Recombinant Rat Protein MIS12 homolog (Mis12)

What is MIS12 and what is its function in cellular processes?

MIS12 is a highly conserved kinetochore protein essential for equal segregation of sister chromatids during mitosis. It serves as a component of the MIS12 complex (MIS12C), which also includes Pmf1, Nsl1, and Dsn1 subunits. Together, these proteins form a crucial part of the KMN network (Knl1, MIS12, Ndc80 complexes) that mediates chromosome attachment to the spindle microtubules during cell division .

The primary functions of MIS12 include:

• Facilitating proper kinetochore-microtubule attachments

• Ensuring normal chromosome alignment and segregation

• Contributing to kinetochore formation during mitosis

• Serving as a binding hub that connects other kinetochore components

Research has established that the absence or dysfunction of MIS12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei, indicating its critical role in maintaining genomic stability .

How should recombinant rat MIS12 be stored and handled for optimal stability?

Based on manufacturer recommendations for rat MIS12 recombinant proteins:

Storage Conditions:

• Short-term storage (2-4 weeks): 4°C

• Long-term storage: -20°C to -80°C

• Avoid repeated freeze-thaw cycles

Buffer Recommendations:

• PBS buffer is commonly used for commercial preparations

• Some preparations may include stabilizers such as glycerol (10%) and, for certain applications, addition of carrier proteins (0.1% HSA or BSA) is recommended for long-term storage

Handling Precautions:

• Minimize exposure to room temperature

• Aliquot the protein solution to avoid repeated freeze-thaw cycles

• When thawing, maintain protein on ice and use within the same day if possible

• For unstable variants, the addition of reducing agents may help maintain protein integrity

What expression systems are commonly used for producing recombinant rat MIS12?

Several expression systems have been used successfully for recombinant rat MIS12 production, each with specific advantages:

For rat MIS12 specifically, commercial sources use various systems:

• HEK293 cells for His(Fc)-Avi-tagged preparations that maintain native structure

• Mammalian cells for applications requiring proper folding and interactions with binding partners

When selecting an expression system, researchers should consider the downstream applications and whether post-translational modifications are critical for the experimental design .

What are the best methods for detecting rat MIS12 in cellular and tissue samples?

Detecting rat MIS12 in experimental samples requires careful consideration of available tools and methodologies:

Western Blotting:

• Optimal dilution range: 1:1000-1:4000 for commercially available antibodies

• Expected molecular weight: 25-28 kDa (observed), theoretical 24 kDa

• Validated in multiple cell lines (although primarily human: HEK-293, HeLa, HepG2)

• Sample preparation should include phosphatase inhibitors if phosphorylation status is important

Immunofluorescence:

• MIS12 typically shows distinct kinetochore localization during mitosis

• Co-staining with CENP-A can help identify centromeric regions

• Fixation method is critical: paraformaldehyde (4%) is commonly used

• Signal amplification may be necessary due to relatively low abundance

Immunoprecipitation:

• Can be used to study MIS12 interactions with binding partners

• Validated antibodies specific for rat MIS12 should be employed

• Cross-linking prior to lysis may help preserve transient interactions

Mass Spectrometry:

• Useful for identifying post-translational modifications

• Cross-linking and mass spectrometry (XL-MS) has been successfully used to study the architecture of the MIS12 complex

The choice of method depends on the specific research question, with Western blotting being most straightforward for expression analysis and immunofluorescence providing critical insights into localization during cell division .

How can I design experiments to investigate the interaction between rat MIS12 and CENP-C?

Investigating the MIS12-CENP-C interaction requires specialized approaches due to the dynamic nature of kinetochore complexes:

In Vitro Binding Assays:

• Recombinant protein pull-down:

  • Express and purify rat MIS12 complex (full complex recommended over individual subunits)

  • Express the N-terminal fragment of rat CENP-C (containing the MIS12-binding domain)

  • Perform pull-down assays with tagged proteins

  • Analyze binding by SDS-PAGE and Western blotting

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize either MIS12 complex or CENP-C fragment

  • Measure binding kinetics and affinity constants

  • Test the effect of Aurora B phosphorylation on binding affinity

Cellular Approaches:

• Proximity Ligation Assay (PLA):

  • Allows visualization of protein interactions in fixed cells

  • Requires specific antibodies against both rat MIS12 and CENP-C

  • Quantifiable signal represents interactions within 40nm

• FRET/FLIM Analysis:

  • Express fluorescently tagged MIS12 and CENP-C in rat cell lines

  • Measure energy transfer as an indicator of direct interaction

  • Can be performed in living cells to capture dynamic interactions

• Mutations and Deletions:

  • Generate CENP-C constructs lacking the M12BD (Mis12C-binding domain)

  • Express in rat cells and analyze kinetochore formation

  • The M12BD deletion has been shown to reduce centromeric localization of Aurora B during mitosis

Key considerations include examining how Aurora B phosphorylation at specific sites of DSN1 enhances the CENP-C:MIS12C interaction, as this regulatory mechanism appears conserved across species .

What approaches are most effective for studying MIS12 phosphorylation and its functional consequences?

The phosphorylation status of MIS12 complex components, particularly DSN1, significantly impacts function:

Identification of Phosphorylation Sites:

• Mass Spectrometry Analysis:

  • Purify MIS12 complex from rat cells at different cell cycle stages

  • Perform phosphopeptide enrichment (TiO₂ or IMAC)

  • Use LC-MS/MS to identify phosphorylation sites

  • Compare with known sites in human DSN1 (Ser100, Ser109)

• Phospho-specific Antibodies:

  • Develop or obtain antibodies against specific phosphorylation sites

  • Validate specificity using phosphatase treatments

  • Use for Western blotting and immunofluorescence applications

Functional Analysis:

• Phosphomimetic and Phospho-deficient Mutants:

  • Generate DSN1 constructs with S→E mutations (phosphomimetic) or S→A mutations (phospho-deficient)

  • Express in cells depleted of endogenous DSN1

  • Analyze kinetochore assembly, chromosome alignment, and microtubule attachments

• In Vitro Reconstitution:

  • Purify recombinant MIS12 complex components

  • Perform in vitro phosphorylation with Aurora B kinase

  • Assess binding to CENP-C and other partners before and after phosphorylation

• Inhibitor Studies:

  • Treat rat cells with Aurora B inhibitors

  • Analyze MIS12 complex assembly and function

  • Monitor chromosome segregation errors

The relationship between Aurora B phosphorylation and MIS12 function is particularly important, as studies have shown that Aurora B phosphorylation at specific DSN1 sites enhances the CENP-C:MIS12C interaction, impacting kinetochore assembly and function .

How does the structure of the rat MIS12 complex compare with that of other species, and what are the functional implications?

The MIS12 complex has a highly conserved structure across species, though with some notable variations:

Structural Characteristics:

• MIS12C forms an extended rod approximately 200 Å in length

• The four subunits (MIS12, PMF1, DSN1, NSL1) span the entire length of the complex

• The complex contains distinct subcomplexes: MIS12:PMF1 and DSN1:NSL1

• The "Head1" and "Head2" domains are critical for interactions with other proteins

Species Comparisons:

Functional Implications:

• The high conservation of MIS12 complex structure suggests critical functional constraints

• The MIS12:PMF1 subcomplex forms the "backbone" of MIS12C, essential for stability

• NSL1 subunit appears adaptable, maintaining significant stability even in the absence of DSN1 (as observed in Drosophila)

• The relative positions of domains are crucial for high-affinity binding of CENP-C to MIS12C

Understanding these structural features provides valuable insights for designing experiments and interpreting results when using rat models for kinetochore research .

What are the current challenges in studying MIS12 function in rat models, and how might they be addressed?

Several challenges exist when studying MIS12 in rat models, requiring specialized approaches:

Technical Challenges:

• Limited Rat-Specific Resources:

  • Fewer validated antibodies and reagents compared to human or mouse systems

  • Solution: Cross-validate antibodies raised against human MIS12 for rat specificity

  • Develop new rat-specific tools using recombinant proteins as immunogens

• Complex Formation and Stability:

  • The MIS12 complex is most stable and functional as a tetrameric complex

  • Individual subunits may not fold properly when expressed alone

  • Solution: Co-express all four subunits or use bicistronic/multicistronic expression systems

• Dynamic Nature of Kinetochore Assembly:

  • Kinetochore components assemble and disassemble throughout the cell cycle

  • Solution: Synchronize cells or use live-cell imaging with fluorescently tagged proteins

Experimental Approaches:

• CRISPR/Cas9 Gene Editing in Rat Cells:

  • Generate endogenously tagged MIS12 to study native expression and localization

  • Create conditional knockout models to study loss-of-function phenotypes

  • Engineer specific mutations to investigate phosphoregulation

• Single-Molecule Approaches:

  • Super-resolution microscopy (STORM, PALM) to visualize kinetochore architecture

  • Single-molecule tracking to study dynamics of MIS12 recruitment and turnover

• Proteomics Approaches:

  • BioID or APEX proximity labeling to identify interaction partners

  • Quantitative proteomics to measure stoichiometry of kinetochore complexes

• Integrative Structural Biology:

  • Combine crystallography, cryo-EM, and cross-linking mass spectrometry

  • Build comprehensive structural models of rat kinetochore assemblies

Addressing these challenges will require interdisciplinary approaches and adaptation of techniques developed for other model systems to rat cells and tissues .

How does MIS12 dysfunction contribute to chromosomal instability, and what experimental approaches can reveal these mechanisms?

MIS12 dysfunction has significant consequences for chromosomal stability, with several experimental approaches available to study these mechanisms:

Consequences of MIS12 Dysfunction:

• Misaligned metaphase chromosomes

• Lagging anaphase chromosomes

• Interphase micronuclei formation

• Abnormally extended metaphase spindle length

• Compromised kinetochore-microtubule attachments

Experimental Approaches:

• RNA Interference Studies:

  • siRNA or shRNA targeting rat MIS12

  • Analysis of mitotic defects using high-content imaging

  • Quantification of chromosome missegregation rates

  • Assessment of spindle checkpoint activation through hMad2 localization

• Live Cell Imaging:

  • Time-lapse microscopy of fluorescently labeled chromosomes

  • Measurement of mitotic timing and chromosome dynamics

  • Tracking of individual kinetochore movements

  • Correlative light and electron microscopy for ultrastructural analysis

• Chromosome Segregation Assays:

  • Fluorescence in situ hybridization (FISH) to detect aneuploidy

  • Micronucleus assays to quantify segregation errors

  • Single-cell sequencing to detect copy number variations

• Molecular Mechanism Studies:

  • Analysis of Aurora B activity and localization

  • Examination of kinetochore-microtubule attachment stability

  • Assessment of error correction pathways

  • Evaluation of spindle assembly checkpoint function

• Disease Model Relevance:

  • Connection to FTO-mediated stabilization of MIS12 and cellular senescence

  • Potential role in atherosclerosis through VSMC senescence

  • Links to other age-related pathologies

Understanding these mechanisms is particularly important given emerging evidence connecting MIS12 to cellular senescence pathways through FTO-mediated stabilization, potentially linking chromosome segregation fidelity to broader cellular aging processes .

How does MIS12 interact with the broader kinetochore network in rat cells, and what methodology best captures these interactions?

MIS12 serves as a central hub within the kinetochore network, with numerous interactions that can be studied through complementary approaches:

Key Interaction Partners:

• CENP-C: Direct binding through the N-terminal region of CENP-C

• Ndc80 complex: Forms part of the KMN network for microtubule attachment

• Knl1 complex: Together with MIS12 and Ndc80, completes the KMN network

• Aurora B: Regulatory kinase that phosphorylates DSN1 to enhance CENP-C binding

• NSL1, ZWINT, CASC5, PMF1, NDC80, DSN1, and CBX5: Directly interact with MIS12

Methodological Approaches:

• Biochemical Reconstitution:

  • Stepwise assembly of rat kinetochore subcomplexes in vitro

  • Measurement of binding affinities and kinetics

  • Analysis of cooperative assembly mechanisms

• Cross-linking Mass Spectrometry (XL-MS):

  • Identifies protein-protein interactions with residue-specific resolution

  • Provides distance restraints for structural modeling

  • Has been successfully applied to human MIS12 complex

• Proximity-dependent Labeling:

  • BioID or TurboID fusion to MIS12 to identify proximal proteins

  • APEX2 for temporal control of labeling during specific cell cycle stages

  • Comparison of interactomes across different mitotic phases

• Fluorescence Microscopy Techniques:

  • FRET pairs to measure direct interactions

  • FRAP to assess dynamic exchange of components

  • Three-dimensional structured illumination microscopy (3D-SIM) to resolve kinetochore subdomains

• Integrated Network Analysis:

  • Computational integration of interaction data

  • Network modeling to predict functional dependencies

  • Comparison with human kinetochore networks to identify conserved and divergent features

These approaches collectively provide a comprehensive view of the rat kinetochore interactome centered on MIS12, revealing both structural organization and dynamic regulation throughout the cell cycle .

What is the relationship between MIS12 function and cellular senescence pathways, and how can this be experimentally investigated?

Recent research has revealed an unexpected connection between MIS12 and cellular senescence pathways:

Emerging Research Findings:

• FTO (Fat Mass and Obesity-associated protein) may stabilize MIS12 protein via a proteasome-mediated pathway

• This stabilization appears to inhibit senescence in human mesenchymal progenitor cells (hMPCs)

• Similar mechanisms may operate in vascular smooth muscle cells (VSMCs)

• This pathway potentially links MIS12 to age-related diseases like atherosclerosis

Experimental Approaches to Investigate this Relationship:

• Protein Stability and Turnover:

  • Cycloheximide chase assays to measure MIS12 half-life

  • Proteasome inhibitors to assess degradation pathways

  • Ubiquitination assays to identify specific regulation mechanisms

  • Investigation of potential deubiquitinases that might counteract degradation

• FTO-MIS12 Interaction Studies:

  • Co-immunoprecipitation to confirm physical interaction

  • Domain mapping to identify interaction interfaces

  • CRISPR-based disruption of the interaction

  • Small molecule modulators of FTO activity

• Senescence Markers and Pathways:

  • β-galactosidase staining to identify senescent cells

  • Analysis of senescence-associated secretory phenotype (SASP)

  • Cell cycle analysis with MIS12 overexpression or depletion

  • Transcriptomic profiling to identify senescence pathways affected by MIS12

• Disease Model Approaches:

  • ApoE^-/- mice fed with high-fat diet as atherosclerosis models

  • Analysis of MIS12 and FTO levels in atherosclerotic plaques

  • Vascular smooth muscle cell culture systems

  • Genetic manipulation of MIS12 levels in relevant cellular and animal models

This emerging research direction suggests that MIS12 functions extend beyond its canonical role in mitosis, potentially influencing cellular aging processes with implications for age-related diseases .

How can advanced imaging techniques be optimized to study the dynamics of rat MIS12 during mitosis?

Advanced imaging techniques offer powerful tools for studying MIS12 dynamics, requiring specific optimization for rat systems:

Superresolution Microscopy Approaches:

• Structured Illumination Microscopy (SIM):

  • Achieves ~100 nm resolution, sufficient to resolve kinetochore subdomains

  • Optimal fluorophores: Alexa Fluor 488, 568, 647

  • Sample preparation: Critical to minimize spherical aberration with proper mounting media

  • Can be combined with live-cell imaging for dynamic studies

• Stochastic Optical Reconstruction Microscopy (STORM):

  • Provides ~20 nm resolution to distinguish individual proteins within complexes

  • Requires photoswitchable dyes (e.g., Alexa 647, Cy5.5)

  • Buffer optimization: Oxygen scavenging system with appropriate thiol concentration

  • Best for fixed samples to reveal precise spatial organization

Live-Cell Imaging Strategies:

• Fluorescent Protein Tagging:

  • Endogenous tagging via CRISPR/Cas9 preferred over overexpression

  • mNeonGreen or HaloTag provide superior brightness and photostability

  • Position of tag critical: C-terminal tagging generally preferred for MIS12

  • Validation that tagging doesn't disrupt function is essential

• Multi-dimensional Imaging:

  • 4D imaging (x,y,z,t) to capture complete kinetochore dynamics

  • Dual-color imaging to correlate MIS12 with binding partners

  • Spinning disk confocal provides optimal balance of speed and resolution

  • Deconvolution to enhance spatial resolution

Quantitative Analysis Methods:

• Tracking Algorithms:

  • Spot detection optimized for kinetochore size (~200 nm)

  • Tracking parameters adjusted for different mitotic phases

  • Analysis of inter-kinetochore distance as measure of tension

  • Statistical analysis of trajectory populations

• Fluorescence Correlation Techniques:

  • FRAP to measure residence time of MIS12 at kinetochores

  • FCS to determine local concentration and diffusion properties

  • Single-particle tracking for individual molecule dynamics

  • Correlation of dynamics with mitotic progression

Optimizing these techniques specifically for rat cells requires careful attention to sample preparation, imaging parameters, and analysis protocols tailored to the specific properties of rat kinetochores and cell division dynamics .

What are the most common issues when working with recombinant rat MIS12, and how can these be addressed?

Researchers frequently encounter several challenges when working with recombinant rat MIS12:

Protein Expression and Purification Challenges:

Functional Assay Challenges:

• Loss of Activity:

  • Ensure proper phosphorylation status is maintained

  • Include phosphatase inhibitors in lysis buffers

  • Consider the tetrameric nature of the complex for functional studies

• Non-specific Interactions:

  • Optimize salt concentration in binding assays

  • Include competing proteins (BSA) to reduce background

  • Validate interactions with multiple techniques

• Inconsistent Localization:

  • Verify cell cycle stage (MIS12 recruitment varies throughout cell cycle)

  • Ensure fixation method preserves kinetochore structure

  • Consider pre-extraction to reduce cytoplasmic background

Quality Control Approaches:

• Analytical Methods:

  • Size exclusion chromatography to verify complex formation

  • Mass spectrometry to confirm protein identity and modifications

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to optimize buffer conditions

• Functional Validation:

  • In vitro binding assays with known partners (CENP-C)

  • Microtubule co-sedimentation assays

  • Activity-based assays when appropriate

These troubleshooting strategies address the most common issues encountered when working with recombinant rat MIS12, particularly focusing on maintaining the integrity of the complex and ensuring functional activity .

How can contradictory experimental results regarding MIS12 function be reconciled and investigated further?

When facing contradictory results in MIS12 research, systematic approaches can help resolve discrepancies:

Common Sources of Contradictory Results:

• Cell Type and Species Differences:

  • MIS12 function may vary between species (human vs. rat)

  • Different cell types may have varying dependencies on MIS12

  • Example: The M12BD of CENP-C appears dispensable in some cell types but not others

• Experimental Conditions and Timing:

  • Cell cycle synchronization methods affect results

  • Acute vs. chronic protein depletion yields different phenotypes

  • Partial knockdown vs. complete knockout gives varying results

• Interacting Partners and Compensation:

  • Redundant mechanisms may mask phenotypes

  • Adaptive responses in stable cell lines

  • Context-dependent interactions with regulatory factors

Systematic Resolution Approaches:

• Standardized Experimental Design:

  • Direct side-by-side comparison of conditions

  • Consistent cell lines, antibodies, and reagents

  • Quantitative measurements rather than qualitative assessments

  • Blinded analysis to prevent bias

• Multi-pronged Technical Approaches:

  • Combine genetic approaches (RNAi, CRISPR) with different mechanisms

  • Use both fixed and live cell imaging

  • Employ biochemical and cellular assays in parallel

  • Determine dose-response relationships rather than single-point measurements

• Targeted Hypothesis Testing:

  • Design experiments specifically addressing the contradiction

  • Test boundary conditions where differences emerge

  • Identify variables that could explain divergent results

  • Consider kinetic aspects and temporal dynamics

Case Study Approach:
For example, if contradictory results exist regarding MIS12's role in Aurora B localization:

• Systematically vary Aurora B activity levels

• Test in multiple cell types and species

• Examine effects of MIS12 phosphorylation status

• Consider cell cycle stage and kinetochore attachment status

By adopting these systematic approaches, researchers can resolve contradictions and develop more nuanced understanding of context-dependent aspects of MIS12 function .

What quality control measures are essential when evaluating commercial recombinant rat MIS12 for research applications?

When selecting and validating commercial recombinant rat MIS12 for research, several quality control measures are essential:

Pre-Purchase Evaluation:

• Protein Specifications Review:

  • Expression system: Mammalian cells preferred for proper folding

  • Purity level: Minimum ≥80%, with ≥95% preferred for functional studies

  • Endotoxin levels: <1.0 EU per μg for cell culture applications

  • Tags: Consider impact of tags on function (His tags generally minimal impact)

• Documentation Assessment:

  • Certificate of analysis with actual test results

  • Lot-specific data on purity, activity, and endotoxin

  • Sequence verification information

  • Stability data and recommended storage conditions

Post-Purchase Validation:

• Physical Characterization:

  • SDS-PAGE to confirm size and purity (expected 25-28 kDa)

  • Western blot with anti-MIS12 antibody

  • Mass spectrometry to verify identity and detect modifications

  • Dynamic light scattering to assess aggregation state

• Functional Validation:

  • Binding assays with known partners (e.g., CENP-C N-terminus)

  • Complex formation with other MIS12C components

  • Phosphorylation status assessment if critical for experiments

  • Cell-based activity assays when appropriate

Storage and Handling Validation:

• Stability Testing:

  • Aliquot and test protein functionality after different storage durations

  • Compare fresh vs. freeze-thawed samples

  • Assess impact of different buffer conditions on stability

  • Document lot-to-lot variation if using multiple purchases

• Application-Specific Quality Control:

  • For structural studies: Verify monodispersity by SEC-MALS

  • For interaction studies: Confirm binding parameters match literature values

  • For cell-based assays: Test for endotoxin contamination

  • For in vivo applications: Additional sterility testing