Recombinant Danio rerio Centrosomal protein of 19 kDa (cep19)

What is the molecular structure and function of CEP19 in Danio rerio?

CEP19 is a 19 kDa centrosomal protein that localizes to the distal end of the mother centriole. In vertebrates, it forms a functional module with FOP and CEP350 to drive the early steps of ciliogenesis . The C-terminus of CEP19 is crucial for both its localization to centrioles and its function in ciliogenesis, as this region mediates the interaction between CEP19 and its binding partners FOP/CEP350 .
While specific structural data for Danio rerio CEP19 is limited, the protein likely shares considerable homology with human CEP19 given the conservation of centrosomal proteins across vertebrate species. Functional studies suggest that CEP19 plays a critical role in the docking of ciliary vesicles to the distal end of mother centrioles, a crucial step in cilia formation .

How does the expression pattern of cep19 vary during zebrafish development?

Methodological answer: To determine cep19 expression patterns during zebrafish development, researchers typically employ:

• Quantitative RT-PCR at different developmental stages (0-72 hpf)

• Whole-mount in situ hybridization to visualize spatial expression

• Immunofluorescence with anti-CEP19 antibodies
  While the search results don't provide specific data on zebrafish cep19 expression patterns, researchers should examine expression in ciliated tissues such as pronephric ducts, neural tube, and sensory organs like the otic vesicle, which typically express centrosomal proteins involved in ciliogenesis.

What expression systems are optimal for producing recombinant Danio rerio CEP19?

For efficient production of recombinant Danio rerio CEP19, bacterial expression systems using E. coli are commonly employed for initial characterization studies. Based on established protocols for recombinant protein expression in zebrafish, the following methodological approach is recommended:

• Clone the cep19 coding sequence into an in vivo/in vitro compatible vector such as pIVEX (similar to approaches used for other Danio rerio proteins)

• Transform into an E. coli expression strain (BL21(DE3) or Rosetta)

• Induce expression with IPTG (0.5-1 mM) at lower temperatures (16-18°C) to enhance proper folding

• Include a fusion tag (His6, GST, or MBP) to facilitate purification and potentially improve solubility
  For functional studies requiring post-translational modifications, consider:

• Baculovirus expression systems in insect cells

• Mammalian expression systems (HEK293T, CHO cells)

What are the key challenges in purifying functional recombinant CEP19?

Purification of functional recombinant CEP19 presents several challenges that researchers should address:

• Solubility issues: CEP19 may form inclusion bodies in bacterial systems. To overcome this:

  • Use solubility-enhancing fusion partners (MBP, SUMO)

  • Optimize induction conditions (lower temperature, reduced IPTG concentration)

  • Employ specialized E. coli strains designed for difficult-to-express proteins

• Maintaining protein-protein interaction capabilities: Since CEP19 functions through interactions with partners like FOP and CEP350 , preserving the native conformation is crucial:

  • Include stabilizing agents in purification buffers (glycerol 5-10%, reducing agents)

  • Consider mild detergents if membrane-association is suspected

  • Validate functionality through binding assays with known partners

• Preserving the critical C-terminal domain: The C-terminus of CEP19 is essential for its localization and function . Ensure that:

  • C-terminal tags don't interfere with function

  • Proteolytic degradation is minimized during purification

  • The integrity of the C-terminus is verified by mass spectrometry

How can researchers assess CEP19's role in ciliogenesis in zebrafish models?

Methodological approach for investigating CEP19's role in ciliogenesis:

• Generate CEP19-deficient zebrafish models:

  • CRISPR/Cas9 knockout targeting the cep19 gene

  • Morpholino-based knockdown for transient loss of function

  • Rescue experiments using wild-type and mutant CEP19 constructs

• Analyze cilia formation and function:

  • Immunofluorescence microscopy using antibodies against:

    • Acetylated α-tubulin (ciliary axoneme marker)

    • γ-tubulin (basal body marker)

    • CEP19, FOP, and CEP350 to assess recruitment hierarchy

  • Transmission electron microscopy to examine:

    • Ciliary vesicle docking to mother centrioles

    • Basal body ultrastructure

    • Ciliary transition zone formation

• Assess physiological impact:

  • Examine left-right asymmetry defects (heart looping)

  • Evaluate kidney function (pronephric cysts)

  • Test for sensory deficits (otic vesicle development, lateral line formation)
    These approaches parallel methods used in studies of CEP19 knockout cells, where defects in ciliary vesicle docking to the distal end of mother centrioles were observed .

What methods can be used to study the CEP19-FOP-CEP350 interaction network in zebrafish cells?

To investigate the CEP19-FOP-CEP350 interaction network in zebrafish cells, researchers should employ multiple complementary approaches:

• Biochemical interaction studies:

  • Co-immunoprecipitation of endogenous proteins

  • GST pulldown assays using recombinant proteins

  • Yeast two-hybrid screening to identify additional interactors

  • Proximity labeling methods (BioID, APEX) to identify the broader interactome

• Advanced microscopy techniques:

  • Three-dimensional structured-illumination microscopy (3D-SIM) to map the relative spatial distribution of CEP19, FOP, and CEP350 at the distal end of the mother centriole

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

  • Live-cell imaging with fluorescently tagged proteins to track recruitment dynamics

• Functional dissection:

  • Sequential knockdown/knockout experiments to establish the recruitment hierarchy

  • Expression of deletion mutants to identify critical interaction domains

  • Point mutations in key residues to disrupt specific interactions
    Studies in other systems have shown that CEP350/FOP act upstream of CEP19 in their recruitment hierarchy, and the C-terminus of CEP19 mediates the interaction between CEP19 and FOP/CEP350 .

How can Danio rerio CEP19 models contribute to understanding obesity mechanisms?

Zebrafish models of CEP19 deficiency can provide valuable insights into obesity mechanisms based on the association between CEP19 mutations and morbid obesity in humans :

• Generation of disease-relevant models:

  • CRISPR/Cas9 knock-in of the human R82* mutation associated with obesity

  • Analyze phenotypic consequences throughout development and in adult fish

• Metabolic assessments:

  • Quantify adipose tissue accumulation using:

    • Oil Red O staining

    • Fluorescent lipid reporters

    • Micro-CT imaging for 3D visualization

  • Measure metabolic parameters:

    • Glucose tolerance tests

    • Lipid profiles (triglycerides, cholesterol)

    • Oxygen consumption and energy expenditure

• Molecular pathway analysis:

  • RNA-seq to identify dysregulated metabolic pathways

  • Phosphoproteomics to detect altered signaling cascades

  • Investigate ciliary signaling pathways (Hedgehog, Wnt) that may link ciliary dysfunction to metabolic disorders
    This approach leverages the benefits of zebrafish models (rapid development, optical transparency, genetic tractability) while addressing the clinical features observed in patients with CEP19 deficiency, including decreased HDL cholesterol, hypercholesterolemia, and hypertriglyceridemia .

What is the relationship between CEP19 mutations and ciliopathies in vertebrate models?

The relationship between CEP19 mutations and ciliopathies can be explored through comparative analysis across vertebrate models, including zebrafish:

• Phenotypic spectrum analysis:

  • Compare phenotypes of CEP19-deficient zebrafish with known ciliopathy models

  • Assess specific tissues where primary cilia are critical:

    • Neural tube (Hedgehog signaling)

    • Kidney (fluid flow sensing)

    • Eye (photoreceptor development)

    • Brain (neuronal migration)

• Mechanistic investigations:

  • Examine mother centriole ultrastructure by electron microscopy

  • Analyze ciliary vesicle docking defects, which are observed in CEP19 knockout cells

  • Investigate the functional relationships between CEP19 and other ciliopathy-associated proteins

• Rescue experiments:

  • Test whether human CEP19 can rescue zebrafish cep19 deficiency

  • Introduce known human mutations (e.g., R82*) and assess their ability to complement loss of function

  • Evaluate whether overexpression of interacting partners (FOP, CEP350) can compensate for CEP19 deficiency
    This research is particularly relevant given that ciliopathies represent a growing spectrum of disorders, and CEP19's role in ciliary vesicle docking positions it as a potential contributor to this disease category .

How does post-translational modification affect CEP19 function and localization?

While specific data on post-translational modifications (PTMs) of zebrafish CEP19 is limited, researchers should consider the following methodological approaches to study PTMs:

• Identification of PTMs:

  • Mass spectrometry analysis of immunoprecipitated endogenous CEP19

  • Site-directed mutagenesis of predicted modification sites

  • Phospho-specific or ubiquitin-specific antibodies

• Cell cycle regulation:

  • Synchronize cells using methods such as double thymidine block or RO-3306 treatment

  • Examine changes in CEP19 PTMs throughout the cell cycle, particularly during the G2/M transition when centrosome maturation occurs

• Functional consequences:

  • Generate phosphomimetic and phospho-dead mutants

  • Assess impact on:

    • Centrosomal localization

    • Interaction with FOP and CEP350

    • Ciliary vesicle docking
      Researchers should note that other centrosomal proteins undergo significant regulation via PTMs during the cell cycle, particularly during centrosome maturation in early mitosis .

What roles does CEP19 mRNA localization play in centrosomal protein delivery?

Based on studies of other centrosomal proteins like pericentrin (PCNT), researchers should investigate whether CEP19 mRNA undergoes spatial localization:

• mRNA localization analysis:

  • Fluorescence in situ hybridization (FISH) to visualize cep19 mRNA distribution

  • Live-cell imaging with MS2-tagged cep19 mRNA

  • Cell cycle-dependent analysis, as some centrosomal mRNAs show enrichment during specific cell cycle phases

• Translation-dependent localization mechanisms:

  • Inhibition of translation with puromycin and cycloheximide to test if cep19 mRNA localization requires active translation

  • Microtubule depolymerization with nocodazole to test cytoskeletal dependence

  • Dynein inhibition with ciliobrevin D to assess motor protein requirements

• Co-translational targeting hypothesis testing:

  • Polysome profiling to detect actively translating cep19 mRNA

  • Proximity-specific ribosome profiling near centrosomes

  • Pulse-chase experiments to track newly synthesized CEP19 protein
    This investigation would parallel findings for pericentrin, where mRNA is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, facilitating efficient protein incorporation during centrosome maturation .

How conserved is CEP19 structure and function across vertebrate species?

To assess the evolutionary conservation of CEP19 across vertebrates, researchers should employ:

• Sequence analysis methods:

  • Multiple sequence alignment of CEP19 orthologs from diverse vertebrates

  • Phylogenetic tree construction to visualize evolutionary relationships

  • Conservation mapping onto predicted structural domains

• Functional conservation testing:

  • Cross-species complementation assays (can human CEP19 rescue zebrafish phenotypes?)

  • Domain swapping experiments between zebrafish and human CEP19

  • Identification of conserved interaction interfaces with FOP and CEP350

• Structural comparison:

  • Homology modeling of Danio rerio CEP19 based on structural data from other species

  • Comparison of predicted C2 domains, similar to those found in other centrosomal proteins

  • Analysis of conserved regions that might be critical for ciliary vesicle docking
    This comparative approach would provide insights into which domains of CEP19 are under evolutionary constraint, potentially highlighting functionally critical regions beyond the known C-terminal domain that mediates interactions with FOP and CEP350 .

What are the divergent features of Danio rerio CEP19 compared to mammalian orthologs?

A comprehensive analysis of divergent features between zebrafish and mammalian CEP19 would include: