Biology subjects

Roth, R.

Publications and source records attributed to Roth, R..

14 recordsLinked to original sources

bioRxiv2026

The proximal N-terminus of IRAG is required for potentiation of HCN4 channels

The inositol triphosphate-associated, ER transmembrane proteins IRAG and LRMP are isoform specific regulators of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, while IRAG mimics the effect of cAMP on the channel. We previously showed that regulation by LRMP requires both the N-terminus of HCN4 and a unique orientation of the HCN4 cAMP transduction center, which is comprised of the N-terminal HCN domain, the C-linker, and the S4-S5 linker. However, it remains unknown if the homologous IRAG requires similar structural features to mimic cAMP-dependent potentiation, or if the site and mechanism of action are different between the two regulators. Using patch clamp electrophysiology, we determined that the initial 43 amino acids of IRAG are necessary and sufficient to confer regulation of HCN4. Similar to LRMP, IRAG also requires a portion of the N-terminus of HCN4 to confer its regulatory effects. Also similar to LRMP, two point mutations in the C-linker region, which are the only sequence differences in that region between HCN4 and the other HCN isoforms, were able to eliminate the effect of IRAG suggesting the unique orientation of the cAMP transduction center in HCN4 is likely important for IRAG function. Taken together, these findings suggest a model whereby IRAG and LRMP interact with the channel in similar regions, although potentially in unique ways, and act on the cAMP transduction center with LRMP inhibiting the coupling of this region to gating and IRAG strengthening it. SUMMARYThe ER transmembrane protein IRAG binds to and potentiates HCN4 channels. This study demonstrates that IRAG regulation of HCN4 requires only the first 43 amino acids of IRAG and involves contributions from the N-terminus and cAMP transduction center of HCN4.

biophysics↗

bioRxiv2026

Crystalline guanine packed within vacuoles serves as nitrogen store in Chromera velia

Nitrogen is an important element for all living organisms. Photoautotrophic organisms need to assimilate nitrogen from the environment, therefore changes in nitrogen availability have a strong influence on their growth and metabolism. Many microalgae have been known to contain crystalline inclusions, and recently, it has been shown that many of these consist of purines like guanine and thus must be linked to the cellular nitrogen metabolisms. The alveolate alga Chromera velia contains such guanine crystals, and during its lifecycle, the alga is thought to be subjected to strong changes in external nitrogen availability. Here, we investigated the formation or decline of crystalline guanine in dependence of the availability of inorganic nitrogen in the growth medium. Cells were examined using polarised light microscopy, Raman micro-spectroscopy, chromatography (HPLC), transmission and scanning electron microscopy. The cellular guanine crystal content decreased during nitrogen starvation and increased upon transfer of the cells back to standard growth medium containing nitrate. Raman micro-spectroscopy showed that the crystals were composed of anhydrous guanine in beta-polytype. They appear in unspecific positions throughout the cell, and staining with the green dye Lysotracker DND-26 suggests that they are within vacuoles. Stacks of crystals could be observed in cells via freeze fracture and freeze etching electron microscopy, which unambiguously showed a membrane around the crystal aggregates, in a similar arrangement as has been shown for guanine storage vacuoles (GSV) in Chlamydomonas reinhardtii. We developed a method to isolate the guanine crystals from whole cells, and were able to obtain crystals which retained their flat, plate-like structure, matching the electron microscopic observations from whole cells. The isolated crystals were shown to consist of nitrogen rich compounds via energy-dispersive X-ray (EDX) analysis, and Raman micro-spectroscopy confirmed that they consist of guanine.

cell biology↗

bioRxiv2026

PUFA modulation of ASIC3 involves both specific and lipid solvent-like interactions

AbstractInflammatory mediators including polyunsaturated fatty acids (PUFAs) potentiate ASIC3 by altering channel gating, yet the molecular basis for their interactions remains poorly defined. Using all-atom MD simulations and electrophysiology, we show that DHA accumulates around ASIC3 through interactions with a membrane-facing electropositive region along the outer leaflet of TM1. In the open state, a more buried binding site for the lipid is exposed that allows the carboxylate head group to bind a critical arginine along with nearby polar residues. Mutation of this site prevents the slowing of channel desensitization but only reduces the effect PUFAs have on the pH dependence of activation. The same mutations do not prevent regulation by other lipid classes like N-acyl amino acids or lysophosphatidyl choline. Our results provide the first detailed description of a PUFA binding site on ASICs, offering new insights into lipid modulation and potential strategies for developing novel treatments for inflammatory pain.

biophysics↗

bioRxiv2025

Functionalized Calcium Carbonate Microparticles in Ethyl Cellulose Films: A Vehicle for Sustained Amoxicillin Release for Medical Applications

The continuous quest for materials capable of providing extended release of antimicrobial drugs is particularly important for indwelling medical applications. In this study, we utilized amoxicillin as a model active pharmaceutical ingredient (API) to investigate the feasibility of using porous media - specifically, functionalized calcium carbonate (FCC) microparticles - as a primary drug carrier embedded within an ethyl cellulose (EC) polymer film. Our main objective was to prolong and sustain the release of the API. The fabrication process of the microparticle containing film involved two key steps: loading the model API into the FCC particles and then embedding these loaded particles into the polymeric film. Amoxicillin was loaded into the FCC particles using a solvent evaporation method. Detailed characterization through Scanning Electron Microscopy (SEM), lab- and synchrotron-based XRD revealed that amoxicillin precipitated both inside and on the surface of the FCC particles, predominantly in an amorphous form. Additionally, ultraviolet-visible (UV-vis) spectroscopic data demonstrated an increased release rate from the particles compared to pure amoxicillin powder. Embedding amoxicillin pre-loaded porous FCC particles in the EC film led to a more rational sustained release compared with powder amoxicillin embedded directly in the film, advantageously delivering the same amount of amoxicillin over a longer period; a result that may be relevant for indwelling medical devices such as urinary catheters, vascular access devices or wound drains.

bioengineering↗

bioRxiv2024

Elucidating the Differential Impacts of Equivalent Gating-Charge Mutations in Voltage-Gated Sodium Channels

Voltage-gated sodium (Nav) channels are pivotal for cellular signaling and mutations in Nav channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSDI and VSDII of the cardiac sodium channel Nav1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 {micro}s molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our s-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure- function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.

biophysics↗

bioRxiv2024

Molecular Insights into Single Chain Lipid Modulation of Acid-Sensing Ion Channel 3

Polyunsaturated fatty acids (PUFAs) and their analogs play a significant role in modulating the activity of diverse ion channels, and recent studies show that these lipids potentiate acid-sensing ion channels (ASICs), leading to increased activity. The potentiation of the channel stems from multiple gating changes, but the exact mechanism of these effects remains uncertain. We posit a mechanistic explanation for one of these changes in channel function, the increase in the maximal current, by applying a combination of electrophysiology and all-atom molecular dynamics simulations on the open-state hASIC3. Microsecond-scale simulations were performed on open-state hASIC3 in the absence and presence of a PUFA, docosahexaenoic acid (DHA), and a PUFA analog, N-arachidonyl glycine (AG). Intriguingly, our simulations in the absence of PUFA or PUFA analogs reveal that a tail from the membrane phospholipid POPC inserts itself into the pore of the channel through lateral fenestrations on the sides of the transmembrane segments, obstructing ion permeation through the channel. The binding of either DHA or AG prevented POPC from accessing the pore in our simulations, relieving the block of ionic conduction by phospholipids. Finally, we use the single-channel recording to show that DHA increases the amplitude of the single-channel currents in ASIC3, which is consistent with our hypothesis that PUFAs relieve the pore block of the channel induced by POPCs. Together, these findings offer a potential mechanistic explanation of how PUFAs modulate ASIC maximal current, revealing a novel mechanism of action for PUFA-induced modulation of ion channels.

biophysics↗

bioRxiv2023

Physiological Profiles of Male and Female CrossFit Athletes

ObjectiveTo 1) establish extensive physiological profiles of highly-trained CrossFit athletes using gold-standard tests and 2) investigate which physiological markers best correlate with CrossFit(R) Open performance. MethodsThis study encompassed sixty participants (30 males and 30 females), all within the top 5% of the CrossFit(R) Open, including 7 CrossFit(R) Semi-finalists and 3 Games finalists. Isokinetic dynamometers were employed to measure maximum isometric and isokinetic leg and trunk strength. Countermovement jump height and maximum isometric mid-thigh pull strength were assessed on a force plate. V{square}O2peak was measured by a cardiopulmonary exercise test, while critical power and W were evaluated during a 3-minute all-out test, both on a cycle ergometer. ResultsMale and female athletes median (IQR) V{square}O2peak was 4.64 (4.43, 4.80) and 3.21 (3.10, 3.29) (L{middle dot}min-1), critical power 314.5 (285.9, 343.6) and 221.3 (200.9, 238.9) (W) and mid-thigh pull 3158 (2690, 3462) and 2035 (1728, 2347) (N). Linear regression analysis shows strong evidence for associations between different anthropometric variables and CrossFit(R) Open performance in men and women, whereas for markers of cardiorespiratory fitness such as V{square}O2peak this was only true for women but not men. Conventional laboratory evaluations of strength, however, manifest minimal evidence for associations with Open performance across both sexes. ConclusionsThis study provides the first detailed insights into the physiology of high-performing CrossFit(R) athletes and informs training optimization. Further the results emphasize the advantage of athletes with shorter limbs and suggests potential modifications to Open workout designs to level the playing field for athletes across different anthropometrics characteristics.

physiology↗

bioRxiv2021

Structures of TRPM5 channel elucidate mechanism of activation and inhibition

The Ca2+-activated TRPM5 channel plays an essential role in the perception of sweet, bitter, and umami stimuli in type II taste cells and in insulin secretion by pancreatic beta cells1-3. Interestingly, the voltage dependence of TRPM5 in taste bud cells depends on the intracellular Ca2+ concentration4, yet the mechanism remains elusive. Here we report cryo-electron microscopy structures of the zebrafish TRPM5 in an apo closed state, a Ca2+-bound open state, and an antagonist-bound inhibited state, at resolutions up to 2.3 [A]. We defined two novel ligand binding sites: a Ca2+ binding site (CaICD) in the intracellular domain (ICD), and an antagonist binding site in the transmembrane domain (TMD) for a drug (NDNA) that regulates insulin and GLP-1 release5. The CaICD site is unique to TRPM5 and has two roles: shifting the voltage dependence toward negative membrane potential, and promoting Ca2+ binding to the CaTMD site that is conserved throughout Ca2+-sensitive TRPM channels6. Replacing glutamate 337 in the CaICD site with an alanine not only abolished Ca2+ binding to CaICD but also reduced Ca2+ binding affinity to CaTMD, suggesting a cooperativity between the two sites. We have defined mechanisms underlying channel activation and inhibition. Conformational changes initialized from both Ca2+ sites, 70 [A] apart, are propagated to the ICD-TMD interface and cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the TMD in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents to treat metabolic disorders.

biophysics↗

bioRxiv2021

3D Visualization of the Podocyte Actin Network using Integrated Membrane Extraction, Electron Microscopy, and Deep Learning

Although actin stress fibers are abundant in cultured cells, little is known about these structures in vivo. In podocytes of the kidney glomerulus, much evidence suggests that mechanobiological mechanisms underlie injury, with changes to actin stress fiber structures potentially responsible for pathological changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. We therefore developed the first visualization technique capable of resolving the three-dimensional (3D) podocyte actin network with unprecedented detail in healthy and injured podocytes, and applied this technique to reveal the changes in the actin network that occur upon podocyte injury. Using isolated glomeruli from healthy mice as well as from three different mouse injury models (Cd2ap-/-, Lamb2-/- and the Col4a3-/- model of Alport syndrome), we applied our novel imaging technique that integrates membrane-extraction, focused ion bean scanning electron microscopy (FIB-SEM), and deep learning image segmentation. In healthy glomeruli, we observed actin cables that link the interdigitating podocyte foot processes to newly described actin structures located at the periphery of the cell body. The actin cables within the foot processes formed a continuous, mesh-like, electron dense sheet that incorporated the slit diaphragms required for kidney filtration. After injury, the actin network was markedly different, having lost its organization and presenting instead as a disorganized assemblage of actin condensates juxtaposed to the glomerular basement membrane. The new visualization method enabled us, for the first time, to observe the detailed 3D organization of actin networks in both healthy and injured podocytes. Shared features of actin condensations across all three injury models further suggested common mechanobiological pathways that govern changes to podocyte morphology after injury.

cell biology↗

bioRxiv2020

Dach1 extends artery networks and protects against cardiac injury

Coronary artery disease (CAD) is the leading cause of death worldwide, but there are currently no available methods to stimulate growth or regeneration of artery networks in diseased hearts. Studying how arteries are built during embryonic development could illuminate strategies for re-building these vessels in the setting of ischemic heart disease. We previously found, using loss-of-function experiments, that the transcription factor Dach1 is required for coronary artery development in mouse embryos. Here, we report that Dach1 overexpression in endothelial cells (ECs) extended coronary arteries and improved survival and heart function in adult mice following myocardial infarction (MI). Dach1 overexpression increased the length and number of arterial end branches, in both heart and retinal vasculature, by causing additional capillary ECs to differentiate into arterial ECs and contribute to growing arteries. Single-cell RNA sequencing (scRNAseq) of ECs undergoing Dach1-induced arterial specification indicated that it potentiated normal artery differentiation, rather than functioning as a master regulator of artery cell fate. ScRNAseq also showed that normal arterial differentiation is accompanied by repression of lipid metabolism genes, which were also repressed by Dach1 prior to arterialization. Together, these results demonstrate that increasing the expression level of Dach1 is a novel pathway for driving specification of artery ECs and extending arterial vessels, which could be explored as a means of increasing artery coverage to mitigate the effects of CAD.

developmental biology↗

bioRxiv2020

Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular non-compaction

Non-compaction cardiomyopathy is a devastating genetic disease caused by insufficient consolidation of ventricular wall muscle that can result in inadequate cardiac performance. Despite being the third most common cardiomyopathy, the mechanisms underlying the disease, including the cell types involved, are poorly understood. We have previously shown that endothelial cell-specific deletion of the chromatin remodeler gene Ino80 results in defective coronary vessel development that leads to ventricular noncompaction in embryonic mouse hearts. Here, we used single-cell RNA-sequencing to characterize endothelial and endocardial defects in Ino80-deficient hearts. We observed a pathological endocardial cell population in the non-compacted hearts, and identified multiple dysregulated angiocrine factors that dramatically affected cardiomyocyte behavior. We identified Col15A1 as a coronary vessel-secreted angiocrine factor, downregulated by Ino80-deficiency, that functioned to promote cardiomyocyte proliferation. Furthermore, mutant endocardial and endothelial cells (ECs) upregulated expression of secreted factors, such as Tgfbi, Igfbp3, Isg15, and Adm, which decreased cardiomyocyte proliferation and increased maturation. These findings support a model where coronary ECs normally promote myocardial compaction through secreted factors, but that endocardial and ECs can secrete factors that contribute to non-compaction under pathological conditions.

developmental biology↗

bioRxiv2020

Visualizing synaptic plasticity in vivo by large-scale imaging of endogenous AMPA receptors

Elucidating how synaptic molecules such as AMPA receptors mediate neuronal communication and tracking their dynamic expression during behavior is crucial to understand cognition and disease, but current technological barriers preclude large-scale exploration of molecular dynamics in vivo. We have developed a suite of innovative methodologies that break through these barriers: a new knockin mouse line with fluorescently tagged endogenous AMPA receptors, two-photon imaging of hundreds of thousands of labeled synapses in behaving mice, and computer-vision-based automatic synapse detection. Using these tools, we can longitudinally track how the strength of synapses changes during behavior. We used this approach to generate an unprecedentedly detailed spatiotemporal map of synaptic plasticity underlying sensory experience. More generally, these tools can be used as an optical probe capable of measuring functional synapse strength across entire brain areas during any behavioral paradigm, describing complex system-wide changes with molecular precision.

neuroscience↗

bioRxiv2019

Contacting domains that segregate lipid from solute transporters in malaria parasites

While membrane contact sites (MCS) between intracellular organelles are abundant1, and cell-cell junctions are classically defined2, very little is known about the contacts between membranes that delimit extracellular junctions within cells, such as those of chloroplasts and intracellular parasites. The malaria parasite replicates within a unique organelle, the parasitophorous vacuole (PV) but the mechanism(s) are obscure by which the limiting membrane of the PV, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the host erythrocyte (RBC). Here, we demonstrate the existence of multiple micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localized the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We found that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. Thus, these two different transport systems handling hydrophilic and hydrophobic substances, respectively, assume complementary and exclusive distributions. This new structural and molecular data assigns a functional significance to a macroscopic membrane domain.

microbiology↗

bioRxiv2019

Encephalitic alphaviruses exploit caveolae-mediated transcytosis at the blood-brain barrier for CNS entry

Venezuelan and Western equine encephalitis viruses (VEEV and WEEV) invade the CNS early during infection, via neuronal and hematogenous routes (1, 2). While viral replication mediates host-shut off, including expression of type I interferons (IFN) (3, 4), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin (Cav)-1-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and non-replicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1-association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS, and that IFN differentially restricts this process at the BBB.\n\nImportanceVEEV, WEEV and EEEV are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the blood-stream, however the underlying molecular mechanisms have remained largely unknown. Here we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPAses within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.

microbiology↗