Recombinant Xenopus laevis Kinetochore protein Nuf2-A (nuf2-a)

What is Xenopus laevis Nuf2-A and what is its role in kinetochore function?

Xenopus laevis Nuf2-A (xNuf2) is a 462-amino-acid protein with a predicted molecular mass of 54.4 kD that functions as an essential component of the outer kinetochore. xNuf2 is part of the highly conserved Ndc80 complex, which plays crucial roles in chromosome congression, kinetochore-microtubule attachment, and spindle assembly checkpoint signaling. Through immunolocalization studies, xNuf2 has been shown to associate with kinetochores from prometaphase through anaphase, consistent with its role in mediating kinetochore-microtubule interactions during these critical phases of mitosis .

Function-blocking experiments using antibodies against xNuf2 have demonstrated that the protein is required for proper chromosome congression and for cells to maintain mitotic arrest in response to microtubule drugs, indicating its essential function in the spindle checkpoint . When the xNdc80 complex (which includes xNuf2) is depleted from Xenopus extracts, kinetochores fail to recruit key components including xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating the complex's foundational role in functional kinetochore assembly .

How does xNuf2 interact with other kinetochore components?

xNuf2 forms a stable biochemical complex with xNdc80 (Xenopus homolog of Ndc80/HEC1), with co-immunoprecipitation experiments confirming this physical interaction. The xNdc80-xNuf2 complex has an approximate size of 190 kD and represents the primary functional form of these proteins in vivo . Immunodepletion of xNdc80 from Xenopus egg extracts also removes xNuf2, demonstrating that the majority of xNuf2 is physically associated with xNdc80 .

The interaction between Nuf2 and Ndc80 occurs through an extended coiled-coil region, with the N-terminal CH domains of both proteins forming a globular head that directly contacts microtubules. Structural studies of homologous proteins show that Nuf2's coiled-coil region forms a continuous helix that pairs with the Ndc80 polypeptide chain, including through a specialized "switchback" region in Ndc80 (commonly referred to as the Ndc80 loop) .

What is the estimated concentration of xNuf2 in Xenopus egg extracts?

Based on quantitative immunoblotting comparing extract signals with known amounts of recombinant protein, the concentration of xNuf2 in Xenopus egg extracts has been estimated at approximately 53 nM. For comparison, xNdc80 is present at about 66 nM in the same extracts . These measurements provide important reference points for researchers designing experiments with physiologically relevant protein concentrations.

What are the optimal expression systems for recombinant Xenopus Nuf2-A?

For expressing recombinant xNuf2, bacterial expression systems using E. coli strains specifically designed for expression of eukaryotic proteins are most commonly employed. Based on protocols developed for homologous proteins, specialized strains such as Rosetta2(DE3)pLysS or Shuffle T7 E. coli are recommended as they provide the tRNAs for rare codons and enhance disulfide bond formation, respectively .

For optimal expression:

• Clone codon-optimized synthetic genes coding for xNuf2 into expression vectors such as pETDuet-1

• Add a TEV protease-cleavable 6-His tag to facilitate purification

• Co-express with binding partners (particularly xNdc80) to enhance stability and solubility

• Induce expression at reduced temperatures (18°C) after cultures reach optimal density (OD 0.8)

• Include cysteine residues at strategic positions (N and C termini) to facilitate heterodimerization with binding partners via disulfide bonds

What purification strategies yield highest purity and activity of recombinant xNuf2?

Based on successful protocols for similar kinetochore proteins, a multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography using Ni-NTA resin to capture the His-tagged protein

• Tag removal: Treatment with TEV protease to cleave the affinity tag

• Secondary purification: Ion exchange chromatography to remove contaminants with different charge properties

• Final polishing: Size exclusion chromatography in a buffer containing 2 mM TCEP or another reducing agent

For highest biological activity, co-expression and co-purification of xNuf2 with xNdc80 is strongly recommended, as these proteins form a functional complex in vivo and may have limited stability when purified individually .

How can researchers verify the integrity and activity of purified recombinant xNuf2?

Additionally, functional activity can be assessed in Xenopus egg extract systems by testing whether the recombinant protein can rescue phenotypes caused by immunodepletion of endogenous xNuf2 .

How can function-blocking antibodies against xNuf2 be utilized to study kinetochore function?

Function-blocking antibodies against xNuf2 have been successfully used to disrupt kinetochore function in both Xenopus tissue culture cells and egg extract systems . Key methodological considerations include:

• Antibody preparation: Affinity-purify antibodies raised against recombinant xNuf2 to ensure specificity

• Cell culture applications: Microinject purified antibodies into XTC cells during interphase before cells enter mitosis

• Expected phenotypes: Cells injected with anti-xNuf2 antibodies show premature exit from mitosis without proper chromosome congression or anaphase movements

• Checkpoint analysis: Treated cells fail to arrest in response to microtubule drugs, indicating disruption of the spindle checkpoint

• Egg extract applications: Add antibodies to egg extracts containing assembled chromosomes

• Controls: Always include non-immune IgG as a negative control and validate antibody specificity by Western blotting

This approach provides a powerful alternative to protein depletion, allowing for acute inhibition of xNuf2 function at specific cell cycle stages .

How can researchers use Xenopus egg extracts to study xNuf2 function?

Xenopus egg extracts provide a versatile biochemical system for studying kinetochore assembly and function. For xNuf2 studies, the following approaches are particularly valuable:

• Immunodepletion: Use anti-xNuf2 antibodies coupled to protein A beads to remove endogenous xNuf2 from egg extracts. This approach also removes xNdc80, as these proteins exist in a complex .

• Add-back experiments: Supplement immunodepleted extracts with purified recombinant xNuf2 (ideally in complex with xNdc80) to rescue function and test mutant proteins.

• Kinetochore assembly assay: Add sperm chromatin to xNuf2-depleted extracts and assess kinetochore assembly by immunofluorescence for other kinetochore markers.

• Checkpoint analysis: Test whether xNuf2-depleted extracts can establish and maintain a spindle checkpoint arrest in response to microtubule-disrupting drugs.

In contrast to antibody inhibition, immunodepletion allows for complete removal of the xNdc80 complex, revealing its requirement for recruiting multiple downstream kinetochore components including checkpoint proteins xMad1, xMad2, xBub1, and xBub3 .

How do the structural features of xNuf2 compare to homologs in other species?

While direct structural data for Xenopus Nuf2 is limited, comparative analysis with homologs provides valuable insights:

• The core structure of Nuf2 is highly conserved across species, consisting primarily of an N-terminal globular CH (calponin homology) domain followed by an extended coiled-coil region .

• The CH domain partners with the corresponding domain in Ndc80 to form a globular "head" that directly contacts microtubules.

• Structural predictions using AlphaFold 2 for human and yeast Nuf2 show that the coiled-coil region forms a continuous helix that pairs with Ndc80, including opposite the specialized "switchback" region in Ndc80 .

• Unlike metazoan and fission yeast sequences, budding yeast Nuf2 shows some sequence divergence, though the predicted structures remain similar .

• The ability of Nuf2 to form a stable complex with Ndc80 is conserved from yeast to humans and Xenopus, suggesting that the binding interface is structurally conserved despite sequence variations .

Given the high level of conservation in kinetochore architecture, structural insights from human and yeast Nuf2 studies are likely applicable to Xenopus Nuf2, particularly regarding core functional domains and interaction surfaces.

What are the challenges in using recombinant xNuf2 for in vitro reconstitution experiments?

Researchers attempting in vitro reconstitution of kinetochore functions with recombinant xNuf2 should consider several technical challenges:

• Protein stability: The coiled-coil structure of Nuf2 makes it prone to aggregation when expressed alone. Co-expression with Ndc80 significantly improves stability and solubility .

• Complex assembly: Full reconstitution requires multiple proteins beyond just xNuf2 and xNdc80, including Spc24, Spc25, and potentially other kinetochore components.

• Post-translational modifications: Recombinant proteins expressed in bacteria lack the post-translational modifications that may be present in vivo and potentially important for function.

• Functional verification: Verifying that reconstituted complexes are functionally equivalent to their endogenous counterparts requires careful activity assays, such as microtubule binding.

• Structural integrity: Ensuring that recombinant proteins adopt the correct conformation may require specialized techniques such as introducing engineered disulfide bonds to stabilize protein-protein interactions .

A recommended approach is to engineer fusion constructs or introduce cysteine residues to stabilize protein complexes, as has been successfully demonstrated with homologous proteins .

How can researchers interpret contradictory data regarding xNuf2 function?

When confronting contradictory data on xNuf2 function, consider these methodological approaches:

• Experimental system differences: Compare results between Xenopus egg extracts and tissue culture cells, as these represent different biological contexts .

• Temporal considerations: Distinguish between immediate effects of xNuf2 inhibition versus long-term consequences of its absence.

• Inhibition method comparison: Compare phenotypes observed with function-blocking antibodies versus protein depletion, as these approaches may affect different protein populations or interaction surfaces .

• Complex integrity analysis: Assess whether experimental manipulations disrupt the entire Ndc80 complex or specifically affect xNuf2 function.

• Crossspecies validation: Test whether findings in Xenopus are consistent with data from other model systems like yeast or human cells, accounting for known differences in kinetochore organization .

A systematic approach combining multiple experimental strategies can help resolve apparently contradictory observations and provide deeper mechanistic insights into xNuf2 function.

What aspects of xNuf2 function remain poorly understood?

Despite significant progress in understanding xNuf2's role in kinetochore assembly and function, several important questions remain:

• Regulatory mechanisms: How is xNuf2 function regulated during the cell cycle, particularly through post-translational modifications?

• Structural dynamics: How does the conformation of the xNdc80-xNuf2 complex change during microtubule capture and chromosome movement?

• Interaction surfaces: Which specific residues in xNuf2 mediate interactions with other kinetochore components beyond xNdc80?

• Developmental regulation: How is xNuf2 expression and function regulated during Xenopus development?

• Species-specific functions: Are there unique aspects of xNuf2 function in Xenopus compared to other vertebrates?

Addressing these questions will require combining structural approaches with functional studies in both Xenopus egg extracts and cell systems .

What emerging technologies might advance xNuf2 research?

These approaches, especially when combined, could provide unprecedented insights into xNuf2 function and regulation in the context of kinetochore assembly and chromosome segregation.