Recombinant Xenopus tropicalis Blood vessel epicardial substance (bves)

Advanced Research Questions

• How can recombinant X. tropicalis BVES be used to study tight junction formation and regulation?

Recombinant X. tropicalis BVES provides a valuable tool for investigating tight junction dynamics through several experimental approaches:

• Functional Rescue Studies:

  • Transfection of recombinant BVES into BVES-deficient cells to restore tight junction formation

  • Measurement of transepithelial electrical resistance (TEER) to quantify barrier function

  • Comparison of rescue efficiency between wild-type BVES (w-BVES) and truncated BVES (t-BVES)

  • Live-cell imaging to visualize junction assembly dynamics in real-time

• Protein Interaction Analysis:

  • Pull-down assays using recombinant BVES to identify binding partners

  • Co-immunoprecipitation to verify interactions with known tight junction proteins

  • FRET or BRET assays to study dynamic protein-protein interactions

  • Domain mapping to identify specific regions required for tight junction association

• Dominant Negative Approaches:

  • Expression of truncated BVES to disrupt endogenous BVES function

  • Analysis of competitive binding between full-length and truncated BVES forms

  • Assessment of tight junction protein mislocalization

Research has demonstrated that human corneal epithelial cells expressing full-length BVES (w-BVES) show increased tight junction function with higher transepithelial electrical resistance, while cells expressing truncated BVES (t-BVES) exhibit disrupted tight junction protein localization and decreased barrier function . These findings highlight how recombinant BVES variants can be used to dissect the structural requirements for proper tight junction assembly.

• What experimental approaches can be used to investigate the role of BVES in RhoA signaling pathway in X. tropicalis?

Investigating BVES-mediated regulation of RhoA signaling requires sophisticated experimental designs:

• RhoA Activity Measurement:

  • FRET-based biosensors for real-time visualization of RhoA activity in living cells

  • Rhotekin-RBD pull-down assays to quantify active GTP-bound RhoA

  • Phosphorylation analysis of RhoA downstream targets (ROCK, LIMK, cofilin)

  • Microscopy-based assessment of stress fiber formation as a readout of RhoA activation

• Molecular Mechanism Investigation:

  • Analysis of GEF-H1 localization in response to BVES manipulation

  • Determination of ZONAB/DbpA transcriptional activity using reporter assays

  • Examination of tight junction protein dynamics and their influence on RhoA regulators

  • Co-localization studies of BVES with RhoA pathway components

• Genetic Approaches in X. tropicalis:

  • CRISPR-Cas9 knockout of BVES in X. tropicalis embryos

  • Morpholino-based knockdown for stage-specific analysis

  • Transgenic expression of constitutively active or dominant negative RhoA

  • Rescue experiments with wild-type or mutant BVES constructs

Studies have shown that expression of truncated BVES (t-BVES) in epithelial cells leads to decreased membrane localization of GEF-H1 (a RhoA activator) and increased RhoA activity, with a significant 30% increase in FRET activity compared to control cells . This indicates that intact BVES normally suppresses RhoA activation, likely by regulating GEF-H1 localization at tight junctions.

• How can researchers investigate the role of BVES in heart development using X. tropicalis embryos?

X. tropicalis provides an excellent model system for studying BVES function in cardiac development:

• Expression Analysis:

  • Whole-mount in situ hybridization to visualize BVES expression during heart development

  • Immunohistochemistry to localize BVES protein in cardiac tissues

  • Single-cell RNA-seq to identify BVES-expressing cell populations

  • Quantitative RT-PCR to measure expression levels across developmental stages

• Loss-of-Function Approaches:

  • CRISPR-Cas9 mediated knockout of BVES

  • Morpholino oligonucleotide injection for targeted knockdown

  • Dominant negative constructs (e.g., truncated BVES)

  • Pharmacological inhibition of BVES-dependent pathways

• Phenotypic Analysis:

  • High-speed video microscopy to assess cardiac contraction and rhythm

  • Optical mapping to visualize cardiac conduction

  • Histological analysis to examine heart morphology

  • Molecular marker analysis to evaluate cardiac differentiation

• Mechanistic Investigation:

  • Analysis of tight junction formation in developing cardiac tissues

  • Assessment of RhoA signaling during cardiogenesis

  • Examination of ZONAB/DbpA-regulated gene expression

  • Investigation of cell adhesion and migration during cardiac morphogenesis

Research in mice has shown that BVES is strongly expressed in the heart, particularly in the atria and cardiac conduction system . BVES knockout mice exhibit stress-induced sinus bradycardia and age-dependent sinoatrial node dysfunction reminiscent of sick sinus syndrome in humans . These findings suggest that X. tropicalis could serve as a valuable model for investigating the developmental origins of cardiac conduction disorders related to BVES dysfunction.

• How does recombinant X. tropicalis BVES compare with human BVES in experimental settings?

Comparing X. tropicalis and human BVES reveals important similarities and differences that impact experimental design and interpretation:

When using X. tropicalis BVES as a model for human BVES function, researchers should:

• Focus on conserved domains and functions

• Validate key findings in human cell systems

• Consider species-specific differences in expression patterns

• Account for potential differences in binding partners

• Perform cross-species rescue experiments to assess functional conservation

Despite these considerations, the core functions of BVES in tight junction regulation and signaling pathway modulation appear conserved between species, making X. tropicalis a valuable model for studying fundamental BVES mechanisms.

• What methodological considerations are important when generating and using recombinant X. tropicalis BVES for research?

Producing and utilizing recombinant X. tropicalis BVES requires careful attention to several methodological aspects:

• Expression System Selection:

  • Bacterial systems (E. coli): Cost-effective but may lack post-translational modifications

  • Insect cells (Sf9, Hi5): Better for membrane proteins with complex folding

  • Mammalian cells (HEK293, CHO): Provide most native-like modifications

  • Cell-free systems: Rapid production but limited scale

• Construct Design:

  • Full-length vs. truncated versions for specific applications

  • Strategic placement of affinity tags to minimize functional interference

  • Inclusion of fluorescent protein fusions for localization studies

  • Codon optimization for the expression system

• Purification Strategy:

  • Detergent selection for membrane protein solubilization

  • Affinity chromatography (His-tag, FLAG-tag, GST)

  • Size exclusion chromatography for removing aggregates

  • Ion exchange chromatography for increasing purity

• Quality Control:

  • SDS-PAGE and Western blotting to verify size and purity

  • Mass spectrometry to confirm protein identity

  • Circular dichroism to assess secondary structure

  • Functional assays to verify activity (e.g., binding to known partners)

• Storage and Handling:

  • Determine optimal buffer conditions to maintain stability

  • Assess freeze-thaw stability

  • Test long-term storage conditions

  • Evaluate the impact of lyophilization if applicable

For functional studies, researchers should compare wild-type BVES (w-BVES) with C-terminus truncated BVES (t-BVES), as previous research has shown distinct functional differences between these forms . The truncated version can serve as a valuable control or tool for dominant-negative approaches in experimental settings.

• What are the current challenges and future directions in BVES research using the X. tropicalis model?

Research on BVES in X. tropicalis faces several challenges but also offers exciting opportunities for future investigation:

• Current Technical Challenges:

  • Limited availability of X. tropicalis-specific antibodies and reagents

  • Need for specialized facilities for amphibian husbandry

  • Longer generation time compared to some other model organisms

  • Limited established transgenic lines for tissue-specific studies

• Biological Complexities:

  • Potential redundancy with other Popeye domain-containing proteins

  • Species-specific differences in BVES expression and regulation

  • Complex tissue-specific roles across development

  • Integration of BVES function with other junctional signaling pathways

• Emerging Research Opportunities:

  • Investigation of BVES in EMT and cancer progression

  • Role in cardiac conduction system development and disease

  • Function in gastrointestinal epithelial homeostasis and inflammation

  • Therapeutic targeting of BVES pathways in human disease

• Methodological Advances:

  • CRISPR-Cas9 genome editing for precise genetic manipulation

  • High-throughput phenotypic screening approaches

  • Advanced imaging techniques for in vivo studies

  • Integration of -omics approaches for systems-level understanding

Resource centers like the National Xenopus Resource (NXR) and the European Xenopus Resource Centre (EXRC) are developing tools including CRISPR-Cas9 resources and transgenic lines that will accelerate BVES research in X. tropicalis . XenoBiores, run by the NBRP X. tropicalis, facilitates the exchange of questions and protocols among researchers , providing valuable support for addressing technical challenges.

Future research directions should leverage the unique advantages of X. tropicalis, including its diploid genome, developmental accessibility, and evolutionary position, to investigate BVES functions that are relevant to human health and disease.