Recombinant Bovine Protein MIS12 homolog (MIS12)

What is the structure and composition of the MIS12 complex?

The MIS12 complex is a four-subunit protein assembly that forms part of the KMN network (KNL1, MIS12, NDC80 complexes), which creates an interface connecting microtubules to centromeric chromatin during cell division. Structural analyses reveal that MIS12 forms an elongated rod-like structure with a long axis of approximately 200 Å (20 nm) . The four MIS12 complex subunits (MIS12, PMF1, DSN1, and NSL1 in humans) have similar topologies and span the entire length of the complex in the same orientation, clustering all N and C termini at opposite ends .
The complex organizes into two distinct subcomplexes: MIS12:PMF1 and DSN1:NSL1, with significant buried surface areas of 4,209 Ų and 3,575 Ų respectively, indicating tight interactions . While working with recombinant bovine MIS12, researchers should expect similar structural features due to the conservation of kinetochore components across vertebrates.

How does the MIS12 complex interact with other kinetochore components?

The MIS12 complex functions as a protein interaction hub at the outer kinetochore. The NSL1 subunit in particular serves as a scaffold supporting interactions with both the NDC80 and KNL1 complexes . Cross-linking studies have identified specific intersubunit contacts, including those between:

• MIS12 and DSN1

• MIS12 and NSL1

• DSN1 or KNL1 and the C-terminal tail of NSL1
  The C-terminal region of NSL1 is necessary for high-affinity binding to KNL1, as evidenced by the inability of truncated MIS12C NSL1-258 to bind KNL1 . At the inner kinetochore, CENP-C interacts with the MIS12 complex, primarily binding to Head1 of the complex and exploiting the interface between Head1 and the helical connector (αC helices of DSN1 and NSL1) .
  When designing interaction studies with bovine MIS12, researchers should focus on these conserved interaction surfaces while accounting for potential species-specific variations in binding affinities.

What phenotypes result from MIS12 complex depletion?

Depletion of MIS12 complex subunits in human or chicken cells results in several characteristic phenotypes that can serve as functional readouts in experimental designs:

• Mitotic delays with misaligned chromosomes

• Defects in chromosome biorientation

• Reduced centromere stretch

• Diminished kinetochore microtubule bundles

• Severe reduction in localization of the outer plate constituent Ndc80/HEC1

• Failure of checkpoint proteins like BubR1 and corona component CENP-E to accumulate to wild-type levels

• Reduced levels of inner kinetochore proteins CENP-A and CENP-H
  These phenotypes reflect the central role of the MIS12 complex in kinetochore assembly. The reduction in Ndc80 complex targeting (61-82% decrease in fluorescence intensity) is likely a direct consequence of the conserved physical interaction between the MIS12 and Ndc80 complexes .

What are optimal methods for reconstituting the recombinant MIS12 complex?

Successful reconstitution of the MIS12 complex requires careful consideration of expression systems and purification strategies. Based on published approaches with human MIS12:
Recommended methodology:

• Co-expression of all four subunits in bacterial systems rather than attempting to reconstitute from individually expressed proteins

• Expression using a polycistronic vector system for balanced stoichiometry

• Addition of solubility-enhancing tags (such as MBP or SUMO) to improve expression yields

• Sequential affinity purification steps followed by size exclusion chromatography
  Initial attempts to reconstitute human MIS12C subcomplexes have shown that NNF1–MIS12 and NSL1–DSN1 form discrete subcomplexes, but these subcomplexes exhibit only moderate solubility and stability . For bovine MIS12, it's advisable to test both full complex reconstitution and subcomplex formation to determine the most stable configurations.
  Analytical ultracentrifugation (AUC) is essential for verifying proper complex assembly and stoichiometry, as shown in the following data for human MIS12C:
  |Construct|Predicted molecular mass (kD)|Observed molecular mass (kD)|Stoichiometry|Sedimentation coefficient (S)|Frictional coefficient (f/f₀)|
  |--|--|--|--|--|--|
  |MIS12C NSL1-258|116.9|115.1|1:1:1:1|4.08|2.079|
  |MIS12C NSL1-258 + SPC24 57–197–SPC25 70–224|151.3|160|1:1:1:1:1:1|4.65|2.26|
  |MIS12C NSL1-258 + HP1|161.3|158|1:1:1:1:2|4.84|2.17|
  |SPC24 57–197–SPC25 70–224|34.4|36|1:1|2.46|1.59|
  |HP1|44.4|45.7|Homodimer|2.69|1.7|

How can researchers assess MIS12 complex interactions with binding partners?

Several complementary approaches can be employed to characterize the interactions of recombinant bovine MIS12 with its binding partners:
Isothermal Titration Calorimetry (ITC):

How does phosphorylation regulate MIS12 complex function?

Phosphorylation plays a critical role in regulating MIS12 complex interactions and function during the cell cycle. For recombinant bovine MIS12 studies, researchers should consider:
Aurora B Kinase Regulation:

• Aurora B phosphorylation enhances the interaction between MIS12C and CENP-C

• When studying recombinant bovine MIS12, consider both phosphorylated and non-phosphorylated forms

• In vitro phosphorylation can be performed using recombinant Aurora B kinase
  Methodological Approaches:

• Generate phosphomimetic mutants (S→D or S→E substitutions) at conserved Aurora B sites

• Compare binding affinities and functional activities of wild-type, phosphomimetic, and phosphodeficient (S→A) variants

• Use mass spectrometry to map and quantify phosphorylation sites

• Employ phospho-specific antibodies (if available) to monitor phosphorylation states
  Timing Considerations:
  MIS12 localization dynamics change throughout the cell cycle - it cycles on interphase kinetochores with a relatively rapid half-time (~7 seconds) but becomes stably associated with kinetochores in mitotic prophase . Experimental designs should account for these cell cycle-dependent changes.

What specialized techniques are useful for studying MIS12 complex structural dynamics?

Given the elongated structure of the MIS12 complex and its role in force transmission during chromosome segregation, several techniques are particularly valuable:
X-ray Crystallography:

• Has been used successfully to determine the structure of human MIS12 complex

• May require optimization for bovine MIS12

• Consider creating deletion constructs (like MIS12C ΔHead2) that may crystallize more readily
  Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Useful for mapping flexible regions and conformational changes upon binding partner interactions

• Can identify regions that become protected or more exposed during complex formation

• Particularly valuable for studying potential tension-dependent conformational rearrangements
  Single-Molecule Techniques:

• FRET-based approaches can monitor conformational changes in real-time

• Optical tweezers can probe force-dependent structural transitions

• Crucial for understanding how mechanical forces affect complex integrity
  Cryo-Electron Microscopy:

• Allows visualization of the complex in different functional states

• Can reveal conformational heterogeneity

• Particularly useful for larger assemblies such as the entire KMN network

How can researchers troubleshoot issues with MIS12 complex reconstitution?

When working with recombinant bovine MIS12, researchers may encounter several challenges:
Problem: Poor solubility or stability of the complex
Solutions:

• Try expression at lower temperatures (16-18°C)

• Include stabilizing agents in buffers (10% glycerol, low concentrations of detergents)

• Test different truncations based on structural information

• Consider co-expression with binding partners like CENP-C fragments that may stabilize the complex
  Problem: Suboptimal stoichiometry
  Solutions:

• Adjust expression vector design to balance expression levels

• Use sequential affinity tags on different subunits

• Apply ion exchange chromatography to separate complexes with different compositions

• Validate final complex composition by mass spectrometry
  Problem: Low activity in functional assays
  Solutions:

• Verify structural integrity by limited proteolysis and circular dichroism

• Check for proper phosphorylation states using Phos-tag gels or mass spectrometry

• Ensure binding partners used in assays are properly folded

• Consider species compatibility issues when using binding partners from different organisms

How can recombinant MIS12 be used to study species-specific kinetochore differences?

While kinetochore components are generally conserved across vertebrates, there may be species-specific adaptations in bovine MIS12 compared to human or other model organisms:
Comparative Binding Studies:

• Express recombinant MIS12 complexes from multiple species (bovine, human, mouse)

• Perform side-by-side binding assays with conserved partners (NDC80 complex, KNL1, CENP-C)

• Quantify differences in binding affinities and kinetics

• Map species-specific interaction interfaces through mutagenesis
  Structural Comparison Approaches:

• Use hydrogen-deuterium exchange mass spectrometry to compare dynamics

• Perform cross-linking mass spectrometry to identify potential differences in spatial arrangements

• Consider species-specific post-translational modifications
  One notable example of species differences comes from Drosophila, where no DSN1 homolog has been identified, yet the remaining three subunits form a stable MIS12 complex . This demonstrates that while the core architecture is conserved, significant adaptations can occur across species.

What controls are essential when performing functional assays with recombinant MIS12?

When designing experiments with recombinant bovine MIS12, include these critical controls:
Structural Integrity Controls:

What cell-based assays are most informative for validating recombinant MIS12 function?

To validate that recombinant bovine MIS12 retains proper functionality:
Complementation Assays:

• Deplete endogenous MIS12 complex components using siRNA or CRISPR in bovine cell lines

• Express siRNA-resistant wild-type or mutant recombinant bovine MIS12

• Assess rescue of phenotypes such as chromosome misalignment, mitotic delay, and kinetochore protein localization

• Quantify kinetochore fluorescence intensities to measure recruitment of downstream components
  Phenotypic Endpoints to Measure:

• Chromosome alignment at metaphase (normal vs. misaligned)

• Mitotic timing (using time-lapse microscopy)

• Kinetochore-microtubule attachment stability

• Localization of outer kinetochore components (especially NDC80 complex)

• BubR1 recruitment (which can be reduced by 42% in MIS12 complex-depleted cells)