Biology subjects

Hudson, R.

Publications and source records attributed to Hudson, R..

7 recordsLinked to original sources

bioRxiv2025

The Lipid-binding PX Domain of RRC-1 (ARHGAP32/33) is Required for Optimal Assembly and Function of Integrin Adhesion Complexes at the Muscle Cell Boundary in C. elegans

Integrin adhesion complexes (IACs) are a network of many proteins that serve as anchors of the cell to the extracellular matrix (ECM). In muscle, IACs located at costameres, also serve to transmit the force of muscle contraction to the outside of the cell. We have reported that IACs, which are found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the RacGEF PIX-1 for their proper assembly or maintenance. We have reported that a RacGAP for the PIX pathway is RRC-1, is in a complex with PIX-1, and that RRC-1 is required for assembly or maintenance of IACs at MCBs. Our previous studies suggested that RRC-1 might be associated with the muscle cell membrane, and here we present evidence that this occurs via its PX domain, a domain that is known to bind to membrane phosphoinositides (PIPs). We predict the existence of a PX domain based on bioinformatic analysis and AlphaFold3, which includes conserved residues characteristic of most PX domains and a PIP binding site. This region of RRC-1 binds to phosphoinositides in vitro. Analysis of a nematode strain that has an in-frame deletion of the PX domain, indicates that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffold protein GIT-1. Lastly, we show that the overexpression of the full length RRC-1, but not RRC-1 with an in-frame deletion of its PX domain, results in reduced accumulation of IAC components and reduced whole animal movement. Our study highlights the importance of RRC-1s lipid interactions at the cell membrane for proper assembly and function of IACs in C. elegans muscle. Article SummaryIntegrin adhesion complexes (IACs) facilitate the transmission of force of muscle contraction to the outside of the cell. IACs, found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the PIX-1 (a RacGEF) signaling pathway for their assembly or maintenance. A RacGAP for the PIX pathway is RRC-1. Here, we show that RRC-1 has a PX domain that binds to membrane phosphoinositides. We also show that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffolding protein GIT-1.

cell biology↗

bioRxiv2025

A single residue determines regulation of TRPV1 by phosphoinositides

The ion channel TRPV1 is expressed in the peripheral nervous system where it mediates heat sensation and pain signaling. How lipids contribute to TRPV1 regulation remains an open topic. Although an inhibitory binding site for phosphoinositides was identified at the vanilloid site in TRPV1 structures, a mechanism for phosphatidylinositol-2-bisphosphate (PI(4,5)P2) to both inhibit and potentiate TRPV1 activity is lacking. This gap in knowledge led us to examine structural and sequence overlap between TRPV1 and the PI(4,5)P2 binding site of TRPV5. In a space located adjacent to the turn from S6 to TRP helices, we identify a second, possible PI(4,5)P2 binding site in TRPV1, which we term the "front porch" site and includes residue H410. On the one hand, we obtained electrophysiological evidence with the TRPV1-H410R mutant that PI(4,5)P2 associates with TRPV1 at the front porch site through an electrostatic interaction to potentiate channel activity. On the other hand, the charge reversal in H410D conferred an increase in channel activity, which was not expected if the electrostatic interaction with PI(4,5)P2 is lost and channel function is crippled. Importantly, capsaicin activated currents from H410D experience significant current run-up, consistent with the slow displacement by capsaicin of a tighter-bound lipid in the vanilloid site, which structures suggest is phosphoinositide (PI). Lastly, we tested TRPV1-H410D sensitivity to PI(4,5)P2 manipulations by dephosphorylating the membrane pool of PI(4,5)P2 to PI with the Pseudojanin rapamycin-phosphatase (PJ) system. Depletion of PI(4,5)P2 with the PJ-system in wild type channels induces 1) a robust increase in baseline activity as channels are released from inhibition and 2) loss of current potentiation, driven by PI(4,5)P2 dissociation from the front porch, both of which were absent in TRPV1-H410D. Based on these findings we hypothesize that plasma membrane TRPV1-H410D maintains PI rather than PI(4,5)P2 in its vanilloid site and does not interact with PI(4,5)P2 in the front porch site. We integrated our discoveries that H410 interacts with PI(4,5)P2 at two distinct sites in a mutually exclusive manner and present a cohesive model for phosphoinositide inhibition and potentiation in TRPV1, addressing a maturation process for TRPV1 that relies on H410 to coordinate the exchange of PI for PI(4,5)P2. Statement of SignificanceA lingering question that hangs over TRPV1 research is how the lipid PI(4,5)P2 regulates the function of this channel. We devised a study based on structural homology between TRPV5 and TRPV1 to identify critical contact points between the lipid and a putative PI(4,5)P2 binding site in TRPV1, which we probed using mutagenesis experiments. The results we obtained lay the groundwork for how a two-site binding mechanism of PI(4,5)P2 couples to TRPV1 gating and address important questions about the channels maturation and association with phosphoinositides as it is transferred through membrane compartments with different phosphoinositide compositions. We conclude that TRPV1 expressed and purified from different compartments may preserve the phosphoinositide character of that compartment (PI in the endoplasmic reticulum, PI(4,5)P2 in the plasma membrane) and bestow on structures a state that is not necessarily relevant to the biological function of the channel in the plasma membrane.

biophysics↗

bioRxiv2025

IPMK-1 (inositol phosphate multikinase) is required for optimal integrin adhesion complex assembly in C. elegans muscle

Integrin adhesion complexes (IACs) are a network of many proteins including the transmembrane protein integrin that anchor cells to the extracellular matrix (ECM). In C. elegans muscle IACs exist at the bases of dense bodies and M-lines, and at the boundaries of adjacent muscle cells (MCBs). We have reported that the proper assembly of IACs requires the Rac GEF PIX-1 and members of the PIX-1 pathway. Here we report that further investigation into the original Million Mutation Project strain that led us to uncover the role of PIX-1 in muscle, revealed a genetic enhancer of the PIX-1 phenotype. Genetic mapping shows that the enhancer is a recessive mutation in a single gene, ipmk-1. ipmk-1, encodes inositol phosphate multikinase and converts PIP2 to PIP3, IP3 to IP4, and IP4 to IP5. Loss of function of pix-1 results in reduced accumulation of IAC proteins at the MCB, and ipmk-1; pix-1 double mutants additionally display a large gap between adjacent muscle cells. ipmk-1 similarly enhances other members of the PIX pathway including PAK-1, and the RacGAP RRC-1. Lack of ipmk-1 itself results in abnormal clumping of IAC components at the MCB, decreased whole animal locomotion, and mild sarcomere disorganization. Analysis of ipmk-1; ipp-5 double mutants suggest that the MCB defect of ipmk-1 is due to decreased PIP3 in the cell membrane rather than the decreased IP3 and Ca2+ signaling. Overall, these studies provide in vivo evidence, specifically in muscle, that the level of PIP3 and PIP2 influence the proper assembly of IACs. Article SummaryC. elegans muscle have integrin adhesion complexes (IACs) at dense bodies, M-lines, and boundaries between adjacent muscle cells (MCBs). The Benian lab reported that assembly of these IACs requires the PIX-1 pathway. Here they report ipmk-1 is a genetic enhancer of the PIX pathway. ipmk-1encodes inositol phosphate multikinase converting PIP2 to PIP3. pix-1 shows reduced accumulation of IAC components; ipmk-1; pix-1 double mutants additionally have large gaps between muscle cells. ipmk-1 mutants display abnormal clumping of IAC components. This is the first report of the importance of these lipids in assembly of IACs in vivo, and in particular, in muscle.

cell biology↗

bioRxiv2025

Population Pharmacokinetics and Probability of Target Attainment Analysis of Vancomycin Following Intermittent and Continuous Infusion in Adults with Cystic Fibrosis

Vancomycin is the drug of choice for treating pulmonary infections caused by methicillinresistant Staphylococcus aureus (MRSA) in people with cystic fibrosis (PwCF). This study characterized the pharmacokinetics (PK) of continuous and intermittent vancomycin infusions following a loading dose using population PK (PopPK) modeling to inform dosing in PwCF. The PopPK model was developed using therapeutic drug monitoring (TDM) data from adult PwCF who received a vancomycin loading dose followed by intermittent, continuous, or both infusion types for MRSA-related pulmonary exacerbations. A total of 212 samples were collected following 90 intermittent and 42 continuous infusions in 21 patients. The final model was a two-compartment model with first-order elimination, incorporating creatinine clearance (CrCL) as a covariate on vancomycin clearance (CL). The estimated CL and volume of distribution were 4.05 L/h/70 kg and 22.5 L/70 kg, respectively. The model was used to predict the probability of target attainment (PTA) following a single intermittent loading dose (500-1500 mg) and continuous infusion (500-6000 mg) over 24 hours. PTA was assessed using efficacy and toxicity thresholds defined by Area Under the Curve0-24 (AUC0-24)/Minimum Inhibitory Concentration (MIC) ratios [≥]400 mg{middle dot}h/L and <650 mg{middle dot}h/L, respectively. At a MIC of 1 {micro}g/mL, a loading dose of 500 mg followed by a 3750 mg continuous infusion achieved PTA targets for efficacy (66.7%) and safety (82.7%). These findings support the use of PopPK modeling to guide vancomycin dosing strategies for MRSA pulmonary infections in PwCF.

pharmacology and toxicology↗

bioRxiv2025

Missense variant analysis in the TRPV1 ARD reveals the unexpected functional significance of a methionine

The Transient Receptor Potential Vanilloid sub-type 1 (TRPV1) is an ion channel that is activated by heat, extracellular protons, oxidation, and it is implicated in various aspects of inflammatory pain. In this study, we uncover that residue M308 in the TRPV1 ankyrin repeat domain (ARD) stands out from most other buried ARD residues because of the greater number of human missense variants at this position while maintaining a high degree of conservation across species and TRPV channel subtypes. We use mutagenesis and electrophysiology to examine this apparent discrepancy and show that substitutions at position M308 that preserve or reduce side-chain volume have no effect on channel function, whereas substitutions with larger or more polar residues increase channel activity in response to capsaicin or temperature. Substitution of M308 with a histidine bestows channels with pH-dependence that is different from wild type, consistent with the side-chain at position 308 exerting an influence on channel gating. We speculate that M308 is highly conserved because its side-chain could serve as a target for oxidation-dependent modification. On the other hand, we show that a previously described splice variant of TRPV1 that relies on M308 as a start codon diminishes surface expression of co-transfected full-length TRPV1 in HEK293 cells. Together, our findings reveal a functionally important conserved site within the ARD of TRPV1 that could have roles in oxidation-dependent channel regulation as well as tuning the number of active channels in the membrane by enabling expression of a shorter dominant-negative splice variant.

biophysics↗

bioRxiv2024

Protein interactions, calcium, phosphorylation, and cholesterol modulate CFTR cluster formation on membranes

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel whose dysfunction leads to intracellular accumulation of chloride ions, dehydration of cell surfaces, and subsequent damage to airway and ductal organs. Beyond its function as a chloride channel, interactions between CFTR, ENaC, and SLC transporter family membrane proteins and cytoplasmic proteins, including calmodulin and NHERF-1, co-regulate ion homeostasis. CFTR has also been observed to form mesoscale membrane clusters. However, the biophysical mechanisms that regulate the formation of CFTR clusters are unknown. Using a combination of computational modeling and complex biochemical reconstitution assays, we demonstrate that multivalent protein-protein interactions with CFTR binding partners, calcium, and membrane cholesterol can induce CFTR cluster formation on model membranes. Phosphorylation of the intracellular domains of CFTR also promotes cluster formation in the absence of calcium, indicating that multiple mechanisms can regulate CFTR cluster formation. Our findings reveal that coupling of multivalent protein and lipid interactions promote CFTR cluster formation consistent with membrane-associated biological phase separation. SIGNIFICANCE STATEMENTMutations in the cystic fibrosis transmembrane conductance regulator (CFTR) membrane protein underlie cystic fibrosis. It is thought that molecular "hubs" of CFTR and its binding partners co-regulate ion homeostasis and that disruption of these clusters can result in disease. However, the physical basis for molecular hub formation is unclear. In this study, we present evidence that multivalent protein and lipid interactions drive the formation of mesoscale CFTR-containing clusters or "hubs" on model membranes in a manner consistent with biological phase separation. These data provide important insights into physical mechanisms that modulate CFTR membrane organization and offer a new lens for the development of corrective therapeutics.

biophysics↗

bioRxiv2020

CO2 reduction driven by a pH gradient

All life on Earth is built of organic molecules, so the primordial sources of reduced carbon are a major open question in studies of the origin of life. A variant of the alkaline-vent theory suggests that organics could have been produced by the reduction of CO2 via H2 oxidation, facilitated by geologically sustained pH gradients. The process would be an abiotic analog--and proposed evolutionary predecessor--of the modern Wood-Ljungdahl acetyl-Co-A pathway of extant archaea and bacteria. The first energetic bottleneck of the pathway involves the endergonic reduction of CO2 with H2 to formate, which has proven elusive in low-temperature abiotic settings. Here we show the reduction of CO2 with H2 at moderate pressures (1.5 bar), driven by microfluidic pH gradients across inorganic Fe(Ni)S precipitates. Isotopic labelling with 13C confirmed production of formate. Separately, deuterium (2H) labelling indicated that electron transfer to CO2 did not occur via direct hydrogenation with H2. Instead, freshly deposited Fe(Ni)S precipitates appear to facilitate electron transfer in an electrochemical-cell mechanism with two distinct half-reactions. Decreasing the pH gradient significantly, or removing either H2 or the precipitate, yielded no detectable product. Our work demonstrates the feasibility of spatially separated, yet electrically coupled geochemical reactions as drivers of otherwise endergonic processes. Beyond corroborating the ability of early-Earth alkaline hydrothermal systems to couple carbon reduction to hydrogen oxidation through geologically plausible and biologically relevant mechanisms, these results may also be of significance for industrial and environmental applications, where other redox reactions could be facilitated using similarly mild approaches.

biochemistry↗