Recombinant Danio rerio Kinetochore protein NDC80 homolog (ndc80), partial

What is NDC80 and what are its primary functions in kinetochores?

NDC80 (also known as HEC1 in humans) is a core component of the kinetochore, the protein complex that connects chromosomes to spindle microtubules during cell division. The NDC80 complex lies at the heart of the kinetochore and directly binds to microtubules . It serves multiple critical functions:

• Acts as the principal link between mitotic spindle microtubules and centromere-associated proteins

• Functions in chromosome congression and spindle checkpoint signaling

• Couples microtubule depolymerization to chromosome movement, essentially acting as a molecular motor

• Stabilizes kinetochore-microtubule attachments through multiple interactions with microtubule lattice

In Danio rerio, as in other vertebrates, the NDC80 complex likely maintains these conserved functions, making it critical for proper chromosome segregation during zebrafish development and cell division.

What is the structure of the NDC80 complex and how does it relate to function?

The NDC80 complex has a distinctive structural organization that directly relates to its function:

• Consists of four protein subunits: NDC80/HEC1, NUF2, SPC24, and SPC25

• Features globular domains at both ends connected by a long coiled-coil region

• Contains microtubule-binding domains at one end (N-terminal CH domains of NDC80 and NUF2), with the NDC80 N-terminal tail contributing to this binding

• Connects to the inner kinetochore through RWD domains of SPC24 and SPC25 at the opposite end

• Contains a distinctive loop structure in the NDC80 protein that plays functional roles in kinetochore-microtubule attachment stability

Structural studies reveal that the NDC80 complex has a flexible "hinge" that allows bending, and the loop region (residues 431-463 in human NDC80) appears to be important for interactions between adjacent NDC80 complexes .

How does recombinant partial NDC80 differ from the full-length protein?

When working with partial recombinant NDC80 from Danio rerio, researchers should consider:

• Partial constructs may lack certain functional domains, potentially altering binding properties

• The absence of specific regions (such as the loop or N-terminal tail) can significantly affect microtubule binding capacity

• Biochemical properties may differ, including stability, solubility, and interaction capabilities

What expression systems are most suitable for producing recombinant zebrafish NDC80?

For successful expression of recombinant Danio rerio NDC80:

• Bacterial expression systems (E. coli): Suitable for producing individual domains but may require optimization for soluble expression of the full complex

• Insect cell systems: Provide eukaryotic processing capabilities, beneficial for expressing the complete tetrameric complex

• Mammalian expression systems: Useful when post-translational modifications are critical

For comprehensive studies, co-expression of all four NDC80 complex subunits is recommended, as this improves stability and functionality. When designing expression constructs, consider adding purification tags that won't interfere with protein function, preferably at termini away from functional domains.

What are the recommended purification strategies for recombinant NDC80?

Purification of recombinant NDC80 complex from zebrafish requires careful planning:

• Affinity chromatography: Using His-tag, FLAG-tag, or GST-tag for initial capture

• Ion-exchange chromatography: To separate based on charge differences

• Size-exclusion chromatography: Critical for isolating properly assembled complexes

For retention of functionality:

• Maintain appropriate salt concentration (typically 100-300 mM)

• Include reducing agents to prevent oxidation of cysteines

• Consider including protease inhibitors to prevent degradation

• Verify complex integrity using analytical techniques such as SEC-MALS and mass photometry

Researchers studying NDC80 have successfully used these approaches to produce functional recombinant complexes for structural and functional studies .

How can I verify the functionality of recombinant zebrafish NDC80?

Multiple assays can verify recombinant NDC80 functionality:

• Microtubule binding assays:

  • Co-sedimentation with polymerized microtubules

  • Total internal reflection fluorescence (TIRF) microscopy to visualize binding

  • Surface plasmon resonance to measure binding kinetics

• Structural verification:

  • Circular dichroism to assess secondary structure

  • Limited proteolysis to confirm proper folding

  • Electron microscopy to visualize complex architecture

• Functional assays:

  • Microtubule tracking assays to assess ability to follow depolymerizing microtubules

  • Force generation measurements using optical tweezers or beads attached to NDC80

  • Cell-based assays following complementation of NDC80-depleted cells

Research has demonstrated that functional NDC80 complexes should effectively bind microtubules and, when present in sufficient numbers, track depolymerizing microtubule ends .

How does the multivalency of NDC80 complexes affect microtubule binding and tracking?

The multivalent nature of NDC80 is critical for its function:

• Single NDC80 complexes do not effectively track depolymerizing microtubules

• Particles with three or more NDC80 complexes track depolymerizing microtubules efficiently

• The residence time of NDC80 on microtubules increases exponentially with the number of complexes

• Modules with two or more complexes can stall and rescue microtubule depolymerization in a force-dependent manner

These findings suggest that:

• Multiple NDC80 complexes work cooperatively at the kinetochore

• This cooperation creates a force-coupling mechanism essential for chromosome movement

• The arrangement of NDC80 complexes likely determines the efficiency of kinetochore-microtubule attachments

This multivalency principle should be considered when designing experiments with recombinant zebrafish NDC80, particularly when studying its force-generation capabilities.

What role does the NDC80 loop play in kinetochore-microtubule attachments?

The NDC80 loop region plays a crucial role in kinetochore function:

This suggests that when working with zebrafish NDC80, preservation of the loop region is critical for studies involving kinetochore-microtubule attachments. Researchers interested in the mechanistic details could design loop mutants to systematically assess its contribution to NDC80 function.

How can I analyze the effects of NDC80 mutations on chromosome segregation?

To analyze effects of NDC80 mutations:

• In vitro approaches:

  • Compare microtubule binding properties of wild-type and mutant proteins

  • Assess ability to track depolymerizing microtubules using TIRF microscopy

  • Measure force generation capabilities using optical traps or bead assays

• Cell-based approaches:

  • Deplete endogenous NDC80 using siRNA or CRISPR

  • Complement with mutant constructs via electroporation

  • Evaluate mitotic progression, spindle checkpoint function, and chromosome segregation

• Analysis techniques:

  • Live-cell imaging to track chromosome dynamics

  • Immunofluorescence to assess kinetochore-microtubule attachments

  • Quantitative measurements of mitotic timing and chromosome segregation errors

Research shows that depletion of endogenous NDC80 ablates the spindle assembly checkpoint and causes cells to exit mitosis prematurely in the presence of microtubule poisons . Functional recombinant NDC80 can restore this checkpoint, providing a clear readout for functionality.

What techniques can be used to study NDC80-microtubule interactions?

Several complementary approaches enable detailed analysis of NDC80-microtubule interactions:

When working with zebrafish NDC80, researchers can adapt these techniques to characterize species-specific properties and compare them with better-studied mammalian systems.

How can phosphorylation affect NDC80 function and how should this be studied?

Phosphorylation significantly modulates NDC80 function:

• Phosphorylation sites on NDC80, particularly in the N-terminal tail, regulate microtubule binding affinity

• Mitotic kinases like Aurora B phosphorylate NDC80 to correct erroneous kinetochore-microtubule attachments

• Phosphorylation of associated proteins, such as Dam1, can release contacts with NDC80 during error correction

To study phosphorylation effects:

• Generate phosphomimetic mutants (S→D or S→E) and phosphoresistant mutants (S→A)

• Use recombinant kinases for in vitro phosphorylation

• Analyze changes in binding affinity using the techniques described above

• Employ mass spectrometry to identify and quantify phosphorylation sites

• Perform functional assays comparing wild-type, phosphomimetic, and phosphoresistant variants

These approaches allow researchers to understand the regulatory mechanisms controlling NDC80 function during the cell cycle.

What buffer conditions optimize stability and functionality of recombinant NDC80?

Optimal buffer conditions for recombinant NDC80:

For long-term storage:

• Flash-freeze in liquid nitrogen and store at -80°C

• Avoid repeated freeze-thaw cycles

• Consider adding additional cryoprotectants like glycerol (up to 20%)

For functional assays, buffer conditions may need adjustment based on the specific requirements of the experimental system.

How does NDC80 integrate with other kinetochore components?

NDC80 functions within a complex network of kinetochore proteins:

• Connects to the inner kinetochore through interactions with CENP-T and the Mis12 complex

• Collaborates with the DASH/Dam1 complex in yeast or the Ska complex in vertebrates for robust microtubule attachment

• Integrates with signaling components of the spindle assembly checkpoint

• Forms part of the KMN network (KNL1, Mis12, Ndc80) critical for kinetochore function

When studying zebrafish NDC80, researchers should consider these interaction partners and potentially include them in reconstitution experiments. Particularly valuable would be co-expression with interaction partners like CENP-T, which has been shown to enhance NDC80's ability to track depolymerizing microtubules .

What evolutionary insights can be gained from comparing NDC80 across species?

Comparative analysis of NDC80 across species yields valuable insights:

• The NDC80 complex is evolutionarily conserved from yeast to humans

• Presence of multiple copies of the NDC80 complex at kinetochores is a conserved feature

• Species-specific adaptations may reflect differences in mitotic spindle architecture

• Zebrafish as a vertebrate model offers insights into NDC80 function in development

By studying the zebrafish homolog in comparison with better-characterized systems (human, yeast), researchers can identify both conserved mechanisms and species-specific adaptations in kinetochore function. This comparative approach can highlight functionally critical regions that have been maintained throughout evolution.

What are the current limitations in NDC80 research and how might they be addressed?

Current research limitations include:

• Incomplete understanding of the mechanism by which NDC80 harnesses force from depolymerizing microtubules

• Limited knowledge of species-specific aspects of NDC80 function in model organisms like zebrafish

• Technical challenges in reconstituting complete kinetochore structures in vitro

• Difficulty in visualizing NDC80 dynamics at high resolution in living cells

Future approaches to address these limitations include:

• Cryo-electron tomography of intact kinetochore-microtubule attachments

• Improved fluorescent tagging strategies for single-molecule imaging in live cells

• More complete in vitro reconstitutions including additional kinetochore components

• Comparative studies across multiple model organisms, including zebrafish

How can recombinant NDC80 contribute to understanding chromosome segregation disorders?

Recombinant NDC80 research can provide insights into chromosome segregation disorders:

• Aneuploidy in cancer cells often correlates with NDC80 dysregulation

• Developmental disorders may involve mutations affecting kinetochore function

• Mechanistic understanding may lead to targeted therapies for conditions involving chromosome instability

By establishing zebrafish NDC80 as a model system, researchers can leverage the advantages of this vertebrate model for developmental studies while exploring fundamental mechanisms of chromosome segregation relevant to human disease.