Recombinant Danio rerio Spindle assembly abnormal protein 6 homolog (sass6), partial

What is the structural organization of Sass6 protein in Danio rerio and how does it compare to other species?

Danio rerio Sass6 forms a dimer through its C-terminal coiled-coil domain (C-C) that can further homo-oligomerize through an N-terminal headgroup interaction (N-N) to form a ring structure. The interface between the headgroup and the C-C is similar to that seen in most other species including Chlamydomonas and Leishmania, suggesting that Danio rerio Sass6 assembles into a canonical flat ring structure rather than the spiral structure observed in C. elegans . Crystallographic studies of Sass6 from various species, including zebrafish, have revealed that the protein can form ninefold symmetric rings that serve as the basic building blocks of the cartwheel structure at the core of centrioles .

The structure of Danio rerio Sass6 N-CC dimer can be superimposed with Sass6 N-CC dimers from other species with average pairwise RMSD 1.87 ± 0.31 Å over 617 ± 47 backbone atom pairs, demonstrating high structural conservation . This structural conservation suggests evolutionary preservation of Sass6 function across species with canonical cartwheel structures.

How does Sass6 contribute to centriole assembly and what happens when this process is disrupted?

Sass6 is essential for establishing the ninefold symmetry of centrioles through its self-assembly properties. It forms the central hub and spokes of the cartwheel structure that initiates centriole assembly . In functional terms, Sass6 serves as a central scaffolding component ensuring the characteristic ninefold symmetry of centrioles .

What are the optimal expression systems for producing recombinant Danio rerio Sass6 protein?

Based on available research data, several expression systems have been utilized for Sass6 production, each with distinct advantages:

Yeast Expression System: The yeast protein expression system is considered the most economical and efficient eukaryotic system for Sass6 production. It allows for post-translational modifications such as glycosylation, acylation, and phosphorylation to ensure native protein conformation . This system integrates the advantages of mammalian cell expression systems while avoiding their disadvantages.

Comparative Expression System Performance:

Researchers should note that for recombinant Danio rerio Sass6 (AA 26-351) with His tag, the yeast expression system has produced protein with >90% purity suitable for ELISA applications and as raw material for downstream preparation of monoclonal antibodies .

What methodologies are most effective for studying Sass6 oligomerization and cartwheel formation?

Several complementary approaches have proven effective for investigating Sass6 oligomerization and cartwheel formation:

X-ray Crystallography: This technique has been instrumental in determining the high-resolution structure of Sass6 oligomers. For example, the crystal structure of Sass6 1–216-F143D mutant was solved to 2.9 Å, revealing detailed information about the dimerization and oligomerization interfaces . Crystallization of Sass6 fragments, particularly the N-terminal domain and portions of the coiled-coil region, has provided critical insights into how these proteins assemble into ring-like structures .

Cryo-Electron Microscopy (Cryo-EM): This technique has been valuable for visualizing larger Sass6 assemblies and cartwheels. Cryo-EM studies have shown that Sass6 rings can stack to form three-dimensional scaffolds with ~4-nm periodicity at their hub . This approach has been particularly useful for understanding how multiple Sass6 rings interact to create the cartwheel structure.

Circular Dichroism (CD) Spectroscopy: CD has been employed to monitor the thermal denaturation of Sass6 fragments, confirming their proper folding . This technique provides valuable information about the secondary structure and stability of Sass6 domains.

Size Exclusion Chromatography–Multiangle Light Scattering (SEC-MALS): This method has been used to determine the oligomeric state of Sass6 fragments in solution, confirming their dimeric nature . SEC-MALS is particularly useful for characterizing the homogeneity and molecular weight of protein complexes.

The most informative studies combine these techniques with functional assays in vivo, such as the analysis of mutant phenotypes in zebrafish or other model organisms .

How can researchers differentiate between the roles of Sass6 in centriole duplication versus cartwheel assembly?

Differentiating between Sass6's roles in centriole duplication versus cartwheel assembly requires sophisticated experimental approaches:

Mutation Analysis with Structure-Function Correlation: Researchers can introduce specific mutations that disrupt either the N-terminal headgroup interactions (N-N) or the C-terminal coiled-coil dimerization (C-C) of Sass6. Studies have shown that mutations disrupting the N-N interface (such as GFP-Sass6-F143D) prevent efficient cartwheel assembly but still allow the protein to localize to centrioles . By comparing the effects of these distinct mutations, researchers can separate the protein's roles in localization versus structural assembly.

Reconstitution Assays: In vitro reconstitution of cartwheel-like structures using purified Sass6 components allows researchers to directly observe the assembly process. For example, cryo-EM reconstitution assays have demonstrated that amino acid substitutions disrupting asymmetric association also impair Sass6 ring stacking . Such assays can isolate the structural role of Sass6 from its cellular interactions.

Comparative Analysis Across Species: By studying Sass6 function in organisms with different cartwheel architectures (such as Drosophila, Chlamydomonas, and C. elegans), researchers can identify conserved versus specialized roles. For instance, while C. elegans SAS-6 forms spiral structures rather than flat rings, other species including Danio rerio form canonical ninefold symmetric cartwheels , suggesting that the core mechanism of cartwheel assembly is conserved but can be modified through evolution.

What are the current challenges in studying the interaction between Sass6 and other centriolar proteins in zebrafish?

Research on Sass6 interactions faces several significant challenges:

Temporal and Spatial Regulation: Centriole duplication occurs in a highly regulated manner during the cell cycle. Capturing the dynamic interactions between Sass6 and other proteins at specific time points requires sophisticated live imaging techniques. Research has shown that in both zebrafish and human spermatocytes, homologous recombination and assembly of the synaptonemal complex (which involves Sass6) initiate predominantly near telomeres , adding another layer of spatial complexity to these studies.

Redundancy and Compensation: Studies have revealed that centriole assembly involves multiple proteins (Plk4, Sass6, Ana2/STIL, Sass4, Cep135/Bld10) that form a complex network of interactions . This redundancy means that perturbing a single protein may lead to compensatory mechanisms that mask the full phenotype. For example, mutations in Sass6 that prevent homo-oligomerization still allow for the assembly of some centrosome-like structures that can recruit other centriole and centrosome proteins .

Technical Limitations in Zebrafish Models: While zebrafish offer advantages for studying subtelomeric bias in meiotic events (which is less obvious in mice) , working with zebrafish proteins presents unique challenges. These include limited availability of zebrafish-specific antibodies and the need to develop specialized genetic tools for this model organism. Researchers have addressed this by developing both forward and reverse genetic approaches, and several meiotic mutant lines have been isolated .

How might small molecule inhibitors of Sass6 oligomerization be developed and applied in research?

Development of small molecule inhibitors targeting Sass6 oligomerization represents an exciting frontier in centriole biology research:

Proof-of-Principle Studies: Research has already demonstrated that oligomerization of Leishmania major SAS-6 can be inhibited by small molecules in vitro . These pioneering studies provide evidence that targeting Sass6 oligomerization with small molecules is feasible and could be extended to Danio rerio Sass6.

Structure-Based Drug Design Approach:

• Target identification: Focus on the N-terminal headgroup interaction (N-N) interface that mediates Sass6 oligomerization, as mutations in this region (e.g., F143D in Drosophila) are known to disrupt cartwheel formation .

• Virtual screening: Utilize the crystal structures of Sass6 N-terminal domains to perform in silico screening of compound libraries against the oligomerization interface.

• Validation assays: Employ biophysical techniques such as thermal shift assays, surface plasmon resonance, and SEC-MALS to confirm binding and inhibition of oligomerization.

• Functional verification: Test promising compounds in cellular assays to confirm their ability to disrupt centriole duplication without affecting other cellular processes.

Research Applications: Such inhibitors would serve as valuable tools for studying centriole biogenesis and function. They could be used to achieve temporal control over Sass6 function, allowing researchers to disrupt centriole assembly at specific stages of the cell cycle. This approach would complement genetic methods and provide insights into the dynamic aspects of cartwheel assembly.

What is the significance of Sass6 ring stacking in centriole polarity, and how can this be investigated?

Recent research has revealed important insights into Sass6 ring stacking and its implications for centriole polarity:

Structural Basis of Ring Stacking: Electron tomographic studies of cartwheels suggest that the scaffold formed by stacked Sass6 rings is polarized along the proximal-distal axis of centrioles . This polarity appears to be mediated by an asymmetric interaction between the coiled-coil domains of Sass6 from adjacent rings. Understanding this asymmetric interaction is crucial for explaining how proximal-distal polarity emerges in centrioles.

Investigation Approaches:

• High-Resolution Structural Studies: Crystal structures of the coiled-coil domains from various species (including fragments from Chlamydomonas reinhardtii, Danio rerio, and human Sass6) have provided insights into the asymmetric association that may impart polarity to the cartwheel . These studies can be extended to capture different states of the stacking interaction.

• Mutational Analysis: By introducing specific mutations that disrupt the asymmetric association between coiled-coil domains, researchers can test the importance of this interaction for ring stacking and centriole polarity. Cryo-EM reconstitution assays have already demonstrated that such mutations impair Sass6 ring stacking .

• Correlative Light and Electron Microscopy: This approach allows researchers to visualize the localization of specifically labeled Sass6 domains within the context of the entire centriole structure, providing insights into how polarity is established and maintained.

The significance of this research extends beyond basic centriole biology. Since centrioles serve as the foundation for cilia and flagella, understanding how their polarity is established may have implications for ciliopathies and other diseases associated with centriole dysfunction.

What are the critical parameters for successfully purifying functional recombinant Danio rerio Sass6 protein?

Purification of functional recombinant Danio rerio Sass6 requires careful attention to several critical parameters:

Expression Construct Design: When designing expression constructs, researchers should consider:

• Domain boundaries based on structural knowledge

• Inclusion of appropriate affinity tags (His tag is commonly used)

• Codon optimization for the expression host

Expression Conditions Optimization:

• For yeast expression systems, which have been successful for producing >90% pure Sass6 protein, careful optimization of induction conditions is essential

• Temperature, induction duration, and media composition significantly impact protein folding and yield

• For Danio rerio Sass6 (AA 26-351), yeast expression systems have proven effective

Purification Strategy:

• Initial capture using affinity chromatography (His tag purification)

• Further purification by ion exchange chromatography

• Final polishing by size exclusion chromatography to ensure homogeneity

Quality Control Assessments:

• Purity assessment by SDS-PAGE (target >90%)

• Proper folding verification by circular dichroism spectroscopy

• Oligomeric state determination by SEC-MALS

• Functionality testing through oligomerization assays

Storage Considerations:

• Lyophilization has been successful for preserving Sass6 protein preparations

• Buffer composition should be optimized to maintain protein stability

• Avoid repeated freeze-thaw cycles

How can researchers address species-specific differences when studying Sass6 across model organisms?

Researchers must consider several approaches when working with Sass6 across different model organisms:

Structural Alignment Analysis: Perform detailed sequence and structural alignments to identify conserved versus divergent regions. For instance, Danio rerio Sass6 N-CC dimer can be superimposed with Sass6 from other species with average pairwise RMSD 1.87 ± 0.31 Å over 617 ± 47 backbone atom pairs , indicating high structural conservation in these regions.

Complementation Studies: Determine whether Sass6 from one species can functionally replace that of another. This approach can identify truly conserved functions versus species-specific adaptations. For example, complementation studies have been used to confirm gene function in zebrafish mutants .

Domain Swapping Experiments: Create chimeric proteins containing domains from different species to identify which regions confer species-specific functions. This can be particularly informative for understanding why C. elegans Sass6 forms spiral structures while Danio rerio and other species form flat rings .

Model-Specific Genetic Tools:

• For zebrafish studies, both forward and reverse genetic approaches are accessible

• Forward genetics through ENU mutagenesis has yielded several meiotic mutant lines

• CRISPR/Cas9 can be employed for targeted gene editing

• When analyzing genetic variants, researchers should calculate logarithm of the odds (LOD) scores to confirm linkage, with a score of three or above considered proof of linkage

By carefully considering these aspects, researchers can leverage the unique advantages of each model organism while accounting for species-specific differences in Sass6 structure and function.