Recombinant Xenopus tropicalis UPF0468 protein C16orf80 homolog

Basic Research Questions

What is Xenopus tropicalis UPF0468 protein C16orf80 homolog and what is its functional significance?

The UPF0468 protein C16orf80 homolog in Xenopus tropicalis is a conserved protein that belongs to the FAP20/GTL3/C16orf80/BUG22 family. This protein plays a crucial role in forming inner junctions in flagella and cilia, which are essential for maintaining the structural integrity of these organelles. Research in Chlamydomonas has demonstrated that this protein, along with PACRG (Parkin co-regulated gene), forms alternating components of the inner junction connecting the A- and B-tubules throughout the length of the flagellum . The protein has approximately 193 amino acids in humans, with a conserved sequence across species.

Functionally, disruption of this protein results in flagellar motility defects, as the connection between A- and B-tubules becomes less stable. In Chlamydomonas, deletion of FAP20 results in the lack of every other inner junction density, indicating its structural importance . This protein is highly conserved across species, suggesting an evolutionarily conserved function in ciliary and flagellar biology.

Why is Xenopus tropicalis preferred as a model organism for studying this protein over Xenopus laevis?

Xenopus tropicalis offers several distinct advantages over Xenopus laevis for studying proteins like UPF0468 C16orf80 homolog:

• Diploid genome: X. tropicalis has a diploid genome, whereas X. laevis has an allotetraploid genome. This makes X. tropicalis more amenable to genetic analysis and manipulation .

• Genomic conservation: The X. tropicalis genome shows remarkable synteny with mammalian genomes, often in stretches of a hundred genes or more, far greater than that seen between fish and mammals . This facilitates the identification of orthologous genes and regulatory elements .

• Shorter generation time: X. tropicalis has a generation time of 4-6 months compared to 1-2 years for X. laevis, making genetic studies more feasible .

• Compatibility with established techniques: Many tools and techniques developed for X. laevis can be applied directly to X. tropicalis without significant modifications, including whole-mount in situ hybridization protocols and many antibodies .

• Temperature tolerance: While X. tropicalis embryos develop at similar rates to X. laevis, they can tolerate warmer temperatures, which can be beneficial for certain experimental setups, particularly when working with mammalian cells or tissues .

What methods are available for genetic manipulation of the C16orf80 homolog in Xenopus tropicalis?

Several genetic manipulation techniques have been successfully applied to study genes in Xenopus tropicalis, including the C16orf80 homolog:

• CRISPR/Cas9 mutagenesis: This technique has been well-established for X. tropicalis and offers efficient targeted mutagenesis . The protocol typically involves:

  • Design of single guide RNAs (sgRNAs) targeting the gene of interest

  • Injection of Cas9 protein or mRNA along with sgRNAs into fertilized eggs

  • Screening for mutations using T7 endonuclease assays or sequencing

  • Generation of F1 offspring for stable lines

• Homologous recombination-mediated targeted integration: This approach allows for precise gene editing and has been successfully implemented in X. tropicalis .

• Antisense morpholino oligonucleotides (MOs): While less specific than CRISPR, MOs can effectively knockdown gene expression and function well in X. tropicalis .

• Transgenesis: Several X. tropicalis transgenic lines have been established using methods like the meganuclease protocol, which has been revised to significantly increase the percentage of transgenic animals that reach adulthood .

• TILLING (Targeting Induced Local Lesions in Genomes): This reverse genetics approach can be used to identify mutations in specific genes through chemical mutagenesis and subsequent screening .

Advanced Research Questions

How does the structure of Xenopus tropicalis C16orf80 homolog differ from its human counterpart, and what are the functional implications of these differences?

Comparative analysis of the Xenopus tropicalis C16orf80 homolog and human UPF0468 protein C16orf80 reveals both conserved domains and species-specific adaptations:

The extended C-terminal region in the X. tropicalis homolog contains additional regulatory sequences that may play a role in developmental timing or environmental adaptations specific to amphibians. Mutation studies suggest that while the core functional domains remain conserved, species-specific adaptations likely reflect the different biological contexts in which this protein functions.

For experimental investigations of these differences, researchers should consider:

• Generating truncation mutants to determine the functional significance of the extended C-terminal region

• Conducting cross-species rescue experiments to assess functional conservation

• Performing co-immunoprecipitation studies to identify differential protein interaction partners

What are the most effective protocols for expression and purification of recombinant Xenopus tropicalis C16orf80 homolog, and how can expression systems be optimized?

Optimized protocols for expression and purification of recombinant Xenopus tropicalis C16orf80 homolog typically follow these methodological steps:

Expression Systems Comparison:

Recommended Protocol for E. coli Expression:

• Vector Selection: Use pET-based vectors with an N-terminal affinity tag (His6 or GST) for easier purification .

• Expression Optimization:

  • Induce with 0.5 mM IPTG at OD600 = 0.6-0.8

  • Lower induction temperature to 16-18°C overnight to reduce inclusion body formation

  • Add 5% glycerol to lysis buffer to enhance protein stability

  • Include mild detergents (0.1% Triton X-100) if membrane association is observed

• Purification Strategy:

  • Initial capture using affinity chromatography (Ni-NTA for His-tagged proteins)

  • Intermediate purification using ion exchange chromatography

  • Polishing step with size exclusion chromatography

  • Final protein should be >85% pure by SDS-PAGE

• Storage Conditions: Store in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, and 10% glycerol at -80°C .

For difficult-to-express variants, codon optimization for E. coli expression and co-expression with chaperones (GroEL/GroES) can significantly improve yields. Alternatively, expression as a fusion protein with solubility enhancers such as SUMO or MBP has proven effective for this class of proteins.

What experimental approaches can be used to investigate the role of C16orf80 homolog in ciliary and flagellar function in Xenopus tropicalis?

Several comprehensive experimental approaches can be employed to investigate the role of C16orf80 homolog in ciliary and flagellar function:

• CRISPR/Cas9-mediated knockout studies:

  • Design sgRNAs targeting conserved regions of the C16orf80 homolog

  • Generate F0 knockouts by injecting Cas9 protein/mRNA with sgRNAs into fertilized eggs

  • For mosaic analysis, inject into one cell at the two-cell stage to create a unilateral mutant with the uninjected side serving as an internal control

  • Analyze phenotypes related to ciliary and flagellar function, such as left-right asymmetry defects, kidney development, and sperm motility

• Ultrastructural analysis using transmission electron microscopy (TEM):

  • Process ciliated tissues (such as epidermal multiciliated cells) from wild-type and mutant embryos

  • Examine the ultrastructure of the axoneme, particularly the inner junction between the A- and B-tubules

  • Quantify structural abnormalities and compare to known phenotypes from studies in Chlamydomonas

• Functional assays:

  • Ciliary motility: Use high-speed videomicroscopy to record and analyze ciliary beating in epidermal multiciliated cells

  • Sperm motility: Assess sperm motility parameters using computer-assisted sperm analysis (CASA)

  • Flow assays: Measure fluid flow generated by cilia using fluorescent beads

• Protein localization and interaction studies:

  • Generate GFP or epitope-tagged versions of the protein for localization studies

  • Perform immunoprecipitation followed by mass spectrometry to identify interaction partners

  • Use proximity labeling techniques like BioID to identify proteins in close proximity within the ciliary structure

• Rescue experiments:

  • Test functional conservation by attempting to rescue mutant phenotypes with the human ortholog

  • Create chimeric proteins to identify functional domains

  • Introduce point mutations based on predicted functional sites

• Transcriptomic and proteomic analyses:

  • Perform RNA-Seq on tissues from wild-type and mutant animals to identify altered gene expression patterns

  • Use quantitative proteomics to assess changes in protein composition of isolated cilia/flagella

These approaches collectively provide a comprehensive understanding of C16orf80 homolog function in ciliary and flagellar biology in Xenopus tropicalis.

How can researchers leverage the genetic advantages of Xenopus tropicalis to model human ciliopathies associated with C16orf80/CFAP20 mutations?

Xenopus tropicalis offers unique advantages for modeling human ciliopathies potentially associated with C16orf80/CFAP20 mutations. The following methodological approach outlines a comprehensive strategy:

• Patient variant modeling:

  • Identify human pathogenic variants in C16orf80/CFAP20 from clinical databases

  • Generate equivalent mutations in X. tropicalis using CRISPR/Cas9 precision editing

  • For complex genomic rearrangements, consider homology-directed repair approaches

• Phenotypic analysis pipeline:

  • Early development: Assess left-right asymmetry defects (heart looping, gut coiling)

  • Renal development: Evaluate pronephric development and function using dye filtration assays

  • Neurological phenotypes: Examine neural tube closure and brain ventricle formation

  • Sensory systems: Assess development of eyes, otic vesicles, and lateral line organs

  • Behavioral assays: Quantify swimming patterns and responses to stimuli

• Multi-level analysis approach:

  • Tissue-specific CRISPR: Target mutations to specific tissues using targeted injections based on the embryonic fate map

  • Explant cultures: Generate tissue explants to study cell-autonomous effects

  • Live imaging: Use transgenic reporter lines to visualize ciliated structures in vivo

  • Multi-omics: Combine transcriptomics, proteomics, and metabolomics data to identify perturbed pathways

• Therapeutic testing platform:

  • Test small molecules or genetic interventions that may rescue ciliopathy phenotypes

  • Use the high-throughput capabilities of X. tropicalis (thousands of embryos per experiment) to screen compound libraries

  • Validate findings in patient-derived cells or other mammalian models

• Comparative analysis with human data:

  • Create a cross-species phenotype map correlating frog phenotypes with human clinical presentations

  • Identify conserved molecular signatures using bioinformatic approaches

  • Develop predictive models for human phenotypes based on X. tropicalis data

This approach leverages the genetic tractability, high fecundity, external development, and evolutionary conservation of X. tropicalis to provide meaningful insights into human ciliopathies . The experimental advantages of X. tropicalis, including the ability to generate unilateral mutants that serve as their own controls, enable efficient screening of potential therapeutic interventions.

Technical and Methodological Questions

What challenges might researchers encounter when expressing recombinant Xenopus tropicalis C16orf80 homolog, and how can these be addressed?

Common challenges in expressing recombinant Xenopus tropicalis C16orf80 homolog and their methodological solutions include:

Additional methodological considerations:

• Construct design optimization:

  • Include flexible linkers (GGGGS) between the protein and affinity tags

  • Remove predicted disordered regions at termini

  • Consider expressing functional domains separately

• Expression screening strategy:

  • Test multiple constructs in parallel using small-scale expression tests

  • Analyze both soluble and insoluble fractions

  • Optimize induction conditions (temperature, time, inducer concentration)

• Alternative expression approaches:

  • Cell-free expression systems for difficult proteins

  • Periplasmic expression for disulfide-containing constructs

  • Secretion-based systems for toxic proteins

By systematically addressing these challenges, researchers can significantly improve the yield and quality of recombinant Xenopus tropicalis C16orf80 homolog for structural and functional studies.

What are the best approaches for studying protein-protein interactions involving the C16orf80 homolog in Xenopus tropicalis?

Multiple complementary approaches can be employed to comprehensively study protein-protein interactions involving the C16orf80 homolog in Xenopus tropicalis:

• In vivo approaches:

  • Bimolecular Fluorescence Complementation (BiFC): Express proteins of interest fused to complementary fragments of a fluorescent protein in developing embryos

  • Förster Resonance Energy Transfer (FRET): Measure energy transfer between fluorophore-tagged proteins to detect interactions within 10 nm

  • Proximity Ligation Assay (PLA): Detect endogenous protein interactions in tissue sections with high sensitivity and specificity

• Biochemical methods:

  • Co-immunoprecipitation (Co-IP): Pull down the C16orf80 homolog from X. tropicalis tissues or embryos and identify interacting partners by Western blot or mass spectrometry

  • Tandem Affinity Purification (TAP): Use a dual-tagged version of the protein for sequential purification steps to reduce background

  • Crosslinking Mass Spectrometry (XL-MS): Identify interaction interfaces by crosslinking proteins prior to digestion and mass spectrometry

• Proximity-based labeling:

  • BioID: Fuse C16orf80 homolog to a biotin ligase (BirA*) that biotinylates nearby proteins, which can then be purified and identified

  • APEX2: Use peroxidase-catalyzed labeling to identify proximal proteins in subcellular compartments

  • TurboID: An evolved biotin ligase for faster labeling kinetics, suitable for developmental studies

• Genetic interaction screens:

  • Synthetic lethality analysis: Identify genes that, when disrupted along with C16orf80 homolog, cause severe phenotypes

  • Modifier screens: Identify mutations that enhance or suppress C16orf80 homolog mutant phenotypes

• Reconstitution systems:

  • In vitro binding assays: Use purified recombinant proteins to assess direct interactions

  • Surface Plasmon Resonance (SPR): Determine binding affinities and kinetics in real-time

  • Isothermal Titration Calorimetry (ITC): Measure thermodynamic parameters of protein-protein interactions

Experimental design considerations:

• For developmental studies, consider using stage-specific protein extraction to capture temporally regulated interactions

• When studying ciliary/flagellar interactions, isolate these structures to enrich for relevant interacting partners

• Use both tagged and endogenous approaches to validate interactions and avoid tag-induced artifacts

• Compare interactions in wild-type and mutant backgrounds to understand the functional significance of identified interactions

• Consider the dynamics of interactions during development using time-resolved approaches

A combination of these methods provides a robust framework for identifying and characterizing the interactome of the C16orf80 homolog in Xenopus tropicalis.

How can researchers effectively use CRISPR-Cas9 genome editing to study the function of the C16orf80 homolog in Xenopus tropicalis?

Effective implementation of CRISPR-Cas9 genome editing to study C16orf80 homolog function in Xenopus tropicalis requires careful experimental design and execution:

Experimental Design and Planning

• Guide RNA (sgRNA) design:

  • Target conserved functional domains based on alignment with human and other species' homologs

  • Select target sites with minimal predicted off-target effects using tools like CHOPCHOP or CRISPRscan

  • Design multiple sgRNAs to increase success rate and provide phenotypic validation

  • Include sgRNAs that create frameshift mutations early in the coding sequence

• CRISPR delivery methods:

  • Cas9 protein with in vitro transcribed sgRNA: Most efficient, reduces off-target effects

  • Cas9 mRNA with sgRNA: Effective but may have higher off-target rates

  • Plasmid-based expression: Less efficient but useful for tissue-specific editing

Protocol Implementation

Standard protocol for embryo injection:

• Prepare injection mix:

  • 500-1000 ng/μL Cas9 protein or 300-500 ng/μL Cas9 mRNA

  • 200-400 ng/μL sgRNA

  • Phenol red (0.05%) as injection tracer

• Injection procedure:

  • Inject 1-4 nL into one-cell stage embryos for complete knockout

  • For mosaic analysis, inject into one blastomere at the two-cell stage (creating internal control)

  • For tissue-specific effects, inject into specific blastomeres based on fate map

• Mutation detection and validation:

  • T7 Endonuclease I assay or TIDE analysis at 24-48 hours post-fertilization

  • Direct sequencing of PCR products or cloned amplicons

  • For precise mutations, confirm by deep sequencing

Advanced Applications

• Homology-directed repair (HDR):

  • Co-inject repair template (ssODN or dsDNA) with Cas9/sgRNA

  • Introduce specific mutations or epitope tags at endogenous loci

  • Efficiency can be improved using chemical enhancers of HDR

• Conditional knockouts:

  • Use tissue-specific promoters to drive Cas9 expression

  • Implement inducible systems (e.g., heat shock promoters)

  • Create floxed alleles for tissue-specific Cre-mediated deletion

• High-throughput phenotyping:

  • Utilize automated imaging platforms for morphological analysis

  • Implement quantitative behavioral assays

  • Develop tissue-specific reporter lines to monitor affected structures

Analytical Strategies

• Phenotypic analysis pipeline:

  • Assess developmental stages for ciliary/flagellar-related defects

  • Examine left-right asymmetry, kidney development, and neural tube formation

  • Quantify ciliary beating and fluid flow in appropriate tissues

• Molecular characterization:

  • Perform RNA-Seq to identify altered gene expression patterns

  • Use ChIP-Seq to examine potential transcriptional regulatory roles

  • Implement proteomics to identify changes in protein interaction networks

• Functional validation:

  • Rescue experiments with wild-type mRNA (resistant to sgRNA)

  • Domain-specific rescue to identify functional regions

  • Cross-species rescue to test evolutionary conservation

This comprehensive approach leverages the unique advantages of Xenopus tropicalis as a model system, including its external development, transparent embryos, and amenability to microinjection, to provide significant insights into C16orf80 homolog function .

Evolutionary and Comparative Questions

How conserved is the C16orf80 homolog across different species, and what does this tell us about its evolutionary importance?

The C16orf80 homolog shows remarkable conservation across evolutionary diverse species, indicating its fundamental importance in cellular function:

Evolutionary analysis reveals several significant patterns:

• Domain conservation: The central region of the protein shows the highest conservation (>80% similarity across vertebrates), suggesting it contains critical functional elements essential for protein-protein interactions or structural roles in cilia and flagella .

• Lineage-specific adaptations: The C-terminal region shows greater divergence, potentially reflecting adaptation to species-specific regulatory mechanisms or interaction partners. The extended C-terminus in X. tropicalis may relate to amphibian-specific functions .

• Co-evolution with interacting partners: Phylogenetic analysis indicates that C16orf80 homologs co-evolved with other ciliary components, particularly those involved in the inner junction structure, such as PACRG . This suggests a conserved structural role throughout eukaryotic evolution.

• Selection pressure analysis: The ratio of nonsynonymous to synonymous substitutions (dN/dS) across species is considerably less than 1 for the core regions, indicating strong purifying selection and highlighting the protein's essential function.

• Functional conservation: Experimental evidence demonstrates that C16orf80/FAP20 forms the inner junction in Chlamydomonas flagella, alternate with PACRG along the entire flagellar length with a periodicity of 8 nm . This structural role appears conserved from single-celled organisms to vertebrates, underscoring its fundamental importance in ciliary/flagellar architecture.

The high degree of conservation, coupled with its presence in the last eukaryotic common ancestor (LECA), indicates that C16orf80/CFAP20 represents an ancient and essential component of the eukaryotic ciliary/flagellar apparatus. This evolutionary conservation makes X. tropicalis an excellent model for studying the fundamental biology of this protein and its relevance to human ciliopathies.

How does the function of CFAP20/C16orf80 in Xenopus tropicalis compare with its role in other model organisms like Chlamydomonas and mammals?

Comparative functional analysis of CFAP20/C16orf80 across model organisms reveals both conserved core functions and species-specific adaptations:

Comparative analysis insights:

• Core structural function: Across all species, CFAP20/C16orf80 maintains a conserved structural role in the ciliary/flagellar axoneme, specifically in forming the inner junction between microtubule doublets . This fundamental function has been maintained throughout evolution from single-celled organisms to vertebrates.

• Developmental context in vertebrates: In Xenopus tropicalis and mammals, the protein has acquired additional roles in developmental processes involving ciliary function, such as left-right patterning, neural tube formation, and kidney development. These roles are not present in Chlamydomonas due to its unicellular nature.

• Regulatory complexity: Mammalian and X. tropicalis homologs show more complex regulation compared to Chlamydomonas, with tissue-specific expression patterns and developmental timing control. This likely reflects the greater complexity of ciliary functions in multicellular organisms.

• Interaction network expansion: While the core interaction with PACRG is conserved across species , vertebrate homologs appear to have evolved additional protein-protein interactions, potentially relating to their expanded functional roles in development and tissue homeostasis.

• Species-specific adaptations: X. tropicalis C16orf80 homolog shows certain amphibian-specific features, possibly related to its unique developmental context or environmental adaptations. These include specific expression patterns during metamorphosis and potential roles in regenerative processes.

The evolutionary conservation of CFAP20/C16orf80 across diverse species makes X. tropicalis an excellent model for studying the fundamental functions of this protein relevant to human health and disease. Its intermediate evolutionary position between invertebrates and mammals provides unique insights into both conserved mechanisms and vertebrate-specific adaptations.

Disease and Applied Research Questions

What is the potential relationship between Xenopus tropicalis C16orf80 homolog research and human ciliopathies?

Research on Xenopus tropicalis C16orf80 homolog offers valuable insights into human ciliopathies through several mechanistic and translational pathways:

• Ciliopathy modeling and disease mechanisms:

  • The C16orf80 homolog in X. tropicalis participates in the formation of inner junctions in cilia and flagella, a conserved function across species

  • Disruption of this protein potentially leads to ciliopathy-like phenotypes affecting multiple organ systems

  • X. tropicalis embryos permit visualization of early developmental defects that may be lethal in mammalian models, providing insights into severe human ciliopathies

• Specific ciliopathy-relevant phenotypes in X. tropicalis models:

  • Left-right asymmetry defects (comparable to human heterotaxy syndrome)

  • Kidney development abnormalities (modeling nephronophthisis or polycystic kidney disease)

  • Neural tube closure defects (relevant to human central nervous system malformations)

  • Eye development issues (modeling retinal ciliopathies)

• Translational advantages of X. tropicalis as a ciliopathy model:

  • High genome conservation and synteny with humans

  • Rapid, external development allowing direct observation of organogenesis

  • Ability to generate large numbers of embryos for high-throughput screening

  • Feasibility of creating patient-specific mutations using CRISPR/Cas9 technology

  • Capacity for unilateral mutations that provide internal controls, reducing experimental variability

• Potential human ciliopathies associated with C16orf80/CFAP20 dysfunction:

  • Primary ciliary dyskinesia (PCD)

  • Male infertility due to sperm motility defects

  • Renal ciliopathies

  • Situs inversus and heterotaxy syndromes

• Therapeutic screening potential:

  • X. tropicalis embryos can be used to screen small molecule libraries for compounds that rescue C16orf80 mutant phenotypes

  • The system allows testing of genetic interventions, including gene replacement strategies

  • High-throughput phenotypic assays can identify modifiers of ciliopathy severity

The X. tropicalis model offers unique advantages for understanding human ciliopathies through its combination of genetic tractability, experimental accessibility, and evolutionary conservation. As noted in the literature, "X. tropicalis has a proven track record of exceptional success in studying autism spectrum disorders (ASD) risk genes, congenital anomalies, cancer, congenital heart disease, kidney disease, and many more" , suggesting its value extends to ciliopathies as well.

What future directions and applications can be anticipated for research on Xenopus tropicalis C16orf80 homolog?

Research on Xenopus tropicalis C16orf80 homolog is poised for significant advances across multiple scientific domains:

• Advanced genetic engineering approaches:

  • Implementation of inducible CRISPR systems for temporal control of gene disruption

  • Development of tissue-specific gene editing using cell-type-specific promoters

  • Creation of patient-specific variants using precision genome editing technologies

  • Integration of genetic targeting with optogenetic control for spatiotemporal manipulation

• Multi-omics integration and systems biology:

  • Comprehensive characterization of C16orf80 interactome across developmental stages

  • Integration of transcriptomic, proteomic, and metabolomic data to construct regulatory networks

  • Machine learning approaches to predict phenotypic outcomes of specific mutations

  • Comparative multi-omics across species to identify conserved regulatory mechanisms

• Advanced imaging and functional analysis:

  • Super-resolution microscopy to visualize ciliary ultrastructure at nanoscale resolution

  • Live imaging of protein dynamics using new fluorescent protein variants

  • Single-cell approaches to understand cell-type-specific functions

  • Mechanical force measurements to assess ciliary function in intact organisms

• Translational research applications:

  • High-throughput drug screening using X. tropicalis embryos with C16orf80 mutations

  • Development of personalized medicine approaches for ciliopathy patients

  • Testing gene therapy strategies before moving to mammalian models

  • Biomarker discovery for early diagnosis of ciliopathies

• Evolutionary developmental biology insights:

  • Comparative analysis of C16orf80 function across evolutionarily diverse species

  • Investigation of the role of C16orf80 in evolutionary adaptations of ciliary functions

  • Understanding the contribution of this protein to species-specific developmental programs

• Emerging technology integration:

  • Application of genetic code expansion to incorporate unnatural amino acids into the protein

  • CRISPR-based lineage tracing to understand developmental contributions of C16orf80-expressing cells

  • Organ-on-chip technologies combining X. tropicalis cells with engineered microenvironments

  • Single-cell genomics to identify cell-type-specific responses to C16orf80 disruption

• Novel therapeutic strategies:

  • Development of antisense oligonucleotides to modulate C16orf80 expression

  • Small molecule screening to identify compounds that stabilize ciliary structure

  • Exploration of genetic suppressor mechanisms that could inform therapeutic approaches