Recombinant Danio rerio Tetratricopeptide repeat protein 30A (ttc30a)

What is TTC30A and what distinguishes it from its paralog TTC30B?

TTC30A is a tetratricopeptide repeat-containing protein that functions as an integral component of the intraflagellar transport complex B (IFT-B). This protein contains eight tetratricopeptide repeat (TPR) motifs that fold together to form a single TPR domain, which mediates protein-protein interactions and facilitates the assembly of multiprotein complexes . In zebrafish and other vertebrates, TTC30A has a paralog called TTC30B that shares highly similar nucleotide sequences, suggesting evolutionary conservation of function. While these paralogs demonstrate some functional redundancy, recent research has identified paralog-specific interactions and functions, particularly in signaling pathways like Sonic hedgehog .

The primary differences between TTC30A and TTC30B appear to be in their interaction patterns with regulatory proteins rather than in their core IFT functions. For instance, TTC30A specifically interacts with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog signaling, which is not observed with TTC30B .

How is the structure of TTC30A organized and what domains are functionally significant?

TTC30A's structure is characterized by:

• Eight tetratricopeptide repeat (TPR) motifs, each consisting of 34 amino acids arranged in a helix-turn-helix configuration

• These TPR motifs collectively fold into a single TPR domain that mediates protein-protein interactions

• The protein has a molecular weight of approximately 76 kDa

• The TPR domain creates a specialized binding interface that enables TTC30A to interact with multiple IFT-B components

Functionally significant regions include specific amino acid residues that mediate interactions with other IFT-B components. For example, the A375 position appears critical, as the A375V mutation significantly decreases interaction with IFT57, a member of the IFT-B2 subcomplex . This suggests that this region is involved in maintaining proper IFT-B complex integrity and function.

What is the expression pattern of TTC30A in Danio rerio development?

While the provided search results don't specifically detail the expression pattern in zebrafish, TTC30A expression in vertebrates generally correlates with ciliated tissues. In zebrafish, expression would be expected in:

• Kupffer's vesicle during early development (8-12 hours post-fertilization)

• Developing neural tube

• Sensory organs including the otic vesicle and developing retina

• Pronephros (embryonic kidney structure)

• Lateral line organs

Researchers investigating zebrafish ttc30a expression should consider whole-mount in situ hybridization techniques using RNA probes designed against conserved regions that distinguish between ttc30a and ttc30b paralogs.

What role does TTC30A play in cilia formation in vertebrate models?

TTC30A serves as a crucial component in cilia formation through several mechanisms:

• It functions as an integral part of the IFT-B complex, which is essential for anterograde intraflagellar transport - the process of moving ciliary precursors from the base to the tip of the cilium

• TTC30A contributes to the stability and integrity of the IFT-B complex, as complete loss of both TTC30A and TTC30B paralogues leads to severe defects in ciliogenesis resulting in a complete failure of cilia formation

• It appears to play a specific role in tubulin polyglutamylation, a post-translational modification critical for ciliary axoneme stability

How does TTC30A contribute to ciliary signaling pathways?

Recent research has revealed that beyond its structural role in ciliogenesis, TTC30A plays specialized roles in ciliary signaling:

• TTC30A specifically interacts with protein kinase A catalytic subunit α (PKA-C), which negatively regulates Sonic hedgehog (Shh) signaling

• Mutations in TTC30A (particularly A375V) can inhibit the ciliary localization of Smoothened, a key component of the Shh pathway

• This effect appears to be independent of Patched1 but is associated with distinct patterns of phosphorylated PKA substrate accumulation when cells are treated with forskolin

• TTC30A likely serves as a molecular bridge between the IFT machinery and signaling components, facilitating their proper localization within the cilium

This TTC30A-specific interaction with PKA suggests a specialized role in regulating Shh signaling that is not shared or is less prominent in its paralog TTC30B, highlighting functional specialization between these otherwise redundant proteins .

What phenotypes result from TTC30A deficiency in zebrafish models?

Based on the conserved function of TTC30A across vertebrates, zebrafish with ttc30a deficiency would likely exhibit:

• Shortened cilia in multiple tissues

• Reduced tubulin polyglutamylation in ciliary axonemes

• Defects in left-right asymmetry determination due to disrupted Kupffer's vesicle function

• Curved body axis, a common phenotype in zebrafish ciliary mutants

• Potential disruption of Sonic hedgehog-dependent developmental processes

• Kidney cyst formation in pronephric ducts

When designing zebrafish ttc30a knockout studies, researchers should consider that complete loss of ciliary function may only occur when both ttc30a and ttc30b are targeted, due to their partial functional redundancy as observed in mammalian models . Single gene knockouts may show more subtle or tissue-specific phenotypes based on differential expression patterns.

What expression systems are optimal for producing functional recombinant Danio rerio TTC30A?

For functional recombinant Danio rerio TTC30A production, consider these approaches:

• Prokaryotic expression systems:

  • E. coli BL21(DE3) with pET vector systems can be used for basic structural studies

  • Cold-shock expression protocols (15-18°C) generally improve solubility of TPR-containing proteins

  • Co-expression with molecular chaperones (GroEL/GroES) may enhance proper folding

• Eukaryotic expression systems:

  • Insect cell (Sf9, Hi5) expression using baculovirus vectors is preferable for functional studies requiring post-translational modifications

  • Mammalian cell expression (HEK293T) is optimal for interaction studies as demonstrated in previous research

• Recommended tags and purification strategies:

  • N-terminal tags (His6, FLAG, or Strep) are preferable as C-terminal modifications may interfere with TPR domain function

  • Tandem affinity tags (Strep/FLAG) have been successfully used for interaction proteomics studies

  • Size exclusion chromatography is essential as a final purification step to isolate properly folded TTC30A

When expressing recombinant zebrafish TTC30A, researchers should verify protein functionality through in vitro binding assays with known interacting partners such as IFT57 or other IFT-B components.

What CRISPR/Cas9 strategies are most effective for generating TTC30A knockouts in zebrafish?

Based on experiences with TTC30A/B knockouts in mammalian cells, effective CRISPR/Cas9 strategies for zebrafish should include:

• Guide RNA design considerations:

  • Target early exons to ensure complete loss of function

  • Design multiple sgRNAs to increase editing efficiency

  • Carefully evaluate potential off-targets, particularly considering the paralog ttc30b

  • Use tools like CCTop software to evaluate sgRNA efficiency and specificity

• Knockout validation methods:

  • Sequence verification of genomic edits using Sanger sequencing

  • Western blot confirmation of protein loss (note: antibodies may cross-react with ttc30b)

  • Immunofluorescence microscopy to evaluate TTC30 localization and abundance in ciliated tissues

  • Functional assessment of cilia formation using ARL13B as a ciliary marker

• Special considerations:

  • Generate both single ttc30a and double ttc30a/ttc30b knockouts to distinguish paralog-specific functions

  • Consider knockin strategies to introduce specific mutations (e.g., A375V) to study their effects in vivo

What are effective protocols for studying TTC30A protein interactions in zebrafish models?

For investigating TTC30A protein interactions in zebrafish:

• In vivo approaches:

  • CRISPR/Cas9-mediated endogenous tagging with FLAG or Strep tags allows physiological-level interaction studies

  • Proximity labeling techniques (BioID or TurboID fused to TTC30A) can identify the spatial interactome in vivo

  • Co-immunoprecipitation from zebrafish embryo lysates using validated antibodies or tagged proteins

• Affinity purification protocols:

  • Sample preparation: Serum starvation (16-24h) to induce ciliary assembly before lysate preparation

  • Affinity purification using anti-FLAG-M2-agarose beads or Strep-Tactin Superflow

  • Multiple washing steps followed by specific elution with Flag-peptide or Strep elution buffer

  • Quantitative mass spectrometry using label-free quantification approaches

• Data analysis considerations:

  • Use MaxQuant software for label-free quantification and Perseus software for statistical analysis

  • Apply Student's t-Test and Significance A statistical filters to identify high-confidence interactors

  • Compare wild-type vs. mutant TTC30A interactions to identify functionally relevant binding partners

This approach has successfully identified differential interactions between wild-type TTC30A and mutant variants (e.g., A375V) in previous studies, revealing IFT57 as a protein with significantly decreased interaction with the mutant form .

How does TTC30A regulate Sonic hedgehog signaling in vertebrate development?

TTC30A plays a specialized role in Sonic hedgehog (Shh) signaling through several mechanisms:

• Regulation of ciliary protein trafficking:

  • TTC30A facilitates the ciliary localization of Smoothened (Smo), a critical positive regulator of Shh signaling

  • Mutations in TTC30A (e.g., A375V) can inhibit Smo ciliary localization, suggesting TTC30A actively participates in Smo trafficking

• Interaction with PKA signaling:

  • TTC30A specifically interacts with protein kinase A catalytic subunit α (PKA-C)

  • PKA is a negative regulator of Shh signaling, and this interaction may modulate PKA activity or localization

  • TTC30A mutants show altered patterns of phosphorylated PKA substrates when treated with forskolin, suggesting disrupted PKA signaling activity

• Patched1-independent regulation:

  • TTC30A's effect on Smoothened localization appears to be independent of Patched1 (Ptch1)

  • This suggests TTC30A acts downstream of or parallel to Ptch1 in the Shh pathway

These findings indicate that beyond its structural role in IFT-B complex assembly, TTC30A has evolved specialized functions in regulating developmental signaling pathways. The paralog-specific nature of these interactions may explain why certain developmental defects persist even when the paralogous protein is present.

What is known about post-translational modifications of TTC30A and their functional significance?

While direct evidence of TTC30A post-translational modifications (PTMs) is limited in the search results, several observations suggest important regulatory modifications:

• Potential phosphorylation:

  • The interaction between TTC30A and PKA catalytic subunit suggests TTC30A may be a phosphorylation target

  • Differential phosphorylation could regulate TTC30A's interaction with other IFT-B components or signaling molecules

• Role in regulating tubulin modifications:

  • TTC30A appears critical for tubulin polyglutamylation in cilia

  • Single knockout of either TTC30A or TTC30B leads to reduced GT335 staining (a marker for polyglutamylated tubulin)

  • The average intensity of polyglutamylated tubulin drops to approximately 62.58% in TTC30A KO cells compared to controls

• Structural implications of modifications:

  • PTMs likely regulate the binding affinity and specificity of the TPR domains

  • Modifications may induce conformational changes that expose or mask binding interfaces

For researchers investigating TTC30A PTMs in zebrafish, phosphoproteomic analysis comparing wild-type and mutant contexts would be valuable for identifying regulatory modifications. Additionally, site-directed mutagenesis of potential modification sites could help establish their functional significance in ciliogenesis and signaling.

How do TTC30A mutations affect IFT-B complex integrity and function?

Research on TTC30A mutations reveals nuanced effects on IFT-B complex formation and function:

What are the current gaps in understanding TTC30A function in zebrafish development?

Several important knowledge gaps remain in understanding zebrafish ttc30a function:

• Tissue-specific requirements:

  • The differential requirements for ttc30a versus ttc30b in specific zebrafish tissues remain undefined

  • Whether compensation mechanisms differ across tissues is unknown

• Developmental timing:

  • The temporal requirements for ttc30a during different developmental stages need clarification

  • Whether maternal contribution of ttc30a mRNA masks early developmental phenotypes should be investigated

• Signaling specificity:

  • While TTC30A has been implicated in Shh signaling in mammalian cells , its role in other zebrafish ciliary signaling pathways (e.g., Wnt, PDGF, Notch) remains unexplored

  • The interplay between ttc30a and other zebrafish-specific developmental pathways requires investigation

• Paralog evolution:

  • The evolutionary forces driving maintenance of both ttc30a and ttc30b in zebrafish genome need examination

  • Whether subfunctionalization has occurred between these paralogs in teleost fishes compared to other vertebrates is unknown

Addressing these gaps would provide valuable insights into both zebrafish development and the broader evolutionary context of cilia-mediated development across vertebrates.

What methodological challenges exist in studying recombinant TTC30A function?

Researchers face several methodological challenges when working with recombinant TTC30A:

• Protein solubility and stability:

  • TPR-containing proteins often face folding challenges in heterologous expression systems

  • The proper folding of the TPR domain is critical for maintaining functional interactions

• Paralog discrimination:

  • High sequence similarity between TTC30A and TTC30B makes specific detection challenging

  • Currently available antibodies may not distinguish between paralogs

  • Development of paralog-specific reagents is crucial for advanced functional studies

• Functional assay limitations:

  • In vitro reconstitution of IFT-B complex assembly with recombinant components remains technically challenging

  • Current assays may not fully recapitulate the dynamics of IFT in the cellular environment

• Interaction characterization:

  • Distinguishing direct from indirect interactions in complex protein assemblies requires sophisticated approaches

  • Many TTC30A interactions may be context-dependent and missed in standard interaction assays

To address these challenges, researchers should consider:

• Combined approaches using both in vitro and cellular systems

• Development of zebrafish-specific reagents and assays

• Application of emerging structural biology techniques (cryo-EM, HDX-MS) to characterize the TTC30A interaction interface

How might emerging technologies advance TTC30A research in zebrafish models?

Several emerging technologies offer promising avenues for advancing ttc30a research:

• Advanced genome editing approaches:

  • Base editing and prime editing technologies enable precise introduction of specific mutations without double-strand breaks

  • CRISPR activation/interference (CRISPRa/CRISPRi) systems allow temporal control of ttc30a expression

  • These approaches could help distinguish between developmental versus homeostatic requirements for ttc30a

• Live imaging innovations:

  • Advanced light sheet microscopy techniques enable long-term imaging of ciliary dynamics in developing zebrafish

  • Fluorescent tagging of endogenous ttc30a using split-GFP approaches can visualize native protein localization

  • These methods could reveal real-time trafficking and interaction dynamics

• Single-cell technologies:

  • Single-cell RNA-seq and ATAC-seq can identify cell type-specific responses to ttc30a deficiency

  • Spatial transcriptomics could map ttc30a expression patterns with unprecedented resolution

  • These approaches would help decipher the tissue-specific roles of ttc30a versus ttc30b

• Proteomics advances:

  • Proximity labeling techniques (TurboID, APEX) can map the spatial interactome of TTC30A

  • Cross-linking mass spectrometry can identify direct binding interfaces

  • These methods would provide deeper insights into TTC30A's role in protein complex assembly

Integration of these technologies will enable researchers to build comprehensive models of TTC30A function in zebrafish development and disease.