Recombinant Xenopus laevis Reactive oxygen species modulator 1 (romo1)

What is Romo1 and what are its structural characteristics?

Romo1 (Reactive oxygen species modulator 1) is a nuclear-encoded mitochondrial inner membrane protein that regulates ROS production and serves as an essential redox sensor in mitochondrial dynamics. In Xenopus laevis, Romo1 (UniProt: Q4V7T9) contains 79 amino acids with a sequence: MPVAVGPYGQSQPNCFDRVKMGFMMGFAVGMAAGALFGTFSCLRFGMRGRELMGGVGKTMMQSGGTFGTFMAIGMGIRC .

Structurally, Romo1 possesses two transmembrane domains (TMDs), each consisting of an α-helix connected by a basic loop. While TMD1 contains a hydrophobic α-helix, TMD2 forms a distinctive amphipathic helix with polar amino acids clustered on one surface. This structural arrangement is highly conserved across eukaryotes and appears critical for its function as an ion channel .

How does Romo1 function as an ion channel?

Romo1 functions as a nonselective cation channel with unique viroporin-like characteristics, distinguishing it from other known eukaryotic ion channels. The protein forms a channel using its amphipathic helical transmembrane domain, which is necessary for its pore-forming activity. Notably, Romo1's channel activity is specifically inhibited by Fe²⁺ ions, which are essential transition metal ions in ROS metabolism .

Methodologically, this channel activity can be confirmed through planar bilayer patch clamp assays using synthesized Romo1. Due to its tendency to aggregate in salt solutions, special targeting methods have been developed to maximize targeting efficiency in bilayer systems, such as using sorbitol solutions for effective protein delivery to preformed planar bilayers .

What techniques are available for measuring ROS in Xenopus laevis models?

Several techniques can be employed to measure ROS in Xenopus models:

• DCFDA Fluorescent Dye: This cell-permeable dye is widely used for ROS detection in cultured cells and has been optimized for monitoring ROS levels in fresh and bench-aged Xenopus oocytes and eggs .

• HyPer Transgenic Reporter System: This genetic sensor is specifically sensitive to hydrogen peroxide (H₂O₂). The HyPer system consists of a prokaryotic OxyR domain that undergoes reversible conformational changes when oxidized, which can be detected through two excitations of fluorescence. By calculating the ratio of HyPer-oxidised and HyPer-reduced signals, researchers can monitor H₂O₂ levels in vivo throughout development .

• Antioxidant Treatment Controls: N-acetyl-cysteine (NAC) and MCI-186 (Edavarone) can be used as experimental controls to reduce oxidative stress. NAC acts as a precursor of glutathione, while MCI-186 functions as a hydroxyl radical scavenger .

How does Romo1 function differ between species, and what are the implications for using Xenopus as a model organism?

Romo1 is remarkably conserved across eukaryotes, with its amino acid sequence being 100% identical among 82 of 247 animal species compared to human Romo1 . This high conservation suggests fundamental importance in cellular function.

In mammals, Romo1 knockout studies show that it is essential for embryonic development, with null mice dying before embryonic day 8.5, even earlier than knockouts of mitochondrial fusion proteins like OPA1 or MFN1/2 . Similarly, in Xenopus, ROS signaling (which Romo1 modulates) plays critical roles in early embryonic development, particularly in mesoderm formation .

When using Xenopus as a model system for Romo1 research, researchers should consider:

• Developmental Timing: Xenopus embryos display elevated and sustained ROS levels throughout early development until neurula stages, after which levels decrease to less than half the initial ROS levels by tadpole stage .

• Tissue-Specific Differences: The major source of ROS in Xenopus oocytes and eggs appears to be plasma membrane NADPH oxidase, with mitochondrial generation contributing to a lesser extent , which may differ from mammalian systems where mitochondrial ROS often predominates.

• Experimental Accessibility: The Xenopus model offers advantages for studying ROS dynamics in vivo due to the accessibility of embryos for manipulation and observation, enabling visualization of ROS throughout development using transgenic reporters .

What methodological approaches can be used to study Romo1's role in mitochondrial dynamics specifically in Xenopus systems?

To investigate Romo1's role in mitochondrial dynamics in Xenopus systems, researchers can employ several approaches:

How can researchers differentiate between direct and indirect effects of Romo1 on ROS production in experimental designs?

Differentiating between direct and indirect effects of Romo1 on ROS production requires multi-faceted experimental approaches:

• Temporal Manipulation: Employ inducible expression or inhibition systems to temporally control Romo1 activity, allowing observation of immediate (likely direct) versus delayed (potentially indirect) effects on ROS levels.

• Domain-Specific Mutations: Introduce mutations in specific domains of Romo1, particularly in the amphipathic helical transmembrane domain necessary for channel activity, to dissect structure-function relationships. This approach can help determine which aspects of ROS modulation are dependent on the ion channel function versus other potential protein-protein interactions .

• Isolated System Assays: Reconstitute purified Romo1 in liposomes or planar lipid bilayers to assess its direct effects on membrane permeability and ion flux in the absence of other cellular components .

• Inhibitor Studies: Use Fe²⁺ ions, which specifically inhibit Romo1 channel activity, as a tool to acutely block function and observe immediate effects on ROS production .

• Epistasis Experiments: Combine Romo1 manipulation with alterations in known ROS-producing (e.g., NADPH oxidase) or ROS-scavenging (e.g., superoxide dismutase) systems to establish hierarchy and interdependence in ROS regulatory pathways.

What are the optimal conditions for expressing and purifying recombinant Xenopus laevis Romo1 for functional studies?

Optimal expression and purification of recombinant Xenopus laevis Romo1 requires careful consideration of its membrane protein nature:

• Expression System Selection: For a small membrane protein like Romo1, bacterial systems (specifically E. coli) with specialized vectors containing fusion tags (such as His6, GST, or MBP) can be effective. Alternatively, insect cell expression systems may provide better folding for functional studies.

• Buffer Optimization: Due to Romo1's tendency to aggregate in salt-containing buffers, consider using non-ionic osmolytes like sorbitol (1M) during purification steps .

• Storage Conditions: Store purified Romo1 in Tris-based buffer with 50% glycerol at -20°C for short-term storage or at -80°C for extended storage. Avoid repeated freeze-thaw cycles which can compromise protein integrity .

• Functional Validation: After purification, validate protein activity through bilayer patch clamp experiments or ROS production assays in reconstituted systems before proceeding to more complex functional studies .

• Quality Control Measures: Confirm protein identity and integrity using mass spectrometry, circular dichroism to assess secondary structure content, and Western blotting with Romo1-specific antibodies.

How can researchers effectively study the relationship between Romo1 and ROS dynamics during Xenopus embryonic development?

To effectively study Romo1-ROS relationships during Xenopus development:

• Temporal Profiling: Use the HyPer transgenic reporter system to establish a detailed temporal profile of H₂O₂ levels throughout development, from unfertilized eggs through tadpole stages, as a baseline for understanding normal ROS dynamics .

• Stage-Specific Manipulations: Employ techniques for stage-specific modulation of Romo1 expression or activity:

  • Microinjection of mRNAs encoding wild-type or mutant Romo1 forms at specific developmental stages

  • CRISPR/Cas9-mediated genome editing to generate Romo1 knockout or knock-in lines

  • Pharmacological approaches using specific inhibitors of Romo1 channel activity

• Functional Readouts: Assess developmental consequences through:

  • Morphological analysis of embryos following Romo1 manipulation

  • Gene expression analysis focusing on mesoderm formation markers, which are known to be sensitive to ROS levels

  • Cell lineage tracing combined with ROS visualization to connect cellular redox states with developmental fates

• Rescue Experiments: Design rescue experiments by co-expressing Romo1 with antioxidants or by modulating specific downstream pathways to establish causality in observed phenotypes.

The table below summarizes the relationship between ROS levels and developmental stages in Xenopus embryos:

What are the key considerations when designing experiments to investigate Romo1's ion channel properties in Xenopus versus mammalian systems?

When investigating Romo1's ion channel properties across species, researchers should consider:

• Comparative Sequence Analysis: Although highly conserved, subtle sequence variations between Xenopus and mammalian Romo1 might affect channel properties. Perform detailed sequence alignments focusing on the amphipathic helical transmembrane domain critical for channel formation .

• Expression System Selection: For direct comparisons:

  • Use identical expression systems for both proteins

  • Consider heterologous expression in Xenopus oocytes, which provides a native-like membrane environment for frog proteins

  • Alternative mammalian cell lines (HEK293, CHO) with controlled expression levels

• Electrophysiological Approaches:

  • Planar bilayer patch clamp assays using purified proteins

  • Whole-cell patch clamp in cells expressing recombinant Romo1

  • Special considerations for targeting Romo1 to bilayers (e.g., sorbitol-based delivery methods)

• Channel Modulation Comparison:

  • Test sensitivity to Fe²⁺ inhibition across species

  • Examine responses to physiological modulators like oxidative stress

  • Assess voltage dependence and ion selectivity properties

• Functional Consequences:

  • Compare effects on mitochondrial membrane potential

  • Assess ROS production in response to channel activity

  • Evaluate mitochondrial morphology changes upon activation/inhibition

What are common challenges in Romo1 protein expression and how can they be addressed?

Common challenges in Romo1 expression and their solutions include:

• Protein Aggregation: Romo1 tends to aggregate when expressed recombinantly.

  • Solution: Use non-ionic osmolytes like sorbitol (1M) in buffers instead of high salt concentrations .

  • Solution: Express with fusion partners known to enhance solubility (MBP, SUMO, or thioredoxin).

• Low Expression Yields: As a small membrane protein, Romo1 may have low expression yields.

  • Solution: Optimize codon usage for the expression system being used.

  • Solution: Use specialized bacterial strains designed for membrane protein expression (e.g., C41(DE3), C43(DE3)).

  • Solution: Test different induction conditions (temperature, inducer concentration, duration).

• Improper Folding: Ensuring proper folding of the transmembrane domains.

  • Solution: Express at lower temperatures (16-20°C) to slow folding and prevent aggregation.

  • Solution: Include appropriate detergents or lipids during extraction and purification.

• Functional Validation: Confirming that recombinant Romo1 retains native activity.

  • Solution: Establish robust functional assays such as ion channel measurements in reconstituted systems.

  • Solution: Compare activity parameters with native Romo1 where possible.

How can researchers accurately distinguish between Romo1-dependent and Romo1-independent ROS production in Xenopus experimental systems?

To distinguish Romo1-dependent from Romo1-independent ROS production:

• Genetic Approaches:

  • Generate Romo1 knockdown or knockout models using morpholinos or CRISPR/Cas9

  • Create rescue lines expressing wild-type or mutant versions of Romo1

  • Employ inducible expression systems to temporally control Romo1 levels

• Pharmacological Approaches:

  • Use selective inhibitors of known ROS sources:

    • Apocynin for NADPH oxidase

    • Respiratory chain inhibitors for mitochondrial ROS

    • Specific Fe²⁺ concentrations that inhibit Romo1 channel activity

• Compartment-Specific Detection:

  • Employ subcellular-targeted ROS sensors (mitochondrial, cytosolic, membrane-associated)

  • Use organelle-specific dyes in combination with ROS indicators

• Biochemical Source Analysis:

  • Isolate subcellular fractions (mitochondria, plasma membrane, endoplasmic reticulum)

  • Measure ROS production capacity in each fraction with and without Romo1 inhibition

  • Reconstitute purified components to assess direct contributions

• Kinetic Analysis:

  • Perform time-course studies following Romo1 manipulation

  • Immediate changes likely reflect direct effects, while delayed responses may indicate secondary mechanisms

What advanced imaging techniques are most effective for studying Romo1 localization and function in Xenopus embryos?

Advanced imaging techniques for studying Romo1 in Xenopus embryos include:

• Super-Resolution Microscopy:

  • Stimulated Emission Depletion (STED) microscopy for visualizing Romo1 distribution within mitochondrial subcompartments

  • Single-molecule localization microscopy (PALM/STORM) to determine precise protein organization

  • Expansion microscopy to physically enlarge specimens for improved resolution

• Live Imaging Applications:

  • Light sheet microscopy for long-term, low-phototoxicity imaging of developing embryos

  • Multiphoton microscopy for deeper tissue penetration in later stage embryos

  • Fluorescence lifetime imaging (FLIM) to detect changes in the microenvironment around Romo1

• Functional Imaging Approaches:

  • FRET-based sensors to detect Romo1 interactions with other proteins

  • Simultaneous imaging of Romo1 localization and ROS production using spectrally distinct fluorophores

  • Genetically encoded redox sensors (like HyPer) combined with tagged Romo1 for correlation studies

• Sample Preparation Considerations:

  • Optimized fixation protocols that preserve mitochondrial morphology

  • Clearing techniques for improved optical access to deep tissues

  • Cryosectioning approaches for high-resolution analysis of specific regions

• Quantitative Analysis Methods:

  • Automated image analysis pipelines for mitochondrial morphology quantification

  • Correlation analysis between Romo1 levels, localization, and ROS production

  • Machine learning approaches for complex pattern recognition in subcellular distribution

How should researchers interpret apparent contradictions in Romo1-related ROS data between cell types or developmental stages?

When facing contradictory ROS data related to Romo1 across different contexts:

What are the implications of Romo1's dual functions as an ion channel and ROS modulator for therapeutic development?

The dual functionality of Romo1 as both an ion channel and ROS modulator has significant implications:

• Precision Targeting Opportunities: The unique viroporin-like structure of Romo1 distinguishes it from other eukaryotic ion channels, potentially allowing for highly specific therapeutic targeting with minimal off-target effects .

• Mitochondrial Medicine Applications: Given Romo1's essential role in:

  • Embryonic development (knockout is embryonic lethal)

  • Mitochondrial dynamics

  • Metabolism (affects glucose homeostasis and insulin secretion in mice)

  Therapeutics modulating Romo1 could potentially address mitochondrial disorders, metabolic diseases, and developmental abnormalities.

• ROS-Related Conditions: Conditions characterized by oxidative stress imbalance could benefit from Romo1-targeted approaches:

  • Neurodegenerative diseases

  • Ischemia-reperfusion injury

  • Aging-related degeneration

• Mechanism-Based Drug Design Strategy:

  • Channel blockers: Compounds mimicking Fe²⁺ inhibition mechanism

  • Allosteric modulators: Molecules affecting the conformation of the amphipathic helix

  • Expression modulators: Agents that regulate Romo1 levels in specific tissues

• Predictive Biomarkers: Romo1 status (expression level, activity) could serve as a biomarker for:

  • Mitochondrial functional capacity

  • Cellular redox state

  • Metabolic health (particularly in tissues like pancreatic beta cells)

How might the evolutionary conservation of Romo1 inform experimental design and data interpretation?

The high evolutionary conservation of Romo1 across eukaryotes provides valuable insights for research:

• Model System Selection: The 100% sequence identity of Romo1 among numerous animal species suggests findings from one model organism may translate well to others. Researchers can strategically select models based on experimental advantages rather than concerns about protein divergence:

  • Xenopus for developmental studies and accessible embryology

  • Mammals for metabolic and physiological relevance to humans

  • Yeast for high-throughput genetic screens

• Functional Domain Analysis: The conservation pattern within Romo1 highlights critical functional regions:

  • The amphipathic helical domain essential for channel formation is highly conserved

  • Any variable regions likely represent species-specific adaptations worth investigating

• Experimental Validation Across Species:

  • Confirm key findings in multiple model systems to establish universal principles

  • Investigate any species-specific differences as potentially informative adaptations

  • Use evolutionary conservation patterns to guide mutagenesis studies (focus on conserved residues)

• Data Interpretation Framework:

  • Core functions (ion channel activity, ROS modulation) likely conserved across species

  • Regulatory mechanisms and contextual roles may show more species-specific variation

  • When contradictions arise between models, focus on environmental or physiological differences rather than assuming protein functional differences

• Translational Research Potential:

  • High conservation suggests Xenopus findings may have direct relevance for human health applications

  • Therapeutic approaches targeting conserved domains likely to have cross-species efficacy

  • Evolutionary medicine perspective may reveal why such conservation exists (fundamental cellular necessity)