Recombinant Xenopus laevis Microtubule-associated protein RP/EB family member 2 (mapre2)

What is the molecular structure of Xenopus laevis MAPRE2 and how does it compare to human MAPRE2?

The full-length Xenopus laevis MAPRE2 (also known as xMad2B) encodes a 211 amino acid protein that shares high homology with human MAPRE2 . The protein contains a conserved calponin homology (CH) domain at the N-terminus, which is responsible for tracking and interacting with the plus-end tips of growing microtubules. This structural conservation points to an important evolutionary function of this protein in higher eukaryotes .

The homology between human and Xenopus MAPRE2 proteins makes X. laevis an excellent model organism for studying the fundamental mechanisms of MAPRE2 function that may translate to human biology. Comparative sequence analysis reveals that MAPRE2 belongs to a family of three proteins (EB1, EB2, and EB3) that share high structural similarity but have distinct functions in microtubule dynamics .

What are the tissue-specific expression patterns of MAPRE2 in Xenopus laevis?

Expression analysis across developmental stages indicates that MAPRE2 may play roles in both embryonic development and adult tissue maintenance. The expression pattern distinguishes MAPRE2 from other members of the EB family, which may have more ubiquitous expression profiles across tissues .

What are the most effective methods for isolating recombinant Xenopus laevis MAPRE2?

For successful isolation of recombinant Xenopus laevis MAPRE2, researchers should consider the following methodological approach:

• cDNA Cloning: The full-length MAPRE2 cDNA can be isolated from Xenopus laevis tissues, particularly from testis or oocytes where expression is highest. This can be achieved by screening a cDNA library or using RT-PCR with gene-specific primers designed based on available sequence information .

• Expression Systems: Several expression systems can be employed:

  • Bacterial expression: Using E. coli with a His-tag for affinity purification (>95% purity can be achieved)

  • Cell-free protein synthesis: Allows for rapid production with approximately 70-80% purity

  • Mammalian cell expression: HEK-293 cells can produce high-quality recombinant protein with >90% purity

• Purification Protocol: For high-purity isolation:

  • Lyse cells in buffer containing 10 mM Hepes, 500 mM NaCl, pH 7.4

  • Perform affinity chromatography using His-tag or Strep-tag

  • Further purify using size-exclusion chromatography

  • Final product can be lyophilized with 5% trehalose for stability

This systematic approach enables isolation of recombinant MAPRE2 suitable for structural and functional studies.

How can researchers verify the functional integrity of purified recombinant Xenopus laevis MAPRE2?

Verification of functional integrity is crucial before proceeding with experiments. The following methodological approaches are recommended:

• Co-immunoprecipitation assays: Test the ability of purified MAPRE2 to interact with known binding partners, particularly PRCC protein. This interaction is a key indicator of proper folding and functional integrity .

• Microtubule binding assays: Assess the protein's ability to bind to microtubules in vitro using purified tubulin. Functional MAPRE2 should specifically associate with the growing plus-ends of microtubules .

• Subcellular localization: When introduced into cells, functional MAPRE2 should exhibit characteristic localization patterns at microtubule plus-ends, which can be visualized using fluorescence microscopy .

• Microtubule dynamics assays: Measuring changes in microtubule growth velocity and distance in the presence of purified MAPRE2 can confirm its functional impact on microtubule dynamics. Typical values for control conditions (8.05±0.18 μm/min velocity and 5.76±0.19 μm growth distance) provide a baseline for comparison .

These validation steps ensure that the recombinant protein maintains its native functions and is suitable for downstream applications.

How can recombinant Xenopus laevis MAPRE2 be used to study microtubule dynamics?

Recombinant Xenopus laevis MAPRE2 provides a powerful tool for studying microtubule dynamics through several experimental approaches:

• In vitro microtubule plus-end tracking: Using total internal reflection fluorescence (TIRF) microscopy, fluorescently labeled recombinant MAPRE2 can be observed tracking growing microtubule plus-ends. This allows direct measurement of:

  • Growth velocity (typically 8-10 μm/min under normal conditions)

  • Growth distance (typically 5-8 μm under normal conditions)

  • Catastrophe frequency

  • Rescue events

• Cell-based assays: Introduction of recombinant MAPRE2 into cells allows for:

  • Complementation studies in MAPRE2-depleted cells

  • Structure-function analysis using mutated versions

  • Competition assays with endogenous MAPRE2

• Xenopus egg extract system: This cell-free system provides a physiologically relevant environment for studying MAPRE2's role in microtubule organization during mitosis and meiosis. The high concentration of cellular components in Xenopus egg extracts makes it particularly suitable for biochemical and microscopy-based studies .

When designing these experiments, researchers should consider using fluorescently tagged versions of MAPRE2 (e.g., GFP-MAPRE2) for live imaging studies, while ensuring that the tag does not interfere with protein function.

What are the key interactions between MAPRE2 and PRCC in Xenopus laevis and how can they be studied?

The interaction between MAPRE2 (xMad2B) and PRCC in Xenopus laevis represents a critical aspect of cell cycle control. To study this interaction, researchers can employ the following methods:

• Co-immunoprecipitation: This remains the gold standard for confirming protein-protein interactions. When performed with Xenopus laevis proteins, this technique has demonstrated that despite its distinct amino terminus, xPRCC still interacts with xMad2B .

• Immunofluorescence analysis: This technique has revealed that xPRCC is capable of shuttling xMad2B to the nucleus where it exerts its mitotic checkpoint function . The methodology involves:

  • Fixation of Xenopus laevis cells

  • Immunostaining with specific antibodies against both proteins

  • Confocal microscopy for co-localization analysis

• Functional translocation assays: These can be designed to test the ability of PRCC to translocate MAPRE2 to the nucleus:

  • Cells can be transfected with recombinant tagged versions of both proteins

  • Nuclear/cytoplasmic fractionation followed by Western blotting

  • Live-cell imaging with fluorescently tagged proteins

• Mutational analysis: Create structure-based mutations in key domains of either protein to map the interaction surfaces and determine their functional significance in cell cycle control .

These approaches can help elucidate the molecular mechanisms by which MAPRE2 and PRCC cooperate to regulate cell cycle progression in Xenopus laevis and potentially inform similar processes in human cells.

What methods are most effective for MAPRE2 gene manipulation in Xenopus laevis?

Given the tetraploid nature of the Xenopus laevis genome and the presence of two copies of most genes, effective genetic manipulation requires specialized approaches:

• CRISPR/Cas9 genome editing: This has emerged as a preferred method for generating MAPRE2 knockout or knock-in models in Xenopus:

  • Design gRNAs targeting conserved regions in both homeologs (L and S chromosomes)

  • For complete knockout, target early exons (e.g., exon 2) as demonstrated in zebrafish studies of MAPRE2

  • Validate editing efficiency using sequencing and protein expression analysis via Western blotting

  • Phenotypic analysis can be performed at various developmental stages

• Morpholino-based knockdown: This approach remains useful for transient knockdown studies:

  • Design morpholinos targeting splice acceptor sites (e.g., exon 2 of MAPRE2)

  • Inject at 1-2 cell stage embryos (typically 5-10 ng)

  • Validate knockdown efficiency by Western blotting

• mRNA overexpression: For gain-of-function studies:

  • Clone MAPRE2 cDNA into appropriate expression vectors

  • Synthesize capped mRNA in vitro

  • Inject 500-1000 pg of mRNA per embryo at early stages

  • Analyze phenotypes and perform rescue experiments

It's important to note that the allotetraploid nature of X. laevis adds complexity to genetic manipulation, making careful design and validation crucial for successful experiments .

How does MAPRE2 function in Xenopus embryonic development and cell division?

MAPRE2 plays critical roles in embryonic development and cell division in Xenopus laevis through several mechanisms:

• Meiotic division: The high expression of MAPRE2 in oocytes suggests a crucial role in meiotic processes. During oocyte maturation, MAPRE2 likely regulates microtubule dynamics essential for proper spindle formation and chromosome segregation .

• Cell adhesion and tissue integrity: Studies in related models have demonstrated that MAPRE2 loss-of-function leads to disruption of adherens junctions:

  • Disorganization of N-cadherin (Ncad) at cell junctions

  • Decreased ratio of stable to nascent Ncad at cell borders

  • This junction disruption impacts tissue integrity during morphogenesis

• Microtubule dynamics regulation: MAPRE2 knockdown results in:

  • Increased microtubule growth velocity (by approximately 11-26%)

  • Increased microtubule growth distance (by approximately 22-34%)

  • Altered post-translational modifications of tubulin

  • These changes impact proper cellular organization during development

The study of MAPRE2 in Xenopus development provides insights into fundamental mechanisms that may be conserved in human development and disease processes. Researchers should consider these developmental functions when designing experiments with recombinant MAPRE2.

How can Xenopus laevis MAPRE2 be used to model human disease mechanisms?

Xenopus laevis MAPRE2 provides a valuable tool for modeling human disease mechanisms due to its high structural and functional conservation with human MAPRE2. Several methodological approaches can be employed:

• Congenital skin disorders modeling: Human mutations in MAPRE2 have been implicated in congenital symmetric circumferential skin creases type 2 . These can be modeled by:

  • Introducing equivalent mutations in Xenopus MAPRE2 via CRISPR/Cas9

  • Expressing mutant human MAPRE2 in Xenopus embryos

  • Analyzing effects on epithelial development and organization

• Cardiac disease modeling: Recent studies have identified MAPRE2 as a novel locus associated with Brugada syndrome through genome-wide association studies :

  • Xenopus cardiac development can be assessed following MAPRE2 manipulation

  • Electrophysiological studies on Xenopus hearts can reveal conduction abnormalities

  • Sodium channel localization and function can be examined

• Cancer cell behavior studies: MAPRE2 has been implicated in tumorigenesis of colorectal cancers :

  • Xenopus cell lines can be manipulated to express oncogenic variants

  • Cell migration, proliferation, and invasion assays can be performed

  • Response to anti-cancer compounds can be evaluated

• Neurological disorder investigations: Given MAPRE2's expression in neural tissues, it may play roles in neurological disorders:

  • Neural crest cell migration studies using Xenopus embryos

  • Xenotransplantation of MAPRE2-modified cells into developing embryos

  • Analysis of neurite outgrowth and axon guidance

The experimental advantages of Xenopus, including large embryos amenable to micromanipulation and high protein yields, make it particularly suitable for disease modeling studies involving MAPRE2 .

What are the methodological considerations for studying MAPRE2's role in microtubule regulation using recombinant protein?

When studying MAPRE2's role in microtubule regulation using recombinant protein, researchers should consider these methodological aspects:

• Protein concentration effects: MAPRE2 function exhibits concentration dependency:

  • Optimal concentration range should be determined empirically (typically 50-500 nM)

  • Both insufficient and excessive MAPRE2 can lead to similar functional outcomes, a "Goldilocks effect"

  • Calibration curves should be established for each experimental system

• Partner protein considerations: MAPRE2 functions in complex with other proteins:

  • Consider co-expression with interacting partners (e.g., PRCC)

  • Evaluate how partner proteins modulate MAPRE2 activity

  • Test combinations of EB family proteins (EB1, EB2/MAPRE2, EB3) to assess redundancy or synergy

• Post-translational modifications: MAPRE2 function is regulated by modifications:

  • Phosphorylation status affects microtubule binding

  • Expression systems may not recapitulate all native modifications

  • Consider using Xenopus egg extracts to maintain physiological modification patterns

• Experimental readouts: Multiple parameters should be measured:

  Parameter	Normal Range	MAPRE2 Knockdown Effect	Measurement Method
  MT growth velocity	8.05±0.18 μm/min	Increased by 11-26%	Live-cell imaging
  MT growth distance	5.76±0.19 μm	Increased by 22-34%	Live-cell imaging
  N-cadherin stability	High ratio at junctions	Decreased stable:nascent ratio	Immunofluorescence
  Nuclear localization	Partial nuclear	Reduced nuclear	Immunofluorescence

• Experimental system selection: Different systems offer complementary advantages:

  • In vitro reconstitution with purified components provides mechanistic insights

  • Xenopus egg extracts offer physiological complexity while maintaining accessibility

  • Cell culture allows for manipulation in a cellular context

  • In vivo studies in Xenopus embryos provide developmental context

These methodological considerations ensure robust and physiologically relevant results when studying MAPRE2's role in microtubule regulation.

How does Xenopus laevis MAPRE2 compare to its orthologs in other model organisms?

Comparative analysis of MAPRE2 across species reveals important evolutionary insights and functional conservation:

• Sequence conservation:

  • Xenopus laevis MAPRE2 shares 87.4% protein similarity with zebrafish MAPRE2

  • High homology exists with human MAPRE2, particularly in the calponin homology domain

  • The C-terminal region of MAPRE2 is highly conserved across vertebrates, while the N-terminal region shows more variability

• Functional conservation:

  • MAPRE2's role in microtubule dynamics appears conserved across vertebrates

  • Studies in zebrafish have shown that MAPRE2 knockdown affects cardiac conduction similar to findings in human studies

  • The interaction with PRCC is maintained despite sequence divergence, suggesting evolutionary pressure to preserve this function

• Species-specific differences:

  • Xenopus laevis contains two copies of MAPRE2 due to its allotetraploid genome, unlike the diploid human and zebrafish genomes

  • Expression patterns vary across species, with Xenopus showing particularly high expression in reproductive tissues

  • The N-terminal region of Xenopus PRCC, which interacts with MAPRE2, is distinct from that in mouse and human, yet the interaction is preserved

This comparative approach provides valuable insights into both conserved and species-specific aspects of MAPRE2 function, aiding in translational research from model organisms to human biology.

What genomic and transcriptomic resources are available for studying MAPRE2 in Xenopus laevis?

Researchers have access to several genomic and transcriptomic resources that facilitate MAPRE2 studies in Xenopus laevis:

• Genome resources:

  • The Xenopus laevis genome has been sequenced and assembled (current version 10.1)

  • Both the L and S chromosomes (from the two subgenomes) have been characterized

  • Genomic data is accessible through Xenbase (www.xenbase.org), the dedicated Xenopus model organism database

• Transcriptomic resources:

  • RNA-seq datasets from various developmental stages and tissues are available

  • The Xenopus Gene Expression Database contains expression data for numerous genes including MAPRE2

  • PHROG (Proteomic Reference with Heterogeneous RNA Omitting the Genome) provides a comprehensive protein reference database derived from RNA data

• Tools for genetic manipulation:

  • CRISPR/Cas9 design tools specific for Xenopus laevis are available through Xenbase

  • Morpholino design resources take into account the unique aspects of Xenopus genome

  • Transcription activator-like effector nucleases (TALEN) protocols have been optimized for Xenopus

• Clones and reagents:

  • The European Xenopus Resource Centre (EXRC) and commercial sources provide sequence-verified clones

  • Catalogues of antibodies and morpholinos specific to Xenopus MAPRE2 are accessible through Xenbase

  • Expression constructs for recombinant protein production are available from multiple sources

These resources enable comprehensive studies of MAPRE2 from genomic characterization to functional analysis in the Xenopus laevis model system.

What are the major challenges in expressing and purifying functional recombinant Xenopus laevis MAPRE2?

Researchers face several technical challenges when expressing and purifying functional recombinant Xenopus laevis MAPRE2:

• Protein solubility issues:

  • MAPRE2 can form aggregates during expression and purification

  • Solution: Use fusion tags (His, GST, or Strep) and optimize buffer conditions (10 mM Hepes, 500 mM NaCl, pH 7.4 with 5% trehalose has shown success)

  • Expression at lower temperatures (16-18°C) can improve solubility

• Maintaining functional conformation:

  • The microtubule-binding domain of MAPRE2 is sensitive to denaturation

  • Solution: Avoid harsh elution conditions; use gradient elution methods and include stabilizing agents like glycerol or trehalose

  • Validate function through microtubule binding assays after purification

• Expression system selection:

  • Different systems yield varying protein quality and quantity

  • Comparison of expression systems for Xenopus MAPRE2:

  Expression System	Typical Yield	Purity	Advantages	Limitations
  E. coli	5-10 mg/L	>95%	High yield, cost-effective	May lack PTMs
  HEK-293 cells	1-2 mg/L	>90%	Proper folding, PTMs	Higher cost, lower yield
  Cell-free system	0.5-1 mg/mL	70-80%	Rapid production	Variable activity
  Xenopus oocytes	Variable	High	Native environment	Labor intensive, lower scale

• Contamination with egg yolk components:

  • When using Xenopus oocytes as an expression system, lipids and lipoproteins from egg yolk are major contaminants

  • Solution: Develop protocols to efficiently discard these components through optimized centrifugation steps and lipid extraction procedures

• Post-translational modifications:

  • Different expression systems may not reproduce the native pattern of modifications

  • Solution: Consider using Xenopus egg extracts or oocytes for expression when modifications are critical

Addressing these challenges requires careful optimization of expression and purification protocols tailored to the specific experimental needs.

How can researchers overcome the challenges of studying MAPRE2 in the tetraploid Xenopus laevis genome?

The allotetraploid nature of the Xenopus laevis genome presents unique challenges for researchers studying MAPRE2. Here are methodological approaches to address these issues:

• Homeolog-specific analysis:

  • Design primers that distinguish between L and S homeologs of MAPRE2

  • Use RNA-seq analysis to quantify expression of each homeolog separately

  • Apply computational approaches to distinguish between highly similar sequences

• Genetic manipulation strategies:

  • Target conserved regions with CRISPR/Cas9 to affect both homeologs simultaneously

  • Design multiple gRNAs to ensure complete knockout

  • Verify editing efficiency for each homeolog separately using specific PCR assays

• Alternative reference databases:

  • Use PHROG (Proteomic Reference with Heterogeneous RNA Omitting the Genome) which combines multiple mRNA sources and outperforms genome-based references for proteomic studies

  • Comparison of database effectiveness for Xenopus proteomic studies:

  Reference Database	Peptides Identified	Proteins Identified	Relative Effectiveness
  Xenbase published proteins	Base value	1,850	Baseline
  X. tropicalis reference	70% less than baseline	-	Poor
  Preliminary gene models	Significant improvement	3,130	Good
  PHROG database	10% more than gene models	3,176	Best
  PHROG + gene models	1% more than PHROG alone	3,098	Marginal improvement

• Expression analysis challenges:

  • The correlation between mRNA and protein abundance in Xenopus laevis is relatively weak (Pearson correlation of 0.32, Spearman correlation of 0.30 in log-log space)

  • Solution: Validate findings at both RNA and protein levels, particularly when studying MAPRE2 expression

• Integration with genomic resources:

  • Utilize the latest genome builds available through Xenbase

  • Consider using RNA-seq based approaches when genome annotation is incomplete

  • Leverage comparative genomics with X. tropicalis (diploid) to inform studies in X. laevis

By implementing these strategies, researchers can effectively navigate the complexities of the tetraploid genome when studying MAPRE2 in Xenopus laevis.

What are the emerging applications of recombinant Xenopus laevis MAPRE2 in structural biology?

Recombinant Xenopus laevis MAPRE2 presents several promising opportunities for structural biology research:

• Cryo-electron microscopy studies:

  • High-purity recombinant MAPRE2 enables single-particle cryo-EM analysis

  • This can reveal the molecular details of MAPRE2 interaction with microtubule plus-ends

  • Structural comparisons with human MAPRE2 can identify conserved functional domains and species-specific differences

• 2D crystallization approaches:

  • Recombinant MAPRE2 can be embedded in lipid bilayers for 2D crystallization

  • This enables structural studies of membrane-associated MAPRE2 complexes

  • The methodology has been successfully applied to other Xenopus membrane proteins and can be adapted for MAPRE2

• Structural analysis of MAPRE2-PRCC complexes:

  • Co-expression and co-purification of MAPRE2 with PRCC enables structural studies of this important complex

  • Understanding the structural basis of this interaction could reveal mechanisms of nuclear translocation and cell cycle regulation

• Structure-based drug design:

  • High-resolution structures of Xenopus MAPRE2 can inform the design of small molecule modulators

  • These could be developed as research tools or potential therapeutic leads for MAPRE2-associated disorders

  • The structural conservation between species makes Xenopus MAPRE2 a valid model for human applications

• In situ structural studies:

  • Emerging techniques like cryo-electron tomography could reveal MAPRE2's organization in its native cellular context

  • The large size of Xenopus cells makes them particularly suitable for these approaches

These structural biology applications will advance our understanding of MAPRE2's molecular mechanisms and potentially lead to new therapeutic strategies for MAPRE2-associated diseases.

How can multi-omics approaches enhance our understanding of MAPRE2 function in Xenopus laevis?

Integrated multi-omics approaches offer powerful strategies to comprehensively understand MAPRE2 function in Xenopus laevis:

• Integrative genomics and transcriptomics:

  • Combine genome sequencing with RNA-seq to identify regulatory elements controlling MAPRE2 expression

  • Analyze transcriptional responses to MAPRE2 manipulation across developmental stages

  • Compare L and S homeolog expression patterns to identify potential subfunctionalization

• Proteomics and interactomics:

  • Apply proximity labeling techniques (BioID, APEX) with MAPRE2 as bait to identify interaction partners

  • Use quantitative proteomics to measure changes in the microtubule-associated proteome following MAPRE2 manipulation

  • Characterize post-translational modifications of MAPRE2 using mass spectrometry

• Structural biology integration:

  • Combine structural data with interactomics to map binding interfaces

  • Use molecular dynamics simulations informed by experimental structures to predict functional consequences of mutations

  • Validate structural predictions through targeted mutagenesis and functional assays

• Spatial transcriptomics and proteomics:

  • Apply emerging spatial omics technologies to map MAPRE2 expression and function across tissues

  • Correlate with developmental phenotypes to understand tissue-specific roles

  • Identify cell types where MAPRE2 function is most critical

• Single-cell approaches:

  • Use single-cell RNA-seq to identify cell populations dependent on MAPRE2 function

  • Apply single-cell proteomics to characterize cell-specific interactomes

  • Correlate with cellular behaviors like migration and division

These multi-omics approaches will provide a systems-level understanding of MAPRE2 function in Xenopus laevis, revealing its role in development, cell division, and disease mechanisms that may be translatable to human biology.