Recombinant Xenopus tropicalis Borealin (cdca8)

What is Borealin/CDCA8 and what is its role in Xenopus tropicalis?

Borealin, also known as Cell Division Cycle Associated 8 (CDCA8), is a critical component of the Chromosomal Passenger Complex (CPC) which plays essential roles in mitosis and cytokinesis. In Xenopus tropicalis, as in other vertebrates, Borealin functions primarily in regulating proper chromosome segregation and cell division.

The protein is part of the Chromosomal Passenger Complex alongside Aurora B kinase, INCENP (Inner Centromere Protein), and Survivin . This complex coordinates key mitotic events including chromosome alignment, spindle assembly checkpoint function, and cytokinesis completion.

The following table shows Borealin/CDCA8 conservation across species:

This high conservation across species suggests that findings in Xenopus tropicalis Borealin research can provide insights applicable to other vertebrate models and human biology .

How is the Chromosomal Passenger Complex (CPC) function regulated in Xenopus?

In Xenopus, as in other organisms, the CPC undergoes precise spatial and temporal regulation to coordinate cell division events. A major regulatory mechanism involves targeted protein degradation at mitotic exit.

Research has demonstrated that APC/CCdh1 (Anaphase-Promoting Complex/Cyclosome with the Cdh1 adaptor) mediates Borealin degradation upon mitotic exit . This degradation is essential for terminating CPC activity, which contributes to proper control of subsequent DNA replication.

The degradation process depends on specific amino acid residues in Borealin that form a recognition site (degron) for the APC/CCdh1 complex. Experimental evidence shows that mutation of conserved hydrophobic residues (positions equivalent to L21E/F24E/L25E/F28E/V32E plus W70E/F74E in human Borealin) significantly reduces Cdh1 binding and subsequent ubiquitylation .

This regulatory mechanism ensures that CPC activity is appropriately terminated at the end of mitosis, preventing improper persistence of mitotic regulatory proteins during G1 phase.

What expression systems are recommended for producing recombinant Xenopus tropicalis Borealin?

For successful production of functional recombinant Xenopus tropicalis Borealin, several expression systems can be employed depending on the experimental requirements:

When selecting an expression system, consider that recombinant ORF sequences can be delivered in standard vectors like pcDNA3.1 . For experiments involving Xenopus egg extracts, bacterial expression is often sufficient as the extracts provide the necessary environment for proper complex formation.

What are the key functional domains of Xenopus tropicalis Borealin?

Xenopus tropicalis Borealin contains several critical functional domains that mediate its role in the Chromosomal Passenger Complex:

• N-terminal CPC localization module: The N-terminal region participates in forming a three-helix bundle with INCENP and Survivin that constitutes the CPC localization module . This structure is essential for proper targeting of the complex to centromeres during mitosis.

• Survivin-binding region: Specific hydrophobic residues (equivalent to human W70 and F74) dock into the hydrophobic pocket present on the BIR (baculovirus inhibitor of apoptosis protein repeat) domain of Survivin . Mutation of these residues disrupts Survivin binding and compromises CPC function.

• APC/CCdh1 degron: Regions comprising amino acids 18-39 and 69-77 (in human Borealin) are required for binding to Cdh1 and subsequent ubiquitylation . This region likely contains a non-canonical D-box motif that mediates protein degradation after mitosis.

• INCENP interaction surface: Borealin binds to INCENP and contributes to targeting the complex to centromeres . This interaction is critical for CPC assembly and function.

Understanding these domains is essential for designing mutation studies and for interpreting the effects of sequence variations on Borealin function.

What phenotypes result from Borealin depletion or mutation in experimental systems?

Depletion or mutation of Borealin results in distinct phenotypes that highlight its crucial role in cell division:

• Mitotic defects: RNA interference experiments show that Borealin depletion increases the percentage of cells in prometaphase , suggesting problems with chromosome alignment or spindle assembly checkpoint satisfaction.

• Kinetochore-microtubule attachment errors: Borealin-depleted cells exhibit dramatic increases in spindle-kinetochore misattachments , leading to chromosome segregation errors.

• Cytokinesis failures: Cells lacking functional Borealin frequently fail to complete cytokinesis , resulting in binucleated or multinucleated cells.

• Embryonic lethality: Complete knockout of Borealin in mice causes embryonic lethality around day E5.5 , highlighting its essential role in early development. This suggests that similar developmental requirements would exist in Xenopus tropicalis.

• Thyroid development impairment: In other contexts, Borealin deficiency has been linked to impaired thyroid development, with heterozygous mice showing thyroid hypoplasia during embryogenesis .

These phenotypes underscore the importance of Borealin in ensuring proper cell division and organismal development.

How can recombinant Xenopus tropicalis Borealin be used to reconstitute CPC function in vitro?

Recombinant Xenopus tropicalis Borealin can be employed in various approaches to reconstitute and study CPC function in vitro:

• Complete CPC reconstitution: By combining purified recombinant Borealin with other CPC components (Aurora B, INCENP, and Survivin), researchers can reconstitute the complete complex for biochemical and functional studies. This approach allows precise control over protein composition and can reveal requirements for auxiliary factors.

• Add-back experiments in Xenopus egg extracts: Xenopus egg extracts provide an excellent system for studying mitotic events. Depletion of endogenous Borealin followed by addition of recombinant wild-type or mutant proteins can reveal structure-function relationships. The cell-free nature of this system facilitates quantitative analysis of CPC activity.

• Kinase activity assays: Measuring Aurora B kinase activity in the presence of different Borealin variants can elucidate how Borealin contributes to kinase regulation. Typical substrates include histone H3 (Ser10) or MCAK (mitotic centromere-associated kinesin).

• Centromere targeting assays: Using fluorescently labeled recombinant Borealin in combination with artificial chromosomes or chromatin beads can reveal mechanisms of CPC targeting to centromeres during mitosis.

For successful reconstitution, it's crucial to verify that all components are properly folded and active. This can be assessed through activity assays, binding studies, and structural analyses such as circular dichroism or limited proteolysis.

What are the critical residues in Xenopus tropicalis Borealin for protein-protein interactions?

Several critical residues in Xenopus tropicalis Borealin mediate its interactions with other proteins:

• Survivin interaction: Based on structural studies of human Borealin, residues equivalent to W70 and F74 are critical for docking into the hydrophobic pocket on Survivin's BIR domain . Mutation of these residues (W70E/F74E in human Borealin) significantly reduces binding to Survivin.

• Cdh1 binding: The regions comprising amino acids 18-39 and 69-77 (in human Borealin) are required for binding to Cdh1 and subsequent ubiquitylation . A non-canonical D-box motif within this region mediates recognition by the APC/CCdh1 complex.

• INCENP binding: The N-terminal region of Borealin participates in forming a three-helix bundle with INCENP and Survivin . Though specific residues aren't detailed in the search results, this interaction is crucial for CPC assembly.

The table below summarizes key mutational studies that have identified critical residues:

These critical residues serve as important targets for mutagenesis studies to dissect the functional consequences of disrupting specific protein interactions.

How does Borealin's role in Xenopus compare to its function in mammalian systems?

While there are core conserved functions of Borealin across vertebrate species, some interesting differences and similarities can be observed when comparing Xenopus tropicalis and mammalian systems:

• Conserved mitotic functions: The fundamental role of Borealin in the CPC for regulating mitosis appears highly conserved. In both Xenopus and mammals, Borealin depletion causes similar mitotic defects, including chromosome alignment problems and cytokinesis failures .

• Protein complex association: The interaction with other CPC components (Aurora B, INCENP, and Survivin) is conserved across species, suggesting similar structural and functional relationships in the complex.

• Degradation mechanism: In both systems, APC/CCdh1-mediated ubiquitylation and degradation appears to be the primary mechanism for regulating Borealin levels after mitosis .

• Developmental requirements: Homozygous Borealin knockout in mice causes early embryonic lethality (E5.5) , suggesting an essential developmental role. Though specific developmental studies in Xenopus tropicalis aren't detailed in the search results, similar requirements likely exist.

• Tissue-specific functions: In mammals, Borealin has been implicated in thyroid development , a specialized function that might reflect evolutionary adaptations. Whether similar tissue-specific roles exist in Xenopus requires further investigation.

The high conservation of Borealin/CDCA8 sequence across species (as shown in the earlier table) supports the notion that findings in Xenopus can inform our understanding of mammalian Borealin function, and vice versa.

What methodological approaches are most effective for studying Borealin-dependent mitotic events?

Several methodological approaches have proven particularly effective for studying Borealin-dependent mitotic events:

• Xenopus egg extract systems: Cell-free extracts from Xenopus eggs provide an excellent system for studying mitotic processes . These extracts contain the complete machinery for cell cycle progression and allow for convenient immunodepletion-reconstitution experiments.

• Immunodepletion followed by add-back: Depleting endogenous Borealin from Xenopus extracts and adding back recombinant wild-type or mutant proteins allows for detailed structure-function analyses .

• Live imaging with fluorescent proteins: Expressing fluorescently tagged Borealin variants in cells or adding them to egg extracts allows visualization of protein dynamics during mitosis.

• In vitro recombination and deletion/substitution mutagenesis: As demonstrated with other Xenopus proteins, in vitro recombination of deletion mutants allows construction of precise substitution mutants to examine the functional importance of specific sequence regions .

• Binding assays with recombinant proteins: Co-immunoprecipitation and pull-down assays with recombinant proteins can reveal the requirements for complex formation and identify interaction surfaces.

• Ubiquitylation assays: In vitro and in vivo ubiquitylation assays can reveal how Borealin is targeted for degradation and how this process is regulated .

These approaches can be combined to provide comprehensive insights into Borealin function throughout the cell cycle.

How can one assess the quality and functionality of purified recombinant Xenopus tropicalis Borealin?

Assessing the quality and functionality of purified recombinant Xenopus tropicalis Borealin requires multiple complementary approaches:

• Biochemical purity assessment:

  • SDS-PAGE with Coomassie staining to assess purity (>90% is typically desired)

  • Western blotting with specific antibodies to confirm identity

  • Mass spectrometry for precise molecular weight determination and identification of any post-translational modifications

• Structural integrity verification:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Limited proteolysis to evaluate proper folding (well-folded proteins show specific digestion patterns)

  • Size exclusion chromatography to verify monomeric state or appropriate oligomerization

• Functional activity testing:

  • Binding assays with other CPC components (Aurora B, INCENP, Survivin)

  • Ability to restore proper mitotic progression when added to Borealin-depleted Xenopus egg extracts

  • Aurora B kinase activation assays (measuring phosphorylation of histone H3 at Ser10)

• Interaction verification:

  • Pull-down assays with known binding partners

  • Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) for quantitative binding measurements

  • Co-immunoprecipitation from mitotic extracts supplemented with recombinant protein

Functionality criteria should include the ability of the recombinant protein to incorporate into the CPC and support proper chromosome alignment and cytokinesis in appropriate experimental systems.

What is the significance of Borealin/CDCA8 in cancer research and how can Xenopus studies contribute?

Borealin/CDCA8 has emerging significance in cancer research, with Xenopus studies offering valuable insights:

• Cancer association: CDCA8 overexpression has been detected in various malignant tumors and is closely associated with tumor growth . In bladder cancer, high CDCA8 expression correlates with poor clinicopathological features and prognosis .

• Prognostic marker potential: Cox univariable and multivariable analyses have shown that CDCA8 expression is an independent factor influencing cancer-specific survival among bladder cancer patients . This suggests potential as a prognostic biomarker.

• Signaling pathway involvement: Gene Set Enrichment Analysis (GSEA) has identified CDCA8-associated pathways including "G2M checkpoint," "E2F targets," "Myc targets," "mTORC1 signaling," "mitotic spindle," and "PI3K-AKT-mTOR signaling" . These pathways are frequently dysregulated in cancer.

• Therapeutic target potential: The essential role of Borealin in cell division makes it a potential therapeutic target. Disruption of the interaction between CDCA8 and Survivin has been shown to inhibit the growth of hepatocellular carcinoma .

Xenopus studies can contribute to cancer research through:

• Providing a clean biochemical system to study CPC assembly and regulation

• Facilitating high-throughput screening of compounds that disrupt Borealin function

• Allowing detailed structure-function analysis of cancer-associated mutations

• Enabling the study of how CPC dysregulation affects genome stability

The high conservation of Borealin across species makes Xenopus an excellent model for studying basic mechanisms that may be relevant to human cancer biology.

What are the current challenges and future directions in Xenopus tropicalis Borealin research?

Current challenges and promising future directions in Xenopus tropicalis Borealin research include:

• Structural characterization challenges:

  • Obtaining high-resolution structures of the complete CPC remains difficult

  • Understanding the conformational changes during CPC activation and inactivation

  • Defining the structural basis for centromere targeting

• Regulatory mechanism questions:

  • Identifying all post-translational modifications that regulate Borealin function

  • Understanding how Borealin contributes to the spatial and temporal regulation of Aurora B activity

  • Elucidating the mechanisms that coordinate CPC activity with other mitotic regulators

• Future methodological approaches:

  • CRISPR/Cas9-mediated genome editing in Xenopus tropicalis to create specific mutations

  • Super-resolution microscopy to visualize CPC dynamics at centromeres and the central spindle

  • Cryo-electron microscopy for structural studies of the complete CPC

• Translational research directions:

  • Development of small molecules targeting Borealin-Survivin interaction

  • Investigation of Borealin as a biomarker for cancer prognosis and treatment response

  • Understanding how Borealin dysregulation contributes to chromosomal instability in cancer

• Evolutionary conservation studies:

  • Comparative analysis of Borealin function across species to identify core mechanisms

  • Investigation of species-specific adaptations in Borealin regulation

  • Understanding how differences in embryonic development affect Borealin requirements

Addressing these challenges will require integrating multiple experimental approaches and leveraging the unique advantages of the Xenopus system for both biochemical and developmental studies.