Recombinant Xenopus laevis Kinetochore protein Spc25 (spc25)

What is SPC25 and what is its role in the kinetochore complex?

SPC25 (SPC25, NDC80 Kinetochore Complex Component, Homolog) is a crucial protein component of the kinetochore complex that plays an essential role in chromosome segregation and spindle dynamics regulation during mitosis. In Xenopus laevis, as in other organisms, SPC25 forms a heterodimer with SPC24, which together constitute part of the larger NDC80 complex at the kinetochore. This complex is vital for establishing and maintaining proper kinetochore-microtubule attachments during cell division .

The SPC24/SPC25 dimer, composed of tightly interacting RWD domains, caps the Mis12 complex near its C-terminal tails, engaging in tight interactions with the C-terminal regions of DSN1 and NSL1 proteins . This structural arrangement is crucial for the proper functioning of the kinetochore during mitosis.

How is SPC25 function conserved across species?

SPC25's function appears to be highly conserved from yeast to vertebrates, including Xenopus and humans. Studies in budding yeast have shown that Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore, and mutations in these proteins lead to defects in chromosome segregation and mitotic checkpoint control .

The conservation of SPC25 across species suggests that findings in one model organism may provide valuable insights applicable to others. For instance, research indicates that Ndc80p, Nuf2p, and Spc24p are conserved proteins, making it likely that similar complexes are part of the kinetochore in multiple organisms including Xenopus laevis .

What are the optimal expression systems for producing recombinant Xenopus laevis SPC25?

Recombinant Xenopus laevis SPC25 can be successfully expressed in several systems, with yeast being a commonly used host for this specific protein. When working with this protein, researchers should consider:

• Yeast expression system: This has been successfully used to produce recombinant Xenopus laevis SPC25 with high purity (>90%) .

• Alternative expression systems: While yeast is common, researchers might consider E. coli, mammalian cells, or baculovirus infection systems depending on their specific experimental requirements .

• Protein tagging: Typically, recombinant Xenopus laevis SPC25 is produced with a His-tag to facilitate purification and detection . Other tags may be considered based on experimental needs.

The choice of expression system should be guided by the intended application, required protein folding, and post-translational modifications needed for functional studies.

What purification methods are most effective for recombinant Xenopus laevis SPC25?

For optimal purification of recombinant His-tagged Xenopus laevis SPC25, a multi-step purification protocol is recommended:

• Affinity chromatography: Using Ni-NTA or similar metal affinity resins to capture His-tagged SPC25.

• Size exclusion chromatography: To separate SPC25 based on molecular size and further improve purity.

• Quality control: Purity assessment using SDS-PAGE and Western blotting, with expected purity >90% for most research applications .

Researchers should be aware that purification conditions might need optimization based on the specific construct and expression system used. For complex formation studies with other kinetochore components, co-expression and co-purification strategies may yield better results.

What applications are suitable for recombinant Xenopus laevis SPC25?

Recombinant Xenopus laevis SPC25 can be utilized in various experimental applications:

• ELISA: For protein-protein interaction studies or antibody validation .

• Structural studies: To investigate the molecular architecture of the NDC80 complex and its interactions within the kinetochore.

• Functional assays: To examine the role of SPC25 in chromosome segregation and spindle dynamics.

• Protein-protein interaction studies: To identify binding partners and characterize interaction domains.

• In vitro reconstitution: For assembling kinetochore subcomplexes to study their properties.

When designing experiments, researchers should consider using appropriate controls and validation methods to ensure the recombinant protein maintains its native function.

How does Xenopus laevis SPC25 contribute to mitotic checkpoint control?

Studies in yeast have shown that SPC25 plays a critical role in the mitotic checkpoint control, particularly in the Mad2p-dependent checkpoint pathway. When SPC25 function is compromised (as in temperature-sensitive mutants), cells fail to arrest in metaphase in response to checkpoint control, despite defects in chromosome segregation .

For researchers studying Xenopus laevis SPC25:

• Checkpoint assays: Monitor the activation of the spindle assembly checkpoint in Xenopus egg extracts with wild-type versus mutant or depleted SPC25.

• Experimental approach:

  • Use immunodepletion of endogenous SPC25 from Xenopus egg extracts

  • Complement with recombinant wild-type or mutant SPC25

  • Assess checkpoint activation through Pds1p degradation kinetics or equivalent Xenopus markers

  • Evaluate chromosome segregation patterns

• Expected outcomes: Functional SPC25 would support proper checkpoint activation when spindle assembly is disrupted (e.g., with nocodazole), while defective SPC25 would show checkpoint bypass .

This research direction is particularly important as it connects structural components of the kinetochore to cell cycle regulatory mechanisms.

What structural features of Xenopus laevis SPC25 mediate its interaction with other kinetochore components?

Structural studies in other organisms indicate that SPC25 forms a tight heterodimer with SPC24 through their RWD domains, which then interact with other kinetochore components. In the human KMN complex, the SPC24/SPC25 dimer caps the Mis12 complex, interacting with the C-terminal regions of DSN1 and NSL1 .

For researchers investigating the structural basis of these interactions in Xenopus laevis SPC25:

• Domain mapping experiments:

  • Generate truncated versions of SPC25 to identify minimal binding domains

  • Use site-directed mutagenesis to modify potential interaction interfaces

  • Perform pull-down assays to quantify binding affinities

• Structural analysis approaches:

  • X-ray crystallography of the SPC24/SPC25 heterodimer

  • Cryo-EM studies of larger assemblies containing SPC25

  • Cross-linking mass spectrometry to identify interaction points

• Functional validation:

  • Express mutant versions in Xenopus egg extracts

  • Assess kinetochore assembly and function

Understanding these structural features will provide insights into how the kinetochore assembles and functions during mitosis.

How can Xenopus egg extracts be used to study SPC25 function in kinetochore assembly?

Xenopus egg extracts provide an excellent system for studying kinetochore assembly and function due to their ability to recapitulate many aspects of cell cycle progression in vitro. To study SPC25's role:

• Experimental workflow:

  Step	Procedure	Outcome Measurement
  1	Prepare CSF-arrested Xenopus egg extracts	Verify M-phase state by H1 kinase activity
  2	Immunodeplete endogenous SPC25	Western blot confirmation of depletion
  3	Add back recombinant wild-type or mutant SPC25	Quantify incorporation by Western blot
  4	Add sperm chromatin for kinetochore assembly	Monitor by immunofluorescence
  5	Assess kinetochore structure and function	Measure microtubule attachment, chromosome alignment

• Advanced applications:

  • Combine with fluorescently labeled components to track kinetochore assembly in real-time

  • Use extracts cycling between interphase and mitosis to study temporal aspects of SPC25 function

  • Introduce specific perturbations (phosphatase inhibitors, kinase inhibitors) to dissect regulatory pathways

This approach allows for manipulation of SPC25 and observation of direct consequences on kinetochore assembly and function in a physiologically relevant context.

How does Xenopus laevis SPC25 compare structurally and functionally to its homologs in other model organisms?

Comparative analysis of SPC25 across species reveals both conservation and divergence:

Despite differences in primary sequence, the core functions in kinetochore assembly and chromosome segregation appear to be conserved across these species. Researchers should note that while fundamental mechanisms are preserved, species-specific differences in regulation and interaction partners may exist .

When designing experiments using Xenopus laevis SPC25, researchers can often draw on findings from other model organisms, particularly regarding structural domains and core protein-protein interactions.

What advantages does the Xenopus laevis model system offer for studying SPC25 and kinetochore biology?

Xenopus laevis provides several unique advantages for studying kinetochore proteins like SPC25:

• Biochemical tractability: Xenopus egg extracts permit biochemical manipulations and reconstitution experiments that are difficult in other systems .

• Cell-free system: The ability to study kinetochore assembly and function outside the constraints of intact cells allows for precise manipulation of components .

• Evolutionary insights: As an amphibian, Xenopus provides an important evolutionary perspective between fish and mammals.

• Experimental versatility: Xenopus embryos can be easily treated with small molecules in multi-well plates, making them amenable to chemical screening and offering opportunities in drug discovery approaches that might target kinetochore function .

• Whole-organism studies: The development and cellular behaviors observed in Xenopus closely mimic those in mammals, allowing for translation of findings to higher vertebrates .

The combination of these advantages makes Xenopus laevis an excellent model for studying fundamental aspects of SPC25 function in kinetochore biology.

What are common challenges in expressing and purifying functional recombinant Xenopus laevis SPC25?

Researchers working with recombinant Xenopus laevis SPC25 often encounter several technical challenges:

• Solubility issues: SPC25 may form inclusion bodies when expressed alone, particularly in bacterial systems.

  • Solution: Co-express with binding partners (e.g., SPC24) to enhance solubility.

• Functional assessment: Confirming that recombinant SPC25 retains native functionality.

  • Solution: Develop functional assays such as binding studies with known interaction partners or activity in reconstituted systems.

• Stability concerns: Maintaining protein stability during purification and storage.

  • Solution: Optimize buffer conditions (pH, salt concentration, glycerol) and store as aliquots at -80°C to avoid freeze-thaw cycles.

• Expression yield variability: Inconsistent protein yields between batches.

  • Solution: Standardize growth conditions, induction parameters, and purification protocols.

For researchers seeking high-quality protein preparations, it's advisable to test multiple expression systems (yeast, E. coli, mammalian cells) as indicated in the product information .

How can researchers validate the functionality of recombinant Xenopus laevis SPC25 in experimental settings?

To ensure that recombinant Xenopus laevis SPC25 is functionally active, researchers should implement a multi-faceted validation approach:

• Structural integrity assessment:

  • Circular dichroism spectroscopy to confirm proper folding

  • Size exclusion chromatography to verify appropriate oligomeric state

  • Thermal shift assays to evaluate stability

• Binding partner interaction verification:

  • Pull-down assays with known partners (SPC24, Mis12 complex components)

  • Isothermal titration calorimetry to quantify binding affinities

  • Surface plasmon resonance for interaction kinetics

• Functional complementation:

  • Depletion-add back experiments in Xenopus egg extracts

  • Rescue of kinetochore assembly defects

  • Restoration of chromosome segregation in depleted systems

• Cellular localization:

  • If using in cell-based assays, confirm proper kinetochore localization by immunofluorescence

These validation steps ensure that experimental outcomes reflect the true biological functions of SPC25 rather than artifacts of recombinant protein preparation.

How can CRISPR/Cas9 genome editing be applied to study SPC25 function in Xenopus laevis?

CRISPR/Cas9 technology has been successfully implemented in Xenopus systems and offers powerful approaches for studying SPC25:

• Gene knockout strategies:

  • Design guide RNAs targeting conserved regions of SPC25

  • Generate mosaic F0 animals for rapid phenotypic assessment

  • Establish stable knockout lines for comprehensive studies

• Experimental considerations for Xenopus laevis:

  • Account for the allotetraploid genome when designing targeting strategies

  • Target both homeologs (L and S chromosomes) for complete functional analysis

  • Use high efficiency methods such as i-CRISPR for insertion of reporters or tags

• Applications for SPC25 research:

  • Generate endogenously tagged SPC25 for live imaging of kinetochore dynamics

  • Create conditional knockouts to bypass early developmental requirements

  • Introduce specific mutations to test structural hypotheses

The successful application of CRISPR/Cas9 in Xenopus for studying disease models suggests this approach would be valuable for investigating SPC25's role in chromosome segregation and potential links to genomic instability .

What is the potential role of post-translational modifications in regulating Xenopus laevis SPC25 function?

Post-translational modifications (PTMs) likely play crucial roles in regulating SPC25 function during the cell cycle, though specific data for Xenopus laevis SPC25 modifications is limited. Researchers investigating this area should consider:

• Potential PTMs based on studies in other organisms:

  • Phosphorylation sites modulating kinetochore assembly

  • Ubiquitination regulating protein stability

  • SUMOylation affecting protein interactions

• Experimental approaches to identify PTMs:

  • Mass spectrometry analysis of SPC25 isolated from different cell cycle stages

  • Phospho-specific antibodies for Western blotting

  • In vitro modification assays with purified kinases/modifying enzymes

• Functional analysis of identified PTMs:

  • Generate non-modifiable mutants (e.g., phospho-null or phospho-mimetic)

  • Test these mutants in egg extract systems for kinetochore assembly

  • Assess effects on protein-protein interactions and complex formation

Understanding how PTMs regulate SPC25 will provide insights into the dynamic regulation of kinetochore function during mitosis and meiosis in Xenopus laevis.

How might studies of Xenopus laevis SPC25 contribute to understanding chromosome segregation disorders?

Research on Xenopus laevis SPC25 could significantly advance our understanding of chromosome segregation disorders through several avenues:

• Mechanistic insights: Detailed understanding of SPC25's role in the kinetochore could illuminate how defects lead to aneuploidy, a hallmark of many cancers and developmental disorders.

• Translational potential: Given the conservation of SPC25 function across species, insights from Xenopus studies may be directly applicable to human conditions associated with chromosome segregation errors.

• Model development: Xenopus embryos with modified SPC25 could serve as models for chromosome instability syndromes, allowing for drug screening and detailed phenotypic analysis.

• Therapeutic targets: Identifying specific functions and interactions of SPC25 might reveal novel therapeutic targets for conditions characterized by chromosome segregation defects.

The connection between SPC25 expression levels and cancer prognosis observed in human studies (such as in lung adenocarcinoma) suggests that fundamental research in model organisms like Xenopus could provide valuable insights into disease mechanisms .

What interdisciplinary approaches might enhance our understanding of SPC25 function?

Advancing research on Xenopus laevis SPC25 would benefit from integrating multiple disciplines:

• Structural biology and biophysics:

  • Cryo-EM structures of the entire kinetochore complex

  • Single-molecule studies of force generation and microtubule attachments

  • Molecular dynamics simulations of SPC25 interactions

• Systems biology:

  • Network analysis of SPC25 interactions throughout the cell cycle

  • Mathematical modeling of kinetochore assembly and function

  • High-throughput genetic interaction screens

• Advanced imaging:

  • Super-resolution microscopy of kinetochore organization

  • Live cell imaging with specific SPC25 probes

  • Correlative light and electron microscopy for ultrastructural context

• Synthetic biology:

  • Engineering minimal kinetochore systems with defined components

  • Creating optogenetic tools to spatiotemporally control SPC25 function

  • Developing biosensors for monitoring SPC25 interactions in vivo

These interdisciplinary approaches would provide complementary perspectives on SPC25 function, moving beyond traditional biochemical and cell biological methods to gain more comprehensive insights into kinetochore biology.