Recombinant Xenopus tropicalis WD repeat-containing protein 92 (wdr92)

What is Xenopus tropicalis WDR92 and why is it significant for research?

WDR92 (also known as Monad) is a WD-repeat protein that has been highly conserved throughout evolution. It forms part of a prefoldin-like cochaperone complex with a phylogenetic signature compatible with roles in motile cilia assembly or function. The significance of X. tropicalis WDR92 stems from several factors:

• It provides a vertebrate model for studying a protein with critical roles in ciliary function

• X. tropicalis offers advantages as a diploid organism with a sequenced genome containing orthologues for approximately 79% of human genes

• Studies in other organisms (like planaria) have demonstrated that WDR92 depletion causes pleiomorphic defects in ciliary architecture, resulting in immotile or poorly motile structures

• Its involvement in a previously unrecognized cytoplasmic chaperone system specifically required for folding key axonemal components makes it relevant for understanding ciliopathies

Research using X. tropicalis WDR92 can have significant translational implications due to the high level of conservation between amphibian and human proteins in this family.

What is the structural composition of WDR92 and how does it influence function?

WDR92 has a distinctive structural arrangement that directly relates to its function:

• The 1.95-Å crystal structure (PDB accession 3I2N) reveals that the human homolog consists of 28 β-strands and a single short α-helix

• It contains seven WD repeats, each forming a four-stranded β-sheet arranged as a classic toroidal β-propeller

• WDR92 features a large protrusion derived from two extended loops located between β20/β21 and β24/β25

• The molecular surface displays several highly charged exposed patches with both positive and negative regions

• The surface structure has been remarkably conserved between humans, planaria (S. mediterranea), and green algae (C. reinhardtii)

This conservation of structure across diverse species underscores its fundamental importance in cellular function. The β-propeller arrangement facilitates protein-protein interactions critical for its cochaperone functions, while the charged surface patches likely mediate specific binding to client proteins involved in ciliary assembly.

How conserved is WDR92 across species and what does this phylogenetic distribution indicate?

WDR92 demonstrates remarkable evolutionary conservation with specific distribution patterns:

This distribution strongly correlates with organisms that build motile cilia/flagella, suggesting WDR92's specialized role in motile cilia assembly or function rather than in general cellular processes . The near-universal conservation in organisms with motile cilia and absence in those without makes WDR92 a signature protein for motile ciliary function.

What are the recommended methods for recombinant expression of X. tropicalis WDR92?

While specific protocols for X. tropicalis WDR92 expression aren't detailed in the provided references, a methodological approach based on established practices would include:

• Gene cloning and vector construction:

  • PCR amplification of the wdr92 gene from X. tropicalis cDNA

  • Insertion into appropriate expression vectors (pET series for bacterial expression; pFastBac for insect cells)

  • Verification by sequencing to confirm correct reading frame and absence of mutations

• Expression system selection:

  • Bacterial systems (E. coli BL21(DE3)) for initial trials

  • Insect cell systems (Sf9/Hi5) for improved folding of complex proteins

  • Mammalian expression systems for applications requiring post-translational modifications

• Optimization parameters:

  • Temperature: Test reduced temperatures (16-20°C) to improve solubility

  • Induction time: Optimize between 4-24 hours

  • Inducer concentration: Titrate IPTG (0.1-1.0 mM) for bacterial systems

  • Fusion tags: Test N-terminal His6, GST, or MBP tags to improve solubility and facilitate purification

• Purification strategy:

  • Initial capture: Affinity chromatography based on fusion tag

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Tag removal: TEV or PreScission protease cleavage if tags interfere with function

Given WDR92's role in prefoldin complexes, special attention to proper folding during expression is essential for obtaining functionally active protein.

How can researchers verify the structural integrity of recombinant WDR92?

Verification of proper folding and structural integrity is crucial for functional studies:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure composition

  • Thermal shift assays to assess stability and proper folding

  • Dynamic light scattering to evaluate homogeneity and aggregation state

• Functional assays:

  • ATPase activity measurements if applicable

  • Protein-protein interaction studies with known binding partners (e.g., RPAP3, components of prefoldin complex)

  • Co-chaperone activity assays

• Structural analysis:

  • Limited proteolysis to assess domain organization and stability

  • Comparison with published crystal structure data (PDB 3I2N)

  • Advanced structural methods (X-ray crystallography or cryo-EM) for detailed structural determination

• Comparative analysis:

  • Parallel characterization of human WDR92 as reference

  • Comparison of key parameters with published data on WDR92 homologs

The β-propeller structure of WDR92 should show characteristic stability and resistance to denaturation in properly folded preparations.

What are the critical factors for ensuring the functional activity of recombinant X. tropicalis WDR92?

Ensuring functional activity requires attention to several key factors:

• Buffer optimization:

  • pH range: Typically 7.0-8.0 for WD-repeat proteins

  • Salt concentration: Usually 100-300 mM NaCl

  • Addition of stabilizing agents: glycerol (5-10%), reducing agents (DTT or TCEP)

• Preventing aggregation:

  • Storage conditions: -80°C for long-term; avoid repeated freeze-thaw cycles

  • Addition of non-ionic detergents below critical micelle concentration

  • Maintaining appropriate protein concentration (typically below 1 mg/ml for complex proteins)

• Co-factors and interaction partners:

  • Consider co-expression or reconstitution with known binding partners

  • Ensure presence of required metal ions if applicable

  • Test functionality in the presence of components of the prefoldin complex

• Functional validation:

  • In vitro binding assays with known interaction partners

  • Chaperone activity assays measuring prevention of client protein aggregation

  • Complementation assays in WDR92-depleted systems

The complex interactome of WDR92 suggests that its full functionality may depend on proper complex formation with other prefoldin and R2TP complex components.

How can researchers effectively design RNAi-mediated knockdown experiments for WDR92 in X. tropicalis?

Based on successful approaches in planaria and adaptation to X. tropicalis systems:

• Design of targeting constructs:

  • Select 2-3 non-overlapping regions (300-500 bp) of X. tropicalis wdr92 mRNA

  • Avoid regions with significant homology to other genes

  • Create both sense and antisense constructs for dsRNA production

  • For morpholinos, target the 5' UTR or translation start site

• Delivery methods for X. tropicalis:

  • Microinjection of dsRNA/morpholinos into fertilized eggs (1-2 cell stage)

  • Optimize concentration through dose-response experiments (typically 5-20 ng)

  • Include traceable markers (e.g., fluorescent dextran) to monitor delivery

• Validation of knockdown efficiency:

  • RT-PCR to assess mRNA levels (as performed in planaria studies)

  • Western blotting to confirm protein reduction

  • Include controls: uninjected embryos, control morpholino/dsRNA injections

• Phenotypic analysis timeline:

  • Monitor development through key stages (gastrulation, neurulation, organogenesis)

  • Examine ciliated tissues (epidermis, nephrostomes, left-right organizer)

  • Perform high-speed videomicroscopy of ciliary beating at stages 28-32

Based on planaria studies, researchers should anticipate potential phenotypes related to ciliary function and structure, particularly in tissues with motile cilia .

What phenotypic assays are most informative for studying WDR92 function in cilia development?

Given WDR92's role in ciliary assembly, several targeted assays provide valuable insights:

• Ciliary motility assessment:

  • High-speed videomicroscopy (500-1000 fps) to measure ciliary beat frequency

  • Particle tracking to assess flow generation and hydrodynamic coupling

  • Analysis of beat pattern using kymograph analysis

• Ciliary ultrastructure examination:

  • Transmission electron microscopy to assess axonemal organization

  • Focus on key structures affected in planaria: dynein arms, B-tubule closure, central pair complex

  • Immunogold labeling to determine WDR92 localization

• Left-right asymmetry evaluation:

  • Assessment of organ situs (heart, gut looping)

  • Expression analysis of laterality markers (nodal, lefty, pitx2)

  • Examination of the gastrocoel roof plate (GRP, equivalent to mammalian node)

• Ciliary component trafficking:

  • Immunofluorescence of intraflagellar transport proteins

  • Live imaging of fluorescently tagged ciliary proteins

  • FRAP (Fluorescence Recovery After Photobleaching) to assess protein turnover

Planaria studies revealed multiple ciliary defects upon WDR92 knockdown, suggesting researchers should examine multiple aspects of cilia structure and function in X. tropicalis .

How does WDR92 depletion affect specific ciliary structures, and what methodologies best capture these defects?

Based on planaria studies, WDR92 depletion causes pleiomorphic defects in ciliary ultrastructure that can be systematically analyzed:

For comprehensive analysis:

• Quantitative TEM analysis:

  • Score multiple axonemal defects in numerous cross-sections (n>100)

  • Calculate frequency of each defect type

  • Compare to control samples

• Correlative microscopy approaches:

  • Combine live imaging of ciliary beating with subsequent TEM of the same samples

  • Link ultrastructural defects to specific motility phenotypes

• Molecular analysis of axonemal components:

  • Immunofluorescence for dynein components, radial spoke proteins, etc.

  • Biochemical fractionation of cilia to assess component incorporation

  • Proximity labeling to identify affected protein interactions

These analyses would reveal whether X. tropicalis exhibits similar defects to those observed in planaria and potentially identify additional affected structures .

How can CRISPR-Cas9 genome editing be optimized for generating WDR92 mutants in X. tropicalis?

For generating precise WDR92 mutants in X. tropicalis:

• Guide RNA design strategy:

  • Target early exons to ensure null mutations

  • Design multiple sgRNAs (3-4) targeting different regions

  • Screen sgRNAs for off-target effects using computational tools

  • Consider conserved functional domains for domain-specific disruptions

• Delivery optimization:

  • Microinjection into fertilized eggs at one-cell stage

  • Cas9 delivery options: mRNA (500 pg) or protein (500-1000 pg)

  • sgRNA concentration: 200-300 pg per guide

  • Co-inject with fluorescent dextran to track delivery

• Mutation detection and validation:

  • T7 endonuclease or heteroduplex mobility assays for initial screening

  • Direct sequencing of PCR amplicons for precise mutation characterization

  • RT-PCR and Western blotting to confirm effect on transcript and protein

• Breeding strategy for stable lines:

  • Raise F0 mosaic animals to sexual maturity (4-6 months)

  • Outcross to wild-type to generate F1 heterozygotes

  • Genotype F1 offspring to identify carriers of frameshift mutations

  • Intercross F1 heterozygotes to generate homozygous mutants

The diploid genome of X. tropicalis provides a significant advantage over X. laevis for genome editing approaches, enabling faster generation of homozygous mutants .

What comparative approaches between X. tropicalis WDR92 and human WDR92 can provide insights into conserved disease mechanisms?

Several strategic approaches can reveal conserved functional mechanisms:

• Structure-function correlation:

  • Mutagenesis of conserved surface residues based on the crystal structure

  • Test functional conservation through cross-species rescue experiments

  • Map human disease-associated variants onto the X. tropicalis protein

• Interactome analysis:

  • Comparative proteomics to identify conserved binding partners

  • Yeast two-hybrid or BioID approaches in both species

  • Focus on interactions with the R2TP complex and components involved in ciliopathies

• Complementation studies:

  • Express human WDR92 in X. tropicalis WDR92 mutants

  • Test specific human variants for rescue efficiency

  • Quantify rescue at structural, functional, and physiological levels

• Domain swap experiments:

  • Create chimeric proteins between human and X. tropicalis WDR92

  • Identify domains responsible for species-specific interactions

  • Map functional conservation at the sub-protein level

These approaches leverage the approximately 79% conservation between human and X. tropicalis genomes to provide insights into pathogenic mechanisms relevant to human ciliopathies.

How can researchers resolve contradictory data regarding WDR92 function in different model systems?

When facing inconsistent results across experimental systems:

• Systematic comparison framework:

  • Create a standardized comparison table of phenotypes across species

  • Record key parameters: knockdown/knockout method, efficiency, developmental timing

  • Document differences in experimental conditions and genetic backgrounds

• Tissue-specific and temporal analysis:

  • Investigate potential tissue-specific roles using conditional approaches

  • Examine temporal requirements through stage-specific manipulations

  • Consider compensatory mechanisms that may differ between systems

• Biochemical validation across systems:

  • Perform parallel biochemical assays using material from different species

  • Compare protein-protein interactions under identical conditions

  • Assess post-translational modifications that might differ between systems

• Integration with evolutionary context:

  • Analyze correlation between phenotypic differences and evolutionary distance

  • Consider species-specific adaptations in ciliary function

  • Examine the co-evolution of WDR92 with interaction partners

For example, if X. tropicalis WDR92 studies reveal phenotypes beyond ciliary defects (unlike planaria), researchers should systematically investigate whether this represents expanded function in vertebrates or context-dependent roles .

What are the most robust approaches for investigating WDR92's role in the prefoldin-like complex in X. tropicalis?

To characterize WDR92's role in the prefoldin-like complex:

• Complex reconstitution and analysis:

  • Recombinant expression of all components of the X. tropicalis prefoldin-like complex

  • In vitro reconstitution of the complex

  • Structural characterization using cryo-EM

  • Functional assays measuring chaperone activity

• Interaction mapping:

  • Co-immunoprecipitation studies with tagged WDR92

  • Proximity labeling (BioID/TurboID) to capture transient interactions

  • Chemical crosslinking mass spectrometry to map interaction interfaces

  • Yeast two-hybrid screening to identify direct binding partners

• Client protein identification:

  • Proteomics analysis of proteins associated with WDR92 in ciliated tissues

  • Focus on dynein components and other ciliary proteins identified in planaria studies

  • Validation of client interactions using in vitro binding assays

• Functional dissection of the complex:

  • Generate selective mutations disrupting specific interfaces

  • Compare phenotypes of different complex component knockdowns

  • Assess complex integrity in the absence of WDR92

These approaches would reveal whether X. tropicalis WDR92 functions primarily through the prefoldin-like complex as suggested by the planaria studies, and identify the key ciliary client proteins requiring this chaperone system .

What are the common pitfalls in recombinant WDR92 expression and how can they be addressed?

Researchers working with WDR92 should anticipate and address these challenges:

• Protein insolubility issues:

  • Problem: WD-repeat proteins often form inclusion bodies

  • Solutions:

    • Reduce expression temperature (16-18°C)

    • Use solubility-enhancing tags (MBP, SUMO)

    • Test co-expression with interaction partners

    • Explore refolding from inclusion bodies if necessary

• Improper folding:

  • Problem: Complex β-propeller structure may not form correctly

  • Solutions:

    • Express in eukaryotic systems (insect or mammalian cells)

    • Include molecular chaperones during expression

    • Verify folding using circular dichroism or limited proteolysis

    • Optimize buffer conditions (add stabilizing agents)

• Protein degradation:

  • Problem: Susceptibility to proteolysis during purification

  • Solutions:

    • Include protease inhibitors throughout purification

    • Minimize purification time

    • Identify and remove flexible regions prone to degradation

    • Test storage conditions systematically

• Loss of binding partners:

  • Problem: WDR92 may require complex formation for stability

  • Solutions:

    • Consider tandem purification of multiple complex components

    • Stabilize with chemical crosslinking

    • Reconstitute complex post-purification

Each challenge should be documented systematically to build best practices for working with this protein.

How can researchers distinguish between direct and indirect effects of WDR92 depletion in ciliary phenotypes?

Differentiating primary from secondary effects requires multiple complementary approaches:

• Temporal analysis:

  • Track the sequence of cellular and molecular changes after WDR92 depletion

  • Identify the earliest detectable changes as likely primary effects

  • Use inducible knockdown/knockout systems for precise temporal control

• Rescue experiments with structure-guided variants:

  • Design WDR92 variants with mutations in specific interaction surfaces

  • Test which protein interactions are essential for rescuing phenotypes

  • Employ domain deletion constructs to map functional regions

• Biochemical interaction validation:

  • Confirm direct interactions between WDR92 and candidate client proteins

  • Perform in vitro assays with purified components

  • Quantify binding affinities and assess effects of disease-associated mutations

• Multi-level analysis pipeline:

  • Compare transcriptome, proteome, and phenotypic changes

  • Construct pathway models to distinguish proximal from distal effects

  • Validate predicted relationships through targeted interventions

This systematic approach can reveal whether WDR92's role in X. tropicalis is primarily through chaperoning specific ciliary components as suggested by planaria studies, or whether it has additional functions .

What statistical approaches are most appropriate for analyzing the pleiomorphic ciliary defects associated with WDR92 dysfunction?

Given the variable phenotypes observed in planaria, appropriate statistical methods are crucial:

• Quantification and categorization strategy:

  • Develop a standardized scoring system for each type of ciliary defect

  • Classify defects into major categories (dynein arm defects, tubule closure, central pair)

  • Blind scoring by multiple observers to minimize bias

  • Report frequency of each defect type per axoneme

• Statistical methods for complex phenotype analysis:

  • Chi-square testing for comparing frequency distributions of defect types

  • Non-parametric methods (Mann-Whitney) for comparing severity scores

  • Principal component analysis to identify patterns in multi-parameter phenotypes

  • Cluster analysis to identify distinct phenotypic groups

• Sample size considerations:

  • Power analysis to determine required sample sizes (typically n≥100 axonemes)

  • Analysis across multiple animals and multiple tissue samples

  • Bootstrap methods for robust confidence interval estimation

• Correlative analysis:

  • Correlation between ultrastructural defects and functional parameters

  • Multivariate analysis to identify relationships between phenotypes

  • Analysis of phenotype co-occurrence to infer mechanistic relationships

These approaches would enable researchers to determine whether X. tropicalis exhibits the same spectrum of pleiomorphic defects observed in planaria WDR92 knockdowns .