is a highly-conserved mechanism that ensures the lysosomal degradation of disposable or potential toxic cytosolic entities in support of cell survival and adaptation to stress. Autophagy is deregulated in various pathological conditions including cardiovascular, neurological, neoplastic-autoimmune and degenerative disorders. However, clinically relevant pharmacological modulators of autophagy remain elusive, calling for the development of novel screening approaches that are amenable to high throughput/content applications. Here, we describe a simple method to detect autophagy in cultured mammalian cells based on the pH-sensitive fluorescent conjugate CalRexin:pHrodo Red and high throughput/content imaging. CalRexin:pHrodo Red is promptly taken up by endosome-amphisome-autolysosome (low pH [~]4.5) pathway, which is connected to canonical autophagy, culminating with a bright red fluorescence. Importantly, this system allows for the discrimination between autophagic responses coupled with the endosome-amphisome-autolysosome pathways and the endosome-lysosome pathway as elicited by receptor-driven endocytosis and phagocytosis. In human cervical carcinoma sHeLa cells, the accumulation of CalRexin:pHrodo Red as elicited by canonical autophagy activators like the mTOR inhibitor rapamycin or serum starvation was suppressed by conventional inhibitors 3-methyladenine or chloroquine. Similarly, RB1CC1-/-(FIP200) as well as ATG5-/- sHeLa cells (which bear genetic defects in two different steps of autophagy) were unable to accumulate CalRexin:pHrodo Red upon exposure to autophagy activators. Thus, CalRexin:pHrodo Red provides a novel approach for imaging autophagy on high throughout/content platforms to screen large libraries for the identification of novel autophagy-targeting agents.

cell biology↗

bioRxiv2023

Re-coding of G protein-coupled receptor signaling enables emergent cellular behavior.

All cells face the challenge of integrating multiple extracellular signals to produce relevant physiological responses. Different combinations of G protein-coupled receptors, when co-expressed, can lead to distinct cellular outputs, yet the molecular basis for this co-operativity is controversial. One such interaction is the reversal, from inhibition to excitation, at the dopamine D2 receptor in the ghrelin receptors presence, relevant for defecation control. Here we demonstrate that this reversal of dopamine D2 activity, to excitatory, occurs through a dominant switch in downstream signaling. This dominant switch, mediated by downstream signaling, enables fidelity in cellular responses not possible under alternative models, and provides an explanation for previously unresolved observations. Importantly, the switch in D2 signaling does not require ghrelin receptor agonism, rather its constitutive activity, thus accounting for the importance of central nervous system-ghrelin receptor in the absence of endogenous ligands. This re-coding has important implications for our understanding of how atypical receptor pharmacology can occur as well as how sequential signaling at individual neurons may be encoded to produce new outputs.

cell biology↗

bioRxiv2021

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins: insight into mechanisms of G protein selectivity

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here we used cryo-electron microscopy to determine structures of the CCK1R bound to the CCK peptide agonist, CCK-8 and two distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-5 helix bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket was essentially equivalent when Gs was bound, with the Gs 5 helix displaying a conformation that arises from "unwinding" of the far C-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein play critical roles in the promiscuous coupling of individual GPCRs. One-Sentence SummaryCryo-EM structures of the CCK-1R reveal key mechanisms for promiscuous G protein coupling.

pharmacology and toxicology↗