Recombinant Danio rerio Centrosomal protein of 63 kDa (cep63), partial

What is the fundamental role of Cep63 in centrosome biology?

Cep63 is a critical centrosomal protein that ensures reliable centriole duplication in dividing mammalian cells . It functions as part of a protein complex that includes other centrosomal proteins such as Cep152, WDR62, and CDK5RAP2 . This complex is essential for proper centrosome function and centriole duplication during cell division. Loss of Cep63 function can lead to defects in these processes, potentially affecting cell cycle progression and cellular development.

The protein primarily localizes to the centrosome, where it helps recruit and organize other centrosomal components. Specifically, Cep63 forms a heterotetrameric complex with Cep152, which self-assembles into a higher-order cylindrical architecture around centrioles . This assembly is crucial for the structural integrity of the centrosome and its ability to function as the main microtubule-organizing center in animal cells.

How does the genetic structure of Cep63 in Danio rerio compare to its human ortholog?

The Cep63 gene in Danio rerio (zebrafish) shares significant homology with its human ortholog, though with species-specific differences. The human CEP63 gene is located on chromosome 3, specifically at the chromosomal band 3q22.1 . While the search results don't provide the exact chromosomal location for the zebrafish cep63 gene, the conservation of centrosomal proteins across vertebrates suggests similar genomic organization.

When working with recombinant Danio rerio Cep63, it's important to consider these species-specific differences in experimental design. The functional domains are generally conserved between species, with key regions involved in protein-protein interactions and centrosomal localization being preserved through evolution. This conservation makes zebrafish an excellent model organism for studying the function of Cep63 in vertebrate development and cellular biology.

What expression systems are optimal for producing functional recombinant Danio rerio Cep63?

Multiple expression systems can be used for producing recombinant Danio rerio Cep63, each with distinct advantages depending on your experimental needs. Based on the search results, common expression hosts include E. coli, yeast, baculovirus, and mammalian cell systems . The choice of expression system should be guided by your specific experimental requirements:

For most recombinant proteins in this category, a purity of greater than or equal to 85% as determined by SDS-PAGE is standardly achievable . This level of purity is generally sufficient for most research applications, including interaction studies and functional assays.

What are the methodological challenges in studying Cep63-Cep152 interactions using recombinant proteins?

Studying the interaction between Cep63 and Cep152 presents several methodological challenges that researchers should address:

• Phase separation properties: Cep63 and Cep152 cooperatively generate amorphous aggregates that undergo dynamic turnover and inter-aggregate fusion in vivo . These phase separation properties can complicate in vitro studies, as the proteins may behave differently depending on concentration, buffer conditions, and the presence of other cellular components.

• Structural considerations: The purified Cep63- Cep152 complex can form either cylindrical structures or vesicle-like hollow spheres in a spatially controlled manner . To properly study these structures, specialized techniques such as correlative light and electron microscopy (CLEM) or three-dimensional structured illumination microscopy (3D-SIM) may be required .

• Hydrophobic interactions: Two hydrophobic motifs, one each from Cep63 and Cep152, are required for generating phase-separating condensates and high molecular-weight assemblies . When designing interaction studies, these motifs must be preserved to maintain physiologically relevant behavior.

• Effects of 1,6-hexanediol: This liquid-liquid phase separation disruptor has been shown to greatly diminish the ability of endogenous Cep63 and Cep152 to localize to centrosomes . Including controls with such disruptors can help validate observed interactions.

To overcome these challenges, a combination of approaches is recommended, including fluorescence recovery after photobleaching (FRAP) to assess protein dynamics, and careful attention to buffer conditions that may affect phase separation behavior.

How can CRISPR-Cas9 gene editing be optimized for studying Cep63 function in Danio rerio?

CRISPR-Cas9 gene editing offers powerful approaches for studying Cep63 function in zebrafish. When designing a CRISPR strategy for Danio rerio cep63, consider the following methodological guidelines:

• Guide RNA design: Target conserved functional domains identified through comparison with mammalian Cep63, particularly regions known to interact with Cep152 or other centrosomal proteins. Avoid regions with high sequence similarity to other genes to minimize off-target effects.

• Knockout validation strategies: Use multiple approaches to validate knockout efficiency:

  • RT-PCR with primers located within different cep63 exons, similar to the approach used in mouse studies

  • Western blotting with specific antibodies against Cep63

  • Immunoprecipitation followed by Western blotting to detect the presence/absence of the protein

• Phenotypic analysis: Focus on centrosome-related phenotypes:

  • Examine centriole duplication using centrosome markers

  • Assess mitotic spindle formation

  • Evaluate embryonic development with particular attention to processes requiring proper centrosome function

• Rescue experiments: Perform rescue experiments with wild-type or mutant versions of recombinant Cep63 to confirm specificity of observed phenotypes and identify functional domains. Consider using constructs similar to those described in previous studies, such as GFP-Cep63 or truncated versions targeting specific domains .

What is the current understanding of the Cep63-Cep152-WDR62-CDK5RAP2 complex in centrosome function?

The Cep63-Cep152-WDR62-CDK5RAP2 complex represents a critical functional unit within the centrosome. Current research indicates that these proteins form an interconnected network essential for centrosome function:

• Complex formation: Co-immunoprecipitation studies have confirmed that CDK5RAP2, CEP152, WDR62, and CEP63 interact with each other, but not with unrelated centriole components like CP110 . Mass spectrometry has further validated these interactions .

• Functional interdependence: Knockdown experiments have revealed:

  • Depletion of CEP152 or CEP63 disrupts centriole duplication

  • Similarly, knockdown of CDK5RAP2 or WDR62 inhibits centriole duplication

  • CEP63 depletion using certain siRNAs (CEP63 NB) can reduce the centrosomal localization of CDK5RAP2, CEP152, and WDR62

  • The same siRNA treatment also reduced the protein levels of these complex partners

• Hierarchical relationships: Analysis of protein stability and localization after depletion of complex components has shown:

  • CDK5RAP2 depletion reduces centrosomal CEP152 without affecting protein stability

  • Contrary to some previous findings, CEP152 levels at the centrosome may not always decrease in CEP63-depleted cells

  • Different siRNAs targeting CEP63 can have varying effects on other complex components

This complex appears to function as a molecular scaffold that promotes CDK2 centrosomal localization and centriole duplication, making it critical for human neurodevelopment .

How does phase separation contribute to Cep63-Cep152 function, and how can this be investigated experimentally?

Phase separation is emerging as a key mechanism by which Cep63 and Cep152 achieve their functional organization at the centrosome. To investigate this phenomenon:

• Characteristics of phase separation: The Cep63-Cep152 complex exhibits several hallmarks of liquid-like assemblies:

  • They cooperatively generate amorphous aggregates

  • These aggregates undergo dynamic turnover

  • They can participate in inter-aggregate fusion in vivo

  • They demonstrate internal rearrangement within condensates in vitro

• Experimental approaches to study phase separation:

  • Disruption with 1,6-hexanediol: This compound, known to disrupt liquid-liquid phase separation, significantly diminishes the localization of endogenous Cep63 and Cep152 to centrosomes .

  • Fluorescence Recovery After Photobleaching (FRAP): Use this technique to measure the dynamic exchange of proteins within condensates, which is characteristic of liquid-like behavior.

  • In vitro reconstitution: Purified Cep63- Cep152 complex can form different structures (cylindrical structures or vesicle-like hollow spheres) depending on spatial constraints .

  • Macromolecular crowding assays: The complex forms condensate-like solid spheres in the presence of a macromolecular crowder .

• Molecular determinants of phase separation:

  • Two hydrophobic motifs, one each from Cep63 and Cep152, are required for generating phase-separating condensates .

  • These same motifs are necessary for high molecular-weight assembly formation .

• Proposed mechanism: The current model suggests that self-assembly of the Cep63- Cep152 complex is triggered by an intrinsic property of the complex to undergo density transition through hydrophobic-motif-mediated phase separation . This provides a mechanism for how these proteins can reach threshold concentrations in the vast intracellular space and generate self-assembled architectures.

What is the connection between Cep63 mutations and microcephaly in vertebrates?

Cep63 has been implicated in microcephaly, a condition characterized by reduced brain size. The search results indicate a connection between Cep63 and a condition called SCKL6 (likely referring to Seckel syndrome type 6), as mentioned in the LOVD gene database . This connection can be explained through several mechanisms:

• Centrosomal role in neurodevelopment: Cep63, along with other centrosomal proteins like CEP152, CDK5RAP2, and WDR62, forms a complex that is critical for human neurodevelopment by promoting CDK2 centrosomal localization and centriole duplication .

• Impact on neural progenitor proliferation: Proper centrosome function is essential for neural progenitor cell division during brain development. Disruption of Cep63 can lead to defects in this process, potentially reducing the neuron population and resulting in microcephaly.

• Animal models: Research using gene-trap approaches in mice has demonstrated that homozygous disruption of Cep63 results in loss of Cep63 mRNA and protein . These models can provide insights into the developmental consequences of Cep63 deficiency.

• Molecular mechanism: Mutations in Cep63 likely disrupt its interaction with Cep152 and other complex partners, affecting centrosome function and cell division particularly in neural progenitors. Given the phase separation properties of Cep63-Cep152 interactions , mutations might also disrupt the biophysical properties necessary for proper complex formation.

How does Cep63 function differ across developmental stages in Danio rerio?

While the search results don't provide specific information about Cep63 function across developmental stages in zebrafish, we can infer potential developmental roles based on what is known about centrosomal proteins in vertebrate development:

• Early embryonic development: During early cleavage divisions in zebrafish embryos, centrosomes are critical for the rapid cell divisions that occur. Cep63 likely plays an essential role in ensuring proper centriole duplication during these stages.

• Neurogenesis: Given the known connection between Cep63 and microcephaly in mammals, Cep63 likely has important functions during zebrafish brain development, particularly in the proliferation and differentiation of neural progenitors.

• Methodological approaches to study developmental roles:

  • Temporal gene knockdown using morpholinos at different developmental stages

  • Heat-shock inducible transgenic lines expressing dominant-negative Cep63 variants

  • Time-lapse imaging of fluorescently tagged Cep63 to track its dynamics during specific developmental processes

• Tissue-specific requirements: Investigation of tissue-specific functions could be performed using conditional knockout strategies or tissue-specific promoters to drive expression of mutant forms of Cep63.

What in vitro assays can best assess the functional activity of recombinant Danio rerio Cep63?

Several in vitro assays can be employed to evaluate the functional activity of recombinant Danio rerio Cep63:

• Protein-protein interaction assays:

  • Co-immunoprecipitation: Use purified recombinant Cep63 with potential binding partners like Cep152, following protocols similar to those described in the literature .

  • Pull-down assays: Employ tagged versions of Cep63 (such as GFP-Cep63) to pull down interacting proteins from cell lysates .

  • Yeast two-hybrid screening: Identify novel interaction partners or confirm known interactions using the Cep63 coding sequence as bait.

• Phase separation assessment:

  • In vitro condensate formation: Mix purified Cep63 and Cep152 under various buffer conditions to observe condensate formation .

  • FRAP analysis: Measure the dynamics of fluorescently labeled Cep63 within reconstituted condensates .

  • 1,6-hexanediol disruption assays: Test the sensitivity of formed condensates to this phase separation disruptor .

• Structural studies:

  • Electron microscopy: Visualize the structures formed by purified Cep63-Cep152 complexes (cylindrical structures or vesicle-like hollow spheres) .

  • 3D-SIM microscopy: Analyze the detailed organization of Cep63 within reconstituted centrosomal structures .

• Functional centrosome assays:

  • In vitro centrosome assembly: Test the ability of recombinant Cep63 to promote centrosome assembly in cell-free systems.

  • Microtubule nucleation assays: Assess the impact of Cep63 on centrosomal microtubule nucleation capacity.

How can researchers effectively troubleshoot expression and purification issues with recombinant Cep63?

Expressing and purifying recombinant Cep63 can present several challenges. Here are methodological approaches to troubleshoot common issues:

• Low expression levels:

  • Optimize codon usage for the expression host

  • Test different promoter strengths

  • Vary induction conditions (temperature, inducer concentration, duration)

  • Consider using fusion tags that enhance solubility (e.g., MBP, SUMO)

  • Try different expression hosts (E. coli, yeast, baculovirus, or mammalian cells)

• Protein insolubility:

  • Express truncated versions of the protein focusing on soluble domains

  • Test expression at lower temperatures (e.g., 16-18°C)

  • Include solubilizing agents in lysis buffers

  • Consider expressing with chaperones

  • Use a denaturing/refolding protocol if necessary

• Protein degradation:

  • Include protease inhibitors during purification

  • Reduce purification time

  • Maintain samples at low temperature

  • Test different buffer compositions for stability

  • Consider protein stabilizing additives

• Purification challenges:

  • For Cep63, which forms complexes with proteins like Cep152, consider co-expression and co-purification approaches

  • Given the phase separation properties of Cep63-Cep152 , be aware that buffer conditions may significantly impact complex formation

  • Validate protein identity using mass spectrometry

  • Assess protein quality using dynamic light scattering to detect aggregation

• Validation of functionality:

  • Verify protein activity through interaction studies with known partners

  • Conduct centrosome localization studies using fluorescently tagged proteins

  • Perform complementation assays in cells depleted of endogenous Cep63

How do the functional properties of Danio rerio Cep63 compare to mammalian orthologs?

Comparative analysis of Cep63 across species provides insights into conserved functions and species-specific adaptations:

• Conserved interactions:

  • The interaction between Cep63 and Cep152 appears to be conserved across vertebrates, suggesting a fundamental role in centrosome biology .

  • The complex formation with CDK5RAP2 and WDR62 observed in mammalian cells likely occurs in zebrafish as well, though specific validation would be required.

• Functional conservation:

  • Cep63's role in ensuring centriole duplication has been established in mammalian cells and is likely conserved in zebrafish.

  • The phase separation properties of the Cep63-Cep152 complex described in mammalian systems may represent an evolutionarily conserved mechanism for organizing centrosomal components.

• Experimental approaches for comparative studies:

  • Cross-species complementation assays (e.g., expressing zebrafish Cep63 in mammalian cells depleted of endogenous Cep63)

  • Comparative structural analysis of the protein domains

  • Analysis of interaction networks across species

  • Assessment of functional conservation in developmental contexts

• Methodological considerations:

  • When using zebrafish Cep63 as a model for human Cep63 function, consider potential differences in post-translational modifications

  • Account for possible species-specific interaction partners

  • Be aware of potential differences in expression patterns during development

What genetic manipulation techniques are most effective for studying Cep63 function in Danio rerio models?

Several genetic manipulation techniques can be employed to study Cep63 function in zebrafish:

• CRISPR-Cas9 genome editing:

  • Generate knockouts by targeting early exons to create frameshift mutations

  • Create precise point mutations to study specific functional domains

  • Develop knock-in reporter lines to visualize Cep63 expression and localization

  • Implement conditional knockout strategies using inducible promoters

• Morpholino-mediated knockdown:

  • For transient loss-of-function studies, especially during early development

  • Target splice junctions to disrupt proper mRNA processing

  • Combine with rescue experiments using recombinant protein to validate specificity

• Transgenic approaches:

  • Develop transgenic lines expressing fluorescently tagged Cep63 for in vivo imaging

  • Create lines expressing dominant-negative forms under tissue-specific or inducible promoters

  • Generate fish expressing truncated versions to analyze domain-specific functions

  • For rescue experiments, use constructs similar to those described in mouse studies, such as GFP-Cep63

• Gene-trap approaches:

  • Similar to the approach used in mouse studies , gene-trap insertions can provide valuable models

  • Verify absence of Cep63, as done in mouse models using RT-PCR and Western blotting

• Methodological validation:

  • For any genetic manipulation approach, validate the impact on Cep63 expression using a combination of RT-PCR, Western blotting, and immunofluorescence

  • Analyze centrosome structure and function through immunofluorescence for centrosomal markers

  • Assess developmental phenotypes, particularly in tissues where centrosome function is critical