Recombinant Danio rerio TBCC domain-containing protein 1 (tbccd1), partial

What is the molecular function of TBCCD1 in Danio rerio?

TBCCD1 (TBCC domain-containing protein 1) in Danio rerio likely functions similarly to its homologs in other species as a centrosomal protein essential for centrosome positioning and microtubule organization. The protein is related to both TBCC (tubulin cofactor C), which acts as a GTPase activating protein for tubulin involved in the tubulin folding pathway, and the GTPase RP2 (retinitis pigmentosa 2) . In vertebrate cells, TBCCD1 localizes at both proximal and distal regions of the two centrioles, forming a complex structure spanning from subdistal appendages (SDA) to distal appendages (DA) and extending inside and outside the centriole lumen . This localization pattern suggests TBCCD1 plays important roles in centriole structure maintenance and function in zebrafish.

To study TBCCD1 function in zebrafish, researchers typically employ techniques such as:

• CRISPR/Cas9 gene editing to create tbccd1 mutant lines

• Morpholino-based knockdown for transient tbccd1 depletion

• Fluorescent protein tagging (e.g., GFP-TBCCD1) for localization studies

• Immunofluorescence microscopy to observe centrosome and cilia phenotypes

What protein interactions are important for understanding TBCCD1 function?

While specific interaction data for zebrafish TBCCD1 is limited, research on related proteins provides valuable insights. TBCCD1 likely interacts with proteins involved in centrosome function and microtubule organization. Based on the STRING database information for related proteins, potential functional partners may include:

Table 1: Predicted functional partners based on STRING database information for related tubulin chaperone proteins

For studying TBCCD1 interactions in zebrafish, researchers should consider:

• Co-immunoprecipitation (Co-IP) assays with tagged TBCCD1

• Proximity-dependent biotin identification (BioID) to identify proximity partners

• Yeast two-hybrid screening for direct interactors

• Fluorescence resonance energy transfer (FRET) for in vivo interaction confirmation

BioID screening in other systems has linked TBCCD1 to ciliopathy-associated protein networks, suggesting similar interactions may exist in zebrafish .

How does TBCCD1 contribute to centrosome and cilia function in zebrafish?

In zebrafish, as in other vertebrates, TBCCD1 likely plays essential roles in centrosome positioning and cilia formation. Based on studies in other systems, TBCCD1 depletion causes centrosome mispositioning, which affects Golgi integrity, cell migration, and primary cilia assembly . In vertebrate cells, TBCCD1 is required for the stability of the subdistal appendages' external module involved in microtubule anchoring to the centrosome .

For zebrafish-specific studies of TBCCD1's role in centrosome and cilia function, researchers should:

• Analyze cilia formation in sensory organs (lateral line, olfactory placodes)

• Examine pronephric duct development, where motile cilia are essential

• Monitor embryonic development processes dependent on proper centrosome function

• Study brain ventricle formation, which depends on functional ependymal cilia

Methodologically, this would involve:

• High-resolution microscopy of fixed embryos using antibodies against acetylated tubulin (cilia marker) and gamma-tubulin (centrosome marker)

• Live imaging of transgenic lines expressing fluorescent centrosome or cilia markers

• Transmission electron microscopy to examine ultrastructural defects in centrioles and cilia

How do TBCCD1 concentration levels affect centriole appendage maintenance and cilia formation in zebrafish?

Studies in human cells have shown that tightly regulated levels of TBCCD1 are critical for proper centrosome and cilia function. Both depletion and overexpression of TBCCD1 have distinct consequences on centriole appendages and cilia . In human cells, TBCCD1 depletion reduces levels of subdistal appendage proteins involved in microtubule anchoring (Centriolin/CEP110, Ninein, CEP170), while TBCCD1 overexpression decreases levels of the distal appendage protein CEP164 and increases CEP350 .

For zebrafish research, investigating concentration dependence would require:

• Generation of conditional expression systems:

  • Heat-shock inducible TBCCD1 expression

  • Tissue-specific TBCCD1 overexpression using Gal4/UAS system

  • Dose-dependent morpholino knockdown strategies

• Quantitative analysis:

  • Measure fluorescence intensity of appendage proteins at centrioles

  • Correlate TBCCD1 levels with cilia length and frequency

  • Assess microtubule organization at various TBCCD1 concentrations

• Functional read-outs:

  • Analyze ciliary signaling pathways (Hedgehog, PDGF, Wnt)

  • Examine left-right asymmetry establishment in embryos

  • Assess kidney function in pronephric ducts

A titratable expression system would be particularly valuable for defining the optimal TBCCD1 concentration range for proper centriole function in vivo.

What methodological approaches should be used to study the localization pattern of TBCCD1 at zebrafish centrioles?

Determining the precise localization of TBCCD1 at zebrafish centrioles requires advanced microscopy techniques. Based on human cell studies showing TBCCD1's complex localization pattern spanning both proximally and distally at centrioles , researchers studying zebrafish TBCCD1 should employ:

• Super-resolution microscopy approaches:

  • 3D-Structured Illumination Microscopy (SIM)

  • Stochastic Optical Reconstruction Microscopy (STORM)

  • Expansion microscopy combined with confocal imaging

• Multi-color co-localization with centriolar markers:

  • Proximal markers: C-NAP1, SAS-6

  • Distal appendage markers: CEP164

  • Subdistal appendage markers: ODF2, CEP128, Ninein, CEP170

• Sample preparation considerations:

  • Microtubule depolymerization with cold shock to reveal centriole-specific signals

  • Isolation of centrioles from zebrafish embryos or cultured cells

  • Careful fixation protocols to preserve centriole ultrastructure

• For in vivo dynamics:

  • Generation of stable transgenic lines expressing fluorescently tagged TBCCD1

  • Live imaging of developing embryos at key developmental stages

  • Correlative light and electron microscopy for ultrastructural context

Given that TBCCD1 forms a complex structure "spanning from SDA to DA and extending inside and outside the centriole lumen" , employing multiple complementary approaches is essential for a complete understanding of its localization in zebrafish.

How can researchers design experiments to determine if TBCCD1 functions differ between motile and primary cilia in zebrafish?

Zebrafish possess both primary (non-motile) and motile cilia in different tissues, making them an excellent model to study potential differential functions of TBCCD1. Studies in other organisms have shown that TBCCD1 affects both primary cilia (through centrosome positioning) and motile cilia (through basal body positioning) . To address potential differential functions:

• Tissue-specific manipulation strategies:

  • Generate conditional knockouts using tissue-specific Cre lines

  • Use cell-type-specific promoters for rescue experiments

  • Employ spatially restricted morpholino delivery

• Comparative analysis across cilia types:

  • Primary cilia: Examine Kupffer's vesicle, neural tube, and notochord

  • Motile cilia: Study pronephric ducts, nasal pit, and lateral line

  • Nodal cilia: Analyze Kupffer's vesicle during early development

• Functional assays:

  Cilia Type	Location	Assay Method	Readout
  Primary cilia	Neural tube	Hedgehog pathway activity	ptc1, gli1 expression
  Motile cilia	Pronephric ducts	High-speed videomicroscopy	Cilia beat frequency
  Nodal cilia	Kupffer's vesicle	Flow visualization	Directional flow patterns
  Sensory cilia	Hair cells	FM1-43 uptake	Mechanosensory function

  Table 2: Functional assays for different cilia types in zebrafish

• Correlative phenotype analysis:

  • Primary cilia defects: Body curvature, somite formation abnormalities

  • Motile cilia defects: Pronephric cysts, hydrocephalus, situs inversus

Researchers should also consider the temporal aspects, as TBCCD1 may function differently during initial ciliogenesis versus maintenance of established cilia.

What experimental approaches should be used to characterize the interplay between TBCCD1 and microtubule dynamics in zebrafish?

Studies in human cells have shown that TBCCD1 depletion affects microtubule anchoring to centrosomes and that microtubule stabilization with taxol can partially rescue centrosome positioning defects caused by TBCCD1 depletion . To investigate this relationship in zebrafish:

These approaches would help elucidate whether zebrafish TBCCD1 directly affects microtubule dynamics or indirectly influences microtubule organization through other centrosomal proteins.

How can researchers best assess the evolutionary conservation of TBCCD1 function between zebrafish and other model organisms?

TBCCD1 is evolutionarily conserved across eukaryotes, with studies showing functional importance in organisms ranging from unicellular eukaryotes to humans . To systematically analyze evolutionary conservation in zebrafish:

• Comparative genomic and proteomic approaches:

  • Multiple sequence alignment of TBCCD1 proteins across species

  • Domain structure analysis focusing on functional motifs

  • Phylogenetic tree construction to map evolutionary relationships

• Cross-species complementation experiments:

  • Expression of zebrafish TBCCD1 in TBCCD1-deficient human cells

  • Expression of human TBCCD1 in zebrafish tbccd1 mutants

  • Domain swapping between orthologues to identify functionally conserved regions

• Comparative phenotyping:

  Organism	TBCCD1 Depletion Phenotypes	Reference
  Human cells	Centrosome mispositioning, Golgi disorganization, defects in cell migration and primary cilia
  Chlamydomonas	Defects in centriole/flagella number, centriole positioning, spindle orientation
  Trypanosoma	Disorganization of bi-lobe structure, loss of connection between centriole and kinetoplast
  Ectocarpus	Increased cell size, modified Golgi architecture, disruption of MT network, abnormal nucleus positioning
  Paramecium	Defects in basal body positioning/anchoring and accessory structures assembly

  Table 3: Comparative phenotypes of TBCCD1 depletion across species

• Network conservation analysis:

  • Compare TBCCD1 interaction partners identified by BioID across species

  • Analyze conservation of ciliopathy-associated protein networks

  • Examine conservation of regulatory pathways controlling TBCCD1 expression and function

These approaches would help determine which aspects of TBCCD1 function are fundamentally conserved and which may have evolved specifically in the vertebrate lineage.

What are the most effective approaches for expressing and purifying recombinant Danio rerio TBCCD1 for functional studies?

For in vitro characterization of zebrafish TBCCD1, researchers need to optimize expression and purification strategies:

• Expression system selection:

  • Bacterial expression (E. coli): Suitable for structural studies but may lack proper folding and post-translational modifications

  • Insect cell expression (Sf9, High Five): Better for preserving protein structure and function

  • Mammalian cell expression (HEK293, CHO): Optimal for maintaining native protein conformation and modifications

  • Cell-free expression systems: Useful for proteins that are toxic to host cells

• Construct design considerations:

  • Full-length vs. partial constructs (domains of interest)

  • N- or C-terminal fusion tags (His, GST, MBP, SUMO)

  • Codon optimization for the expression system

  • Inclusion of protease cleavage sites for tag removal

• Purification strategy:

  • Multi-step purification combining affinity chromatography with size exclusion and/or ion exchange

  • On-column refolding protocols if necessary

  • Inclusion of phosphatase inhibitors to preserve native phosphorylation state

  • Buffer optimization to maintain protein stability

• Quality control assessments:

  • Circular dichroism to verify proper folding

  • Dynamic light scattering to assess homogeneity

  • Thermal shift assays to determine stability

  • Limited proteolysis to identify stable domains

For functional assays, researchers should consider whether post-translational modifications are critical for TBCCD1 function, as these may require expression in eukaryotic systems rather than bacterial systems.

How can researchers effectively screen for TBCCD1 mutations or variants in zebrafish models?

To develop and characterize zebrafish models with altered TBCCD1 function:

• CRISPR/Cas9 genome editing optimization:

  • Design of guide RNAs targeting conserved domains

  • Screening strategies for identifying mutations

  • Methods for establishing stable mutant lines

• Genotyping approaches:

  • High-resolution melting analysis for rapid screening

  • Restriction enzyme digestion-based genotyping if mutations alter restriction sites

  • Allele-specific PCR for known mutations

  • Next-generation sequencing for comprehensive mutation detection

• Phenotypic screening strategies:

  • In vivo imaging of cilia and centrosomes in developing embryos

  • Behavioral assays for ciliary function (e.g., swimming patterns)

  • Organ-specific assessments (kidney, brain ventricle, heart looping)

• Validation methods:

  • Rescue experiments with wild-type mRNA or transgenes

  • RNA-seq to identify downstream effects of mutation

  • Proteomic analysis to detect compensatory mechanisms

When establishing mutant lines, researchers should carefully control for genetic background effects and consider generating multiple alleles with different types of mutations (null, hypomorphic, domain-specific) to fully understand TBCCD1 function.

What imaging techniques are most appropriate for visualizing TBCCD1 dynamics during zebrafish development?

To capture the dynamic behavior of TBCCD1 during zebrafish development, researchers should employ:

• Advanced live imaging approaches:

  • Light sheet microscopy for whole-embryo imaging with reduced phototoxicity

  • Spinning disk confocal microscopy for high-speed subcellular dynamics

  • Two-photon microscopy for deeper tissue penetration

  • Super-resolution techniques (e.g., STED, PALM) for nanoscale details

• Fluorescent labeling strategies:

  • CRISPR knock-in of fluorescent tags at the endogenous tbccd1 locus

  • BAC transgenic approach to maintain native regulatory elements

  • Optogenetic tools to manipulate TBCCD1 function with light

  • Photoconvertible fluorescent proteins to track protein pools over time

• Sample preparation considerations:

  • Embedding techniques that minimize motion artifacts

  • Temperature control systems for stable development

  • Long-term imaging chambers with proper gas exchange

  • Immobilization strategies that don't affect development

• Analysis methods:

  • Automated tracking of centrosome and TBCCD1 movements

  • Quantification of protein dynamics (FRAP, FCS)

  • Correlation with cell cycle and developmental events

  • 3D reconstruction and rendering of complex structures

These approaches enable researchers to connect TBCCD1 dynamics with developmental processes such as cell division, migration, and differentiation in real time.

What experimental controls are essential when studying the effects of TBCCD1 depletion in zebrafish?

When investigating TBCCD1 function through depletion experiments, proper controls are critical:

• Essential genetic controls:

  • Multiple independent mutant alleles or morpholinos to confirm specificity

  • Rescue experiments with wild-type TBCCD1 to verify phenotype causality

  • Tissue-specific rescue to determine site of action

  • Heterozygote analysis to assess dosage sensitivity

• Off-target effect controls:

  • p53 morpholino co-injection to control for non-specific developmental delays

  • Use of validated CRISPR targets with minimal predicted off-targets

  • Whole-genome sequencing of mutant lines to identify unintended mutations

  • Comparison with published phenotypes of related pathway components

• Technical validation controls:

  • qPCR to confirm mRNA knockdown efficiency

  • Western blotting to verify protein depletion

  • Immunofluorescence to confirm loss of protein at expected locations

  • RT-PCR to check for alternative splicing induced by mutations

• Phenotypic analysis controls:

  • Blinded scoring of phenotypes to prevent observer bias

  • Standardized staging of embryos for consistent comparisons

  • Quantitative metrics rather than qualitative assessments

  • Appropriate statistical analysis with sufficient sample sizes

These controls ensure that observed phenotypes are specifically due to TBCCD1 loss rather than experimental artifacts or off-target effects.

How can TBCCD1 research in zebrafish contribute to our understanding of human ciliopathies?

Zebrafish TBCCD1 research offers valuable insights into human ciliopathies due to evolutionary conservation of centrosome and cilia biology:

• Disease modeling approaches:

  • Generation of zebrafish models carrying human ciliopathy-associated TBCCD1 variants

  • Phenotypic comparison between zebrafish models and human patient symptoms

  • Tissue-specific analysis focusing on organs affected in human ciliopathies (kidney, brain, retina)

• Pathway analysis methods:

  • Epistasis experiments with known ciliopathy genes

  • Transcriptomic profiling to identify dysregulated pathways

  • Chemical screening for suppressors of ciliopathy phenotypes

• Translational opportunities:

  • Drug discovery using phenotype-based screening in zebrafish tbccd1 mutants

  • Identification of genetic modifiers that could serve as therapeutic targets

  • Development of in vivo imaging biomarkers for monitoring disease progression

• Integration with human genetics:

  • Analysis of TBCCD1 variants identified in ciliopathy patients

  • Functional validation of variants of uncertain significance

  • Generation of allelic series to correlate genotype with phenotype severity

BioID screening has already linked TBCCD1 to ciliopathy-associated protein networks , suggesting that deeper investigation of TBCCD1 function in zebrafish will provide valuable insights into human ciliopathies.

What are the most promising directions for integrating multi-omics approaches in zebrafish TBCCD1 research?

To comprehensively understand TBCCD1 function, researchers should integrate multiple omics approaches:

• Multi-omics experimental design:

  • Transcriptomics: RNA-seq of tbccd1 mutants at different developmental stages

  • Proteomics: Mass spectrometry of TBCCD1-associated complexes

  • Phosphoproteomics: Analysis of phosphorylation changes in tbccd1 mutants

  • Interactomics: BioID or proximity labeling to identify interaction networks

• Integration strategies:

  • Correlation of transcriptome changes with proteome alterations

  • Mapping of phosphorylation sites to protein-protein interaction networks

  • Temporal analysis across developmental stages

  • Tissue-specific multi-omics to detect context-dependent functions

• Computational approaches:

  • Network analysis to identify key hubs and signaling pathways

  • Pathway enrichment analysis for functional interpretation

  • Machine learning to predict phenotypic outcomes from multi-omics data

  • Cross-species comparison of TBCCD1-associated networks

• Validation methods:

  • CRISPR screening of identified network components

  • Chemical perturbation of predicted pathways

  • In vivo imaging of predicted interaction partners

  • Biochemical validation of key interactions

These integrated approaches would provide a systems-level understanding of TBCCD1 function that goes beyond individual protein activities to encompass its role in broader cellular networks.

How should researchers approach the study of potential redundancy between TBCCD1 and related proteins in zebrafish?

Given that TBCCD1 is related to TBCC and RP2 , researchers should consider potential functional redundancy:

• Comprehensive analysis of the tbcc gene family:

  • Phylogenetic analysis of tbcc, tbccd1, and rp2 in zebrafish

  • Expression profiling across tissues and developmental stages

  • Subcellular localization comparison of family members

  • Structure-function comparison of protein domains

• Genetic interaction studies:

  • Generation of single, double, and triple mutants

  • Analysis of genetic enhancement or suppression effects

  • Tissue-specific knockout combinations

  • Rescue experiments with different family members

• Biochemical function comparison:

  • GTPase activating protein (GAP) activity assays

  • Tubulin binding and folding assays

  • Comparative interactome analysis

  • In vitro reconstitution of activities

• Evolutionary perspective:

  • Comparative analysis across species with different family member compositions

  • Assessment of subfunctionalization or neofunctionalization events

  • Correlation of gene duplication events with evolutionary innovations in centrosome structure