HEI10 Antibody

What is HEI10 and why is it important in meiosis research?

HEI10 (Human Enhancer of Invasion-10) is a conserved E3 ubiquitin ligase that plays a crucial role in meiotic crossover formation. It facilitates the accurate segregation of homologous chromosomes during meiosis by promoting the formation of crossovers at designated sites . The importance of HEI10 lies in its antagonistic relationship with other proteins such as the SUMO ligase RNF212 in mammals, where it helps determine which recombination sites will become crossovers versus non-crossovers . Without proper HEI10 function, organisms typically show severe meiotic defects and reduced fertility, making it a critical protein for studying the molecular mechanisms of meiosis and fertility .

Methodologically, when investigating HEI10's role, researchers should design experiments that track its dynamic localization throughout meiotic prophase I, as it shows a characteristic pattern transitioning from numerous small foci to fewer bright foci that mark eventual crossover sites .

How does HEI10 expression and localization change during meiotic progression?

HEI10 exhibits a highly dynamic localization pattern throughout meiosis. The protein first appears as distinct punctate foci at early leptotene, with almost all foci localizing on chromosomal axes marked by proteins like REC8 . As meiosis progresses to zygotene, these foci increase in number and begin to form linear arrays of dots or short lines along chromosomes .

By late zygotene/early pachytene, HEI10 extends as linear signals along entire chromosomes, though with noticeable discontinuities or gaps . During mid-pachytene, these linear signals largely split into arrays of dots. The most distinctive change occurs at late pachytene, when certain foci become significantly brighter while others remain faint, with approximately 24-25 bright foci per cell (in rice) . By diplotene, only these bright foci remain, and they precisely mark chiasma positions at diakinesis . HEI10 signals disappear entirely by metaphase I .

This pattern of HEI10 accumulation—transitioning from numerous weak foci to a few bright, enlarged foci—has been used as a quantitative "developmental clock" to study the progression of meiosis .

What is the structure of HEI10 and how does it affect antibody design?

HEI10 is a modular protein containing several functional domains, with the N-terminal RING domain (approximately amino acids 3-41 in rice) being particularly critical for its E3 ubiquitin ligase activity . When designing antibodies against HEI10, researchers must carefully consider which domain to target for optimal specificity and functionality.

For antibody generation, researchers have successfully used fragments of HEI10 cDNA for expression and immunization. In one approach documented in rice studies, a 919 bp fragment of HEI10 cDNA was amplified, ligated into an expression vector (pGEX-4T-2), and transformed into BL21 (DE3) cells . The expressed fusion proteins accumulated primarily as inclusion bodies, which were then isolated by gel electrophoresis, stained with Coomassie blue, and extracted for immunization in rabbits and mice .

When designing HEI10 antibodies, researchers should validate specificity by confirming absence of signal in HEI10-deficient mutants . This approach ensures that the antibody specifically recognizes HEI10 and not other related proteins.

How can HEI10 antibodies be generated for immunolocalization studies?

Generating effective HEI10 antibodies requires careful selection of the antigen sequence and expression system. Based on documented protocols, researchers should consider the following methodological approach:

• Antigen selection: Choose a unique fragment of HEI10 cDNA that will produce a protein fragment specific to HEI10. In published work, a 919 bp fragment has been successfully used .

• Expression vector preparation: Amplify the selected fragment using PCR with primers containing appropriate restriction sites (e.g., BamHI-XhoI) for directional cloning into an expression vector such as pGEX-4T-2 .

• Bacterial expression: Transform the recombinant plasmid into a bacterial expression strain such as BL21 (DE3) and induce protein expression with IPTG (typically 0.2 mM) .

• Protein purification: Since HEI10 fusion proteins tend to form inclusion bodies, use gel electrophoresis followed by Coomassie blue staining to identify and isolate the protein band of interest .

• Immunization: Prepare the isolated protein for immunization in animals such as rabbits and mice to generate polyclonal antibodies .

• Antibody validation: Confirm antibody specificity by testing on tissues from wild-type and hei10 mutant organisms. A specific antibody should show clear signals in wild-type but no signals in hei10 mutants .

This methodology has been proven effective for generating antibodies that can detect both the linear and focal patterns of HEI10 localization during different stages of meiosis.

What are the optimal immunostaining protocols for HEI10 detection in meiocytes?

For optimal HEI10 detection in meiocytes, researchers should follow these methodological steps:

• Sample preparation: Collect appropriate reproductive tissues (anthers in plants, testicular tissue in animals) at stages when meiosis is occurring. Fix tissues promptly in an appropriate fixative such as paraformaldehyde.

• Slide preparation: Prepare chromosome spreads or squashes according to standard cytological techniques for the species being studied.

• Antibody incubation: For dual immunolocalization, which is often necessary to provide context for HEI10 localization, use appropriate combinations of antibodies such as:

  • Anti-HEI10 (rabbit or mouse) typically at 1:500 dilution

  • Anti-REC8 (rat) at 1:500 dilution to mark chromosome axes

  • Anti-SMC3 (rat) at 1:500 dilution as another chromosome axis marker

  • Anti-ZYP1 (guinea-pig) at 1:500 dilution to mark synaptonemal complexes

• Washing steps: Perform washing steps (typically 3 × 5 minutes in 1× PBS with 0.1% Triton) before and after each antibody incubation .

• Secondary antibody selection: Use appropriate fluorescently-labeled secondary antibodies matching the host species of primary antibodies, typically at 1:200 dilution:

  • Anti-rabbit Alexa Fluor 555 for HEI10 detection

  • Anti-rat Alexa Fluor Plus 488 for REC8 or SMC3

  • Anti-guinea-pig Alexa Fluor 647 for ZYP1

• Imaging: For high-resolution imaging of HEI10 foci, 3D structured illumination microscopy (3D-SIM) is recommended, using an appropriate microscope setup such as a Zeiss Elyra PS1 with an EM-CCD camera and Plan-Apochromat ×63, NA 1.40 oil objective .

• Image acquisition parameters: Manually adjust laser power and camera gain values for each cell to optimize image quality and minimize reconstruction artifacts. Use immersion oil with a refractive index of approximately 1.515, optimized for the channels being used .

• Image analysis: Assess bleaching and contrast of raw images using appropriate software tools like the SIMcheck plugin for FIJI .

How can researchers quantify HEI10 foci for accurate data interpretation?

Quantifying HEI10 foci requires careful image analysis and standardized procedures:

• Stage identification: Accurately identify meiotic stages based on chromatin morphology and synaptonemal complex formation using axis markers like REC8 or SMC3 .

• Foci counting strategies:

  • For early stages with numerous foci: Capture z-stack images and use 3D analysis software to identify and count individual foci.

  • For later stages: Distinguish between bright (crossover-designated) and faint (non-crossover) foci based on intensity thresholds .

• Quantification metrics:

  • Total foci number per nucleus (e.g., mean of 297.1 foci at late leptotene in rice)

  • Number of bright foci per nucleus at late pachytene/diplotene (e.g., 24.5-24.7 in rice)

  • Inter-foci distances along chromosomes to analyze crossover interference

• Statistical analysis: Apply appropriate statistical tests when comparing foci numbers between different genotypes or treatments. For variance comparisons, tests like Levene's test for equality of variances can be used .

• Developmental progression analysis: Use HEI10 accumulation patterns as a "developmental clock" to normalize and compare other meiotic events across different cells or genotypes .

This quantitative approach allows researchers to make meaningful comparisons between different experimental conditions and genotypes, providing insights into the regulation of crossover formation.

How does HEI10 dosage affect crossover frequency and distribution?

HEI10 has been identified as a dosage-sensitive regulator of crossing over. Experimental evidence demonstrates that alterations in HEI10 levels directly impact crossover numbers and distribution:

Methodologically, researchers investigating HEI10 dosage effects should:

• Generate systems with precisely controlled HEI10 expression levels, either through genetic manipulation (heterozygotes, overexpression lines) or computational modeling.

• Quantify crossover numbers through direct cytological observation of chiasmata or immunolocalization of late crossover markers like MLH1.

• Analyze not just means but also variance in crossover numbers, as HEI10 overexpression affects both parameters .

• Consider the "coarsening dynamics" model, which explains how HEI10 foci compete for limited resources during meiosis, leading to the designation of some recombination sites as crossovers .

Research indicates that manipulating HEI10 levels could be a potential strategy for modifying recombination patterns in breeding programs, as human recombination rate has been associated with a variant in the 5' UTR of CNNB1IP1 (HEI10) that potentially alters expression levels .

What is the relationship between HEI10 and other meiotic proteins in determining crossover fate?

HEI10 functions within a complex network of proteins that collectively determine crossover versus non-crossover fates at recombination sites. The key interactions include:

Researchers investigating these relationships should design co-immunolocalization experiments with specific antibodies against the relevant proteins and analyze their spatial and temporal relationships throughout meiosis. For accurate interpretation, it's crucial to study these relationships in both wild-type and various mutant backgrounds to understand dependency relationships.

What are the functional implications of the HEI10 RING domain for fertility?

The RING domain of HEI10 is a critical functional element located at the N-terminus (amino acids 3-41 in rice) that enables its E3 ubiquitin ligase activity . Recent research has revealed fascinating insights into the differential requirements of this domain for male versus female fertility:

• Male fertility: The RING domain appears to be absolutely essential for male fertility. In rice, the sh1 mutant, which produces a truncated HEI10 protein missing its N-terminal RING domain, exhibits complete male sterility . In male meiocytes of this mutant, HEI10 foci were detected in only a small portion of cells (3.4%), indicating severely compromised HEI10 function .

• Female fertility: Intriguingly, the RING domain is not absolutely required for female fertility. The same sh1 mutant in rice retains partial female fertility despite lacking the RING domain . Furthermore, expressing the truncated HEI10 (without the RING domain) in hei10 null lines partially restores female fertility .

• Cellular localization: The truncated HEI10 protein without the RING domain can still correctly localize to the nucleus and potentially interact with other meiotic proteins . This suggests that nuclear localization signals and protein interaction domains are located in other regions of the protein.

• Protein structure: SWISS-MODEL analysis has shown that the absence of the N-terminal RING domain does not significantly affect the protein structure of the remaining part of HEI10 .

This differential requirement of the RING domain provides researchers with a valuable tool to explore the similarities and differences between male and female meiosis. The phenomenon also raises important questions about potentially different mechanisms of crossover formation between sexes, a phenomenon known as heterochiasmy .

How can researchers troubleshoot weak or absent HEI10 antibody signals?

When facing issues with HEI10 immunodetection, researchers should methodically address several potential causes:

A systematic approach to testing these variables will help identify and resolve specific issues with HEI10 immunodetection.

How should researchers interpret and address HEI10 localization variability between experiments?

Variability in HEI10 localization patterns between experiments is a common challenge. Researchers should consider these methodological approaches:

• Standardize staging criteria: Establish clear criteria for identifying each meiotic stage based on chromosome morphology and synaptonemal complex formation. Use markers like REC8 or SYCP3 for axis structures and ZYP1 for synaptonemal complexes .

• Quantitative assessment: Rather than relying on qualitative observations, quantify HEI10 parameters:

  • Foci number per nucleus

  • Foci intensity (mean, maximum, distribution)

  • Foci size

  • Co-localization percentages with other proteins

• Internal controls: Include internal controls within each experiment, such as:

  • Wild-type samples processed alongside experimental samples

  • Co-staining with markers of known behavior (e.g., REC8, ZYP1)

  • Multiple meiotic stages on the same slide to confirm stage-specific patterns

• Technical replication: Perform technical replicates within each experiment and biological replicates across independent experiments.

• Statistical analysis: Apply appropriate statistical tests to determine whether observed variations are within expected ranges or represent significant differences.

• Developmental normalization: Use the "developmental clock" approach based on HEI10 accumulation patterns to normalize observations across cells at slightly different sub-stages of meiotic progression .

By implementing these approaches, researchers can distinguish between technical variability and biologically meaningful differences in HEI10 localization.

What imaging techniques provide the most accurate visualization of HEI10 dynamics?

Optimal visualization of HEI10 dynamics requires advanced imaging approaches:

• 3D Structured Illumination Microscopy (3D-SIM): This super-resolution technique is particularly effective for visualizing HEI10 foci and has been successfully employed in recent studies . Key methodological considerations include:

  • Using a microscope system such as Zeiss Elyra PS1 equipped with an EM-CCD camera

  • Employing a high-magnification, high-NA objective (e.g., Plan-Apochromat ×63, NA 1.40)

  • Imaging with multiple stripe angles (three) and phases (five)

  • Capturing z-stacks at small interval sizes (e.g., 0.0909 μm)

  • Using immersion oil with an appropriate refractive index (e.g., 1.515) optimized for the fluorescent channels

• Live imaging approaches: For tracking dynamic changes in HEI10 localization in real-time:

  • Consider fluorescent protein tagging of HEI10 with a tag that minimally disrupts function

  • Use spinning disk confocal microscopy for reduced photobleaching during time-lapse imaging

  • Implement gentle imaging conditions to minimize phototoxicity

• Image processing and analysis:

  • Assess bleaching and contrast of raw images using specialized plugins like SIMcheck for FIJI

  • Implement deconvolution algorithms to improve signal-to-noise ratio

  • Use 3D visualization software for accurate representation of spatial relationships

  • Apply quantitative analysis tools to track changes in foci number, intensity, and size over time

• Multi-channel imaging: For understanding relationships with other proteins:

  • Use fluorophores with minimal spectral overlap (e.g., Alexa Fluor 488, 555, and 647)

  • Implement sequential scanning to minimize channel crosstalk

  • Include appropriate controls for testing antibody specificity and fluorophore performance

These advanced imaging approaches, when implemented correctly, allow researchers to accurately visualize and quantify the dynamic behavior of HEI10 throughout meiosis.

How do researchers reconcile differences in HEI10 behavior across species?

While HEI10's fundamental role in crossover formation is conserved, significant species-specific differences have been documented. Researchers should consider these methodological approaches when comparing across species:

• Systematic comparative analysis: When comparing HEI10 behavior across species, create standardized frameworks for:

  • Temporal dynamics (relative to meiotic stages)

  • Spatial patterns (number and distribution of foci)

  • Molecular interactions (with other recombination proteins)

  • Genetic requirements (dependency relationships)

• Accounting for genomic differences:

  • Genome size and chromosome number affect HEI10 distribution patterns

  • Recombination landscape differences alter HEI10 localization

  • Crossover interference strength varies between species

• Evolutionary context:

  • Consider phylogenetic relationships when comparing HEI10 behavior

  • Recognize that differences may reflect adaptations to species-specific meiotic challenges

  • Account for potential redundancy with paralogous proteins in some species

• Technical considerations:

  • Different antibodies may have varying specificities and sensitivities

  • Fixation and immunodetection protocols optimal for one species may not work well for others

  • Imaging parameters may need adjustment based on cellular architecture differences

• Case comparison example: Mammals vs. Plants

  • In mammals, HEI10 works antagonistically with RNF212 (SUMO ligase)

  • In plants like rice, HEI10's early localization shows high co-localization with MER3

  • In rice, HEI10 appears as early as leptotene, while in mice it was reported to be visible from early-mid pachytene

  • The RING domain shows differential requirements for male versus female fertility in rice , a phenomenon that should be investigated in other species

By carefully considering these factors, researchers can distinguish between true biological differences and technical artifacts when comparing HEI10 behavior across species.

What criteria should be used to assess HEI10 function in novel experimental systems?

When investigating HEI10 function in new experimental systems or under novel conditions, researchers should establish a comprehensive assessment framework:

• Molecular characterization:

  • Confirm HEI10 sequence identity and conservation of key domains (RING domain, coiled-coil regions)

  • Verify expression timing and levels during meiosis

  • Assess post-translational modifications

• Localization analysis:

  • Document the full spatiotemporal pattern throughout meiosis

  • Quantify key parameters: foci number, intensity, dynamics

  • Compare to patterns in well-characterized systems

• Functional analysis:

  • Generate loss-of-function mutants (null and domain-specific)

  • Create gain-of-function variants (overexpression, constitutive activation)

  • Assess fertility phenotypes quantitatively (male and female separately)

• Cytological assessment:

  • Analyze chromosome behavior throughout meiosis

  • Quantify crossover frequency and distribution

  • Evaluate synapsis completion and timing

• Protein interaction network:

  • Identify interaction partners through IP-MS or Y2H approaches

  • Verify co-localization with key meiotic proteins

  • Determine dependency relationships through analysis in various mutant backgrounds

• Standardized metrics for crossover assessment:

  • Total chiasmata count per cell

  • Percentage of chromosome pairs forming at least one chiasma

  • Interference measurements (coefficient of coincidence)

  • Crossover distribution patterns along chromosomes

This framework provides a comprehensive approach to characterizing HEI10 function in any experimental system, facilitating meaningful comparisons across studies.

How can computational modeling inform our understanding of HEI10 dynamics in crossover regulation?

Computational modeling has emerged as a powerful approach for understanding the complex dynamics of HEI10 in regulating crossover formation:

• Coarsening dynamics models: Recent research has employed computational modeling of "coarsening dynamics" to explain how HEI10 foci compete for limited resources during meiosis, leading to the designation of some recombination sites as crossovers . This modeling approach can:

  • Quantitatively reproduce experimental observations of crossover patterns

  • Predict outcomes of genetic manipulations

  • Test hypotheses about regulatory mechanisms

• Key parameters for accurate modeling:

  • Total cellular HEI10 levels and their variance

  • Dynamics of HEI10 exchange between the nucleoplasm and chromosome-bound sites

  • Interaction strengths with partner proteins

  • Spatial constraints along chromosomes

• Model validation approaches:

  • Compare predicted and observed foci number distributions

  • Test model predictions under altered conditions (e.g., HEI10 overexpression)

  • Use statistical measures like variance (σ²) to assess fit to experimental data

• Applications of validated models:

  • Predict effects of novel genetic variations

  • Design targeted interventions to modify recombination patterns

  • Understand evolutionary constraints on crossover regulation

• Integration with other data types:

  • Complement modeling with live imaging to validate dynamic aspects

  • Incorporate structural biology data on protein-protein interactions

  • Include genomic context (recombination hotspots, chromatin features)

When researchers increased total HEI10 within a nucleoplasmic coarsening model to 3.5 times its original amount, it accurately predicted the increase in crossover number but not the observed increase in variance . This led to refinements of the model to include increased standard deviation in total cellular HEI10 levels, which then successfully recapitulated both the experimentally observed increase in crossover number and variance .

This example illustrates how iterative refinement of computational models, guided by experimental data, can provide deeper insights into the mechanisms of HEI10-mediated crossover regulation.

What are the most promising techniques for manipulating HEI10 function in breeding applications?

The dosage-sensitive nature of HEI10 makes it an attractive target for manipulating recombination in plant and animal breeding. Researchers should consider these promising methodological approaches:

• Precise genetic engineering strategies:

  • CRISPR/Cas9-mediated modification of HEI10 coding regions to alter protein activity

  • Targeted modification of regulatory elements to fine-tune expression levels

  • Creation of chimeric HEI10 variants with altered domain functions

• Expression modulation approaches:

  • Inducible overexpression systems for temporally controlled HEI10 elevation

  • RNA interference or antisense strategies for partial knockdown

  • Tissue-specific promoters for localized effects

• Protein engineering tactics:

  • Development of HEI10 variants with modified RING domain activity

  • Creation of separation-of-function mutants that affect male or female meiosis differentially

  • Design of stabilized HEI10 variants with extended half-life

• Combinatorial approaches:

  • Simultaneous manipulation of HEI10 and its antagonists (e.g., RNF212 in mammals)

  • Coordinated modification of multiple crossover regulators

  • Integration with other recombination-modifying strategies

• Experimental design considerations:

  • Careful phenotypic assessment of fertility and viability

  • Comprehensive analysis of crossover number and distribution

  • Evaluation of potential off-target effects on other cellular processes

The discovery that the RING domain of HEI10 is essential for male but not female fertility in rice opens particularly intriguing possibilities for sex-specific manipulation of recombination, which could have valuable applications in hybrid seed production.

How might emerging imaging technologies enhance our understanding of HEI10 dynamics?

Emerging imaging technologies offer exciting possibilities for deeper insights into HEI10 behavior:

• Advanced super-resolution approaches:

  • PALM/STORM techniques for single-molecule resolution of HEI10 distribution

  • Expansion microscopy to physically enlarge specimens for enhanced resolution

  • Lattice light-sheet microscopy for rapid 3D imaging with reduced photobleaching

• Live-cell imaging innovations:

  • Photoactivatable or photoconvertible fluorescent tags for pulse-chase analysis of HEI10 dynamics

  • FRET-based sensors to detect HEI10 interactions with partner proteins in real-time

  • Light-sheet microscopy for long-term imaging with minimal phototoxicity

• Correlative microscopy approaches:

  • Combining fluorescence imaging with electron microscopy for ultrastructural context

  • Integrating super-resolution imaging with chromosome conformation capture techniques

  • Correlating optical imaging with mass spectrometry imaging for protein modification analysis

• Quantitative analysis frameworks:

  • Machine learning algorithms for automated detection and tracking of HEI10 foci

  • Computational modeling to predict and interpret dynamic behaviors

  • Development of standardized metrics for cross-study comparisons

• Multiplexed imaging methods:

  • Exchange-PAINT or DNA-PAINT for highly multiplexed protein detection

  • Sequential imaging approaches to track numerous proteins in the same sample

  • Spectral unmixing for simultaneous visualization of multiple fluorophores

These technological advances will enable researchers to address fundamental questions about HEI10 dynamics with unprecedented precision, potentially revealing new mechanisms of crossover regulation.

What are the unresolved questions regarding HEI10's molecular mechanism of action?

Despite significant progress in understanding HEI10's role in meiosis, several critical questions remain unresolved:

• Substrate specificity:

  • What are the direct ubiquitination targets of HEI10's E3 ligase activity?

  • How does HEI10 distinguish between different potential substrates?

  • What role does the RING domain play in substrate recognition versus catalytic activity?

• Regulatory mechanisms:

  • How is HEI10 expression and localization regulated throughout meiosis?

  • What factors control the transition from numerous weak foci to few bright foci?

  • What role do post-translational modifications play in modulating HEI10 activity?

• Molecular interactions:

  • How does HEI10 interact with the synaptonemal complex?

  • What is the molecular basis for the antagonistic relationship with RNF212 in mammals?

  • How does HEI10 coordinate with other crossover-promoting factors?

• Sex-specific differences:

  • What molecular mechanisms explain the differential requirement of the RING domain for male versus female fertility in rice ?

  • Are there fundamental differences in HEI10 function between male and female meiosis across species?

  • How do these differences contribute to heterochiasmy (different recombination rates between sexes)?

• Evolutionary considerations:

  • How conserved is the molecular function of HEI10 across diverse eukaryotic lineages?

  • What structural features have been maintained or diverged through evolution?

  • How has HEI10 co-evolved with other components of the meiotic recombination machinery?

Addressing these questions will require integrated approaches combining structural biology, biochemistry, genetics, genomics, and advanced imaging techniques. The recent discovery of differential requirements for the RING domain in male versus female fertility provides a particularly intriguing entry point for understanding sex-specific aspects of meiotic recombination regulation.