MAPRE2 Human

What is the basic structure and function of MAPRE2 in human cells?

MAPRE2, also known as EB2 (End-Binding protein 2), is a member of a family of three proteins (EB1, EB2, and EB3) that bind to the growing plus-ends of microtubules via their N-terminal domain and interact with other partners via their C-terminus. MAPRE2 contains a calponin homology (CH) domain responsible for tracking and interacting with the plus-end tip of growing microtubules . Unlike other family members, MAPRE2 has a unique 43 amino acid segment at its N-terminus not present in EB1 or EB3, suggesting specialized functions. The protein demonstrates 87.4% similarity between human and zebrafish homologs, indicating high evolutionary conservation .

The functional significance of MAPRE2 is evident in its regulation of microtubule dynamics. Knockdown experiments have demonstrated that MAPRE2 loss of function leads to increased microtubule growth velocity and distance, affecting microtubule stability and post-translational modifications such as tubulin detyrosination .

How is MAPRE2 expression regulated in different human tissues?

While the search results don't provide comprehensive information on tissue-specific expression patterns, research indicates that MAPRE2 plays critical roles in both cardiac tissue and neural crest cells. In cardiomyocytes, MAPRE2 influences sodium channel function and ventricular conduction . In cranial neural crest cells, MAPRE2 affects migration capabilities and focal adhesion formation .

The regulatory mechanisms controlling MAPRE2 expression remain an active area of investigation. Current research methodologies for studying MAPRE2 expression include RT-qPCR for mRNA quantification and Western blotting for protein expression analysis, as demonstrated in studies of induced pluripotent stem cell (iPSC) lines carrying various MAPRE2 mutations .

What is the evolutionary conservation of MAPRE2 across species?

MAPRE2 demonstrates remarkable evolutionary conservation, particularly at functionally critical residues. Analysis of homologous proteins across diverse organisms, including those with high evolutionary distances from humans such as Arabidopsis thaliana and Saccharomyces cerevisiae, reveals 100% conservation at certain residues (e.g., position 68) .

This high degree of conservation suggests essential structural and functional importance. For research purposes, this conservation validates the use of model organisms like zebrafish for studying MAPRE2 function. The zebrafish homolog shows 87.4% similarity at the protein level compared to human MAPRE2, making it a valuable model for investigating MAPRE2-related phenotypes and mechanisms .

How does MAPRE2 contribute to normal cardiac electrophysiology?

MAPRE2 plays a crucial role in maintaining normal cardiac electrophysiology through its effects on microtubule dynamics and subsequent impacts on sodium channel function and intercellular junctions. Research using mapre2 knockout zebrafish models has revealed that MAPRE2 influences ventricular conduction and voltage-gated sodium channel (Nav) function .

Methodologically, these findings were established through:

• Voltage mapping of isolated hearts, which showed decreased ventricular maximum upstroke velocity of the action potential and reduced conduction velocity in mapre2 knockout models

• Patch clamping experiments demonstrating altered sodium current properties

• Immunocytochemistry showing disruption of adherens junctions

The mechanism linking MAPRE2 to cardiac function involves its regulation of microtubule dynamics, which affects both the subcellular localization of sodium channels and the integrity of adherens junctions essential for proper electrical conduction between cardiomyocytes .

What is the association between MAPRE2 and Brugada syndrome?

Brugada syndrome (BrS) is a cardiac arrhythmia disorder traditionally associated with SCN5A mutations, which account for only about 20% of cases. A genome-wide association study by Bezzina et al. identified a novel locus within intron 2 of MAPRE2, implicating microtubule dynamics in BrS pathophysiology .

Research using zebrafish models with mapre2 loss of function demonstrated a ventricular phenotype consistent with SCN5A loss of function associated with Brugada syndrome. Specifically, mapre2 knockout resulted in:

• Ventricular conduction slowing

• Decreased sodium channel function

• Arrhythmogenesis

• Disruption of adherens junctions

These phenotypes were associated with decreased detyrosinated α-tubulin and altered microtubule dynamics. The mechanistic connection appears to involve MAPRE2's role in regulating microtubule stability, which affects sodium channel density at the cell membrane and results in ventricular conduction defects .

What experimental models are most effective for studying MAPRE2 in cardiac research?

Based on the search results, two complementary model systems have proven effective for studying MAPRE2 function in cardiac research:

• Zebrafish models:

  • Both germline mapre2 knockout zebrafish (using CRISPR/Cas9) and morpholino-based knockdown approaches have been successfully employed

  • Advantages include cardiac electrophysiology similar to humans, optical transparency for imaging, and efficient genetic manipulation

  • Allow for functional studies including ECG recording, voltage mapping, and patch clamping

• Human iPSC-derived cardiomyocytes (iPSC-CMs):

  • Enable MAPRE2 knockdown or knockout in human cells

  • Allow for direct measurement of microtubule dynamics through live-cell imaging with EB3-GFP

  • Permit patch clamping experiments to assess ion channel function

The combination of these models offers complementary strengths. Zebrafish provide an in vivo system for studying cardiac conduction and arrhythmogenesis, while iPSC-CMs allow detailed mechanistic studies of microtubule dynamics in human cells. Both systems support comprehensive functional assessments including electrophysiological measurements, microtubule tracking, and immunocytochemistry .

How does MAPRE2 influence neural crest cell migration?

MAPRE2 plays a significant role in neural crest cell migration, particularly affecting cranial neural crest cells (CNCCs). Research using patient-derived induced pluripotent stem cells (iPSCs) with MAPRE2 mutations demonstrated altered migration capabilities in these cells .

Methodologically, this has been studied through:

• In vitro scratch assays with live imaging: These experiments tracked single CNCCs as they migrated into an open area, revealing that cells with MAPRE2 mutations (either heterozygous or homozygous Q152* stop mutations) displayed significantly lower migration speeds compared to isogenic controls .

• Xenotransplantation experiments: Neural crest progenitors were transplanted into developing chicken embryos, confirming migration defects in vivo .

The mechanism appears to involve focal adhesion (FA) dynamics. Immunohistochemistry and confocal microscopy revealed that MAPRE2-deficient CNCCs had larger vinculin-positive focal adhesion spots compared to control cells, suggesting altered cell-substrate interactions that impair efficient migration .

What craniofacial abnormalities are associated with MAPRE2 mutations?

MAPRE2 mutations have been implicated in Congenital Symmetrical Circumferential Skin Creases type 2 (CSC-KT), a condition characterized by craniofacial dysmorphisms and cleft palate . Research in both human patients and zebrafish models has provided insights into these developmental abnormalities.

Studies in zebrafish embryos demonstrated that MAPRE2 knockdown resulted in:

Interestingly, rescue experiments suggested a "Goldilocks effect" where either overactive or insufficient MAPRE2 leads to similar clinical pathologies in branchial arch maturation . This suggests that precise regulation of MAPRE2 levels is critical for normal craniofacial development.

The connection between MAPRE2 and craniofacial development appears to be mediated through its effects on neural crest cell migration, which is essential for proper formation of craniofacial structures during embryonic development .

What experimental approaches are optimal for studying MAPRE2 in neural development?

Based on the search results, several experimental approaches have proven effective for studying MAPRE2 in neural development:

• Patient-derived iPSC models:

  • Generation of iPSC lines from patients with MAPRE2 mutations

  • Creation of isogenic control lines using CRISPR/Cas9 gene editing

  • Differentiation toward neural crest cells with cranial identity

• CRISPR/Cas9 genome editing:

  • Introduction of specific mutations (e.g., Q152* nonsense mutation)

  • Use of D10A Cas9 mutant nickase with offset sgRNAs to reduce off-target effects

  • Generation of both heterozygous and homozygous mutant lines

• Functional assays:

  • Scratch assays with live imaging to track cell migration

  • Immunohistochemistry to visualize focal adhesion spots

  • Xenotransplantation into chicken embryos for in vivo assessment

• Molecular validation techniques:

  • RT-qPCR for mRNA quantification

  • Western blotting for protein expression analysis

  • Computational modeling for protein structure prediction when 3D structures are unknown

These approaches collectively enable comprehensive investigation of MAPRE2 function in neural crest development, from molecular mechanisms to cellular behaviors and developmental outcomes.

What are the optimal genetic manipulation techniques for studying MAPRE2 function?

Based on the search results, several genetic manipulation techniques have been successfully employed to study MAPRE2 function:

• CRISPR/Cas9 genome editing:

  • For generating germline knockout models in zebrafish

  • For creating patient-specific mutations in human iPSCs

  • For developing isogenic control lines

  Key methodological considerations include:

  • Using the D10A Cas9 mutant nickase with paired sgRNAs on opposite strands to reduce off-target effects

  • Targeting specific exons (e.g., exon 2 in zebrafish mapre2)

  • Validation of editing through sequencing and protein expression analysis

• Morpholino-based knockdown:

  • For acute knockdown in zebrafish embryos

  • Targeting splice acceptor sites (e.g., exon 2 of mapre2)

  • Validation through Western blot analysis

• siRNA-mediated knockdown:

  • For transient knockdown in cell culture models (e.g., human iPSC-derived cardiomyocytes)

  • Validation through Western blot and immunofluorescence

The choice between these approaches depends on research questions, with CRISPR/Cas9 preferred for stable genetic models, morpholinos for developmental studies, and siRNA for acute cellular experiments. Combined approaches provide complementary insights into MAPRE2 function across different temporal and spatial scales.

What imaging and analytical methods best capture MAPRE2-dependent microtubule dynamics?

Advanced imaging techniques have been instrumental in understanding MAPRE2's role in microtubule dynamics. Based on the search results, the following approaches have proven effective:

• Microtubule plus-end tracking with live-cell imaging:

  • Transfection with EB3-GFP to visualize microtubule plus-ends

  • Time-lapse imaging to track individual microtubules

  • Generation of kymographs for quantitative analysis of:

    • Microtubule growth velocity (μm/min)

    • Microtubule growth distance (μm)

• Immunocytochemistry for post-translational modifications:

  • Visualization of detyrosinated α-tubulin

  • Quantification of the ratio of detyrosinated to total α-tubulin

• Confocal microscopy for focal adhesion analysis:

  • Immunohistochemistry to visualize vinculin-positive focal adhesion spots

  • Quantification of focal adhesion size and distribution

These methods collectively enable researchers to characterize how MAPRE2 influences microtubule dynamics, stability, and interactions with cellular structures. The results from these imaging approaches revealed that MAPRE2 loss of function increases microtubule growth velocity and distance, decreases tubulin detyrosination, and affects focal adhesion morphology .

How can researchers effectively measure functional consequences of MAPRE2 alterations?

To measure the functional consequences of MAPRE2 alterations, researchers have employed several sophisticated techniques targeting different cellular and physiological processes:

• Cardiac electrophysiology assessments:

  • Voltage mapping to measure ventricular maximum upstroke velocity and conduction velocity

  • ECG recordings to assess cardiac rhythm and conduction

  • Patch clamping to directly measure ion channel currents (particularly sodium currents)

  • Optical mapping to visualize action potential propagation

• Cell migration and adhesion analyses:

  • Scratch assays with live imaging to track individual cell movement

  • Transfection with H2B-RFP to label nuclei for cell tracking

  • Quantification of migration speed and directionality

  • Analysis of focal adhesion dynamics through immunohistochemistry

• Junction integrity assessments:

  • Immunocytochemistry for adherens junction proteins (e.g., N-cadherin)

  • Use of transgenic lines with fluorescent tags (e.g., cdh2 tandem fluorescent timer)

  • Quantification of junction protein localization

• Rescue experiments:

  • Knockdown of compensatory pathways (e.g., ttl encoding tubulin tyrosine ligase)

  • Assessment of whether interventions restore normal phenotypes

  • Measurement of both molecular (detyrosinated tubulin, adherens junction) and functional (Na+ channel) parameters

These methodologies provide complementary insights into how MAPRE2 affects cellular functions across different biological contexts, from cardiac conduction to neural crest migration.

How should researchers interpret contradictory data on MAPRE2 and microtubule modifications?

A significant challenge in MAPRE2 research involves contradictory findings regarding its effects on microtubule post-translational modifications. The search results highlight one such contradiction:

• In MAPRE2 loss-of-function models: Research showed a decrease in α-tubulin detyrosination .

• In mouse models of cardiomyopathy: Studies found an increase in detyrosinated α-tubulin .

To address such contradictions, researchers should consider:

• Context-specific effects: MAPRE2 may function differently depending on cell type, developmental stage, or disease context. Systematic comparison across multiple models and conditions is essential.

• Compensatory mechanisms: Other microtubule-associated proteins or regulatory pathways may compensate for MAPRE2 loss in different ways across models.

• Methodological approach:

  • Use multiple, complementary methods to measure microtubule modifications

  • Include appropriate controls and isogenic lines to minimize confounding factors

  • Consider the timing of measurements (acute versus chronic effects)

  • Evaluate multiple post-translational modifications simultaneously

• Functional validation: Correlate changes in microtubule modifications with functional outcomes to determine physiological relevance.

The search results suggest a possible resolution through mechanistic research, demonstrating that MAPRE2 loss of function increases microtubule dynamics, potentially leading to decreased microtubule stability and subsequent reduction in detyrosination . This mechanistic insight helps reconcile apparently contradictory observations.

What are the challenges in integrating findings from different model systems for MAPRE2 research?

MAPRE2 research utilizes diverse model systems, each with strengths and limitations. Integrating findings across these systems presents several challenges:

• Species-specific differences:

  • Despite 87.4% protein similarity between human and zebrafish MAPRE2 , species-specific differences in expression patterns, interacting partners, or regulatory mechanisms may exist.

  • Methods for addressing this include: using multiple model organisms, focusing on highly conserved domains, and validating key findings across species.

• In vitro versus in vivo contexts:

  • Cell culture models may not recapitulate the complex three-dimensional environment and multicellular interactions present in vivo.

  • Researchers should combine in vitro mechanistic studies with in vivo functional validation when possible.

• Developmental timing:

  • MAPRE2 functions may vary across developmental stages, making direct comparisons challenging.

  • Careful staging and temporal analyses are essential for meaningful comparisons.

• Genetic background effects:

  • Genetic modifiers may influence MAPRE2-associated phenotypes.

  • Use of isogenic controls and multiple genetic backgrounds can help address this challenge.

• Methodological differences:

  • Variations in experimental protocols, measurement techniques, and data analysis approaches may contribute to apparent discrepancies.

  • Standardization of key methodologies and detailed reporting of experimental conditions are important.

The search results demonstrate successful integration through complementary approaches, such as combining zebrafish models for in vivo cardiac function with human iPSC-derived cardiomyocytes for detailed microtubule dynamics studies , or validating findings from patient-derived iPSCs through xenotransplantation experiments .

What therapeutic approaches might target MAPRE2-related pathways in disease?

While the search results don't explicitly discuss therapeutic interventions targeting MAPRE2, they suggest several potential approaches based on mechanistic insights:

• Modulation of microtubule post-translational modifications:

  • The rescue experiments knocking down ttl (tubulin tyrosine ligase) restored normal detyrosinated tubulin levels, adherens junction formation, and sodium channel function in mapre2 loss-of-function models .

  • This suggests that targeting enzymes involved in tubulin post-translational modifications could represent a novel therapeutic strategy for MAPRE2-related cardiac conduction disorders.

• Stabilization of adherens junctions:

  • MAPRE2 loss disrupts adherens junctions, which affects electrical conduction between cardiomyocytes .

  • Approaches that stabilize these junctions might ameliorate conduction defects associated with MAPRE2 dysfunction.

• Focal adhesion modulation:

  • MAPRE2 mutations affect focal adhesion size and cell migration .

  • Therapies targeting focal adhesion dynamics might address migration defects in neural crest-derived tissues.

• Precision gene editing or gene therapy:

  • For conditions caused by specific MAPRE2 mutations, gene editing approaches might restore normal function.

  • The "Goldilocks effect" observed in rescue experiments suggests that precise regulation of MAPRE2 levels would be critical for therapeutic success.

• Ion channel targeting:

  • Since MAPRE2 dysfunction affects sodium channel function and localization , compounds that modulate these channels might address cardiac conduction defects.

These approaches represent potential "novel therapeutic approaches for treating conduction disorders" and developmental conditions associated with MAPRE2 dysfunction, though significant preclinical research would be needed before clinical translation.

How do experimental data on MAPRE2 compare across different research models?

Table 1: Comparison of Key MAPRE2 Findings Across Research Models

This comparative analysis reveals complementary findings across different model systems, with each providing unique insights into MAPRE2 function. The zebrafish model provides the most comprehensive functional characterization in vivo, while human cell models offer detailed insights into specific cellular processes. The consistency in phenotypes related to cellular adhesion, migration, and cytoskeletal dynamics across models supports conserved functions of MAPRE2 across species and cell types.

What molecular mechanisms link MAPRE2 dysfunction to different disease phenotypes?

Based on the search results, MAPRE2 dysfunction contributes to distinct disease phenotypes through several molecular mechanisms:

Table 2: Molecular Mechanisms Linking MAPRE2 to Disease Phenotypes

These mechanisms highlight how MAPRE2, through its fundamental role in microtubule regulation, can affect diverse cellular processes depending on tissue context. In cardiac tissue, the impact on ion channel function and intercellular junctions primarily affects electrophysiology, while in neural crest cells, the effects on focal adhesions and cell motility disrupt developmental migration patterns.

The search results suggest a "Goldilocks effect" whereby either overactive or insufficient MAPRE2 can lead to similar pathologies , indicating that precise regulation of this protein is critical for normal function across different tissues. This mechanistic understanding provides potential targets for tissue-specific therapeutic interventions.

What emerging technologies might advance MAPRE2 research?

Based on current research approaches identified in the search results, several emerging technologies could significantly advance MAPRE2 research:

• Advanced live-cell imaging techniques:

  • Super-resolution microscopy to visualize microtubule dynamics and interactions at nanoscale resolution

  • Correlative light and electron microscopy (CLEM) to connect molecular-level changes with ultrastructural alterations

  • Lattice light-sheet microscopy for extended live imaging with reduced phototoxicity

• Multi-omics approaches:

  • Spatial transcriptomics to map MAPRE2-dependent gene expression changes within tissue contexts

  • Proteomics to identify the complete interactome of MAPRE2 in different cell types

  • Metabolomics to assess downstream metabolic consequences of altered microtubule dynamics

• Advanced genetic engineering:

  • Base editing or prime editing for precise modification of specific MAPRE2 residues

  • Optogenetic or chemogenetic control of MAPRE2 activity or localization

  • Tissue-specific and inducible MAPRE2 manipulation in animal models

• Organ-on-chip and organoid technologies:

  • Cardiac organoids to study MAPRE2 in three-dimensional tissue architecture

  • Multi-cell type organ-on-chip models to investigate MAPRE2 in complex tissue interactions

  • Patient-specific organoids to model individual disease variants

• Computational approaches:

  • Machine learning for automated analysis of microtubule dynamics and cell migration

  • Molecular dynamics simulations to predict effects of MAPRE2 mutations

  • Network modeling to integrate MAPRE2 into broader cytoskeletal and signaling networks

These technologies would address current limitations in understanding MAPRE2 function across spatial and temporal scales, from molecular interactions to tissue-level phenotypes.

What are the most critical unanswered questions in MAPRE2 research?

Analysis of the search results reveals several critical knowledge gaps in MAPRE2 research that warrant further investigation:

• Tissue-specific functions:

  • How does MAPRE2 function differ across cell types and tissues?

  • What explains the tissue-specific manifestations of MAPRE2 mutations despite its ubiquitous expression?

• Regulatory mechanisms:

  • How is MAPRE2 expression and activity regulated under normal and pathological conditions?

  • What signaling pathways modulate MAPRE2 function?

• Interaction with other microtubule-associated proteins:

  • How does MAPRE2 functionally differ from other EB family members (EB1 and EB3)?

  • What is the significance of the unique 43 amino acid segment at the N-terminus of MAPRE2?

  • How does MAPRE2 coordinate with other microtubule regulatory proteins?

• Mechanism of ion channel regulation:

  • How precisely does MAPRE2 influence sodium channel localization and function in cardiomyocytes?

  • Are other ion channels similarly affected by MAPRE2 dysfunction?

• Developmental dynamics:

  • How does MAPRE2 function change during development and differentiation?

  • What developmental processes beyond cardiac conduction and neural crest migration depend on MAPRE2?

• Therapeutic targeting:

  • Can MAPRE2-related pathways be selectively modulated for therapeutic benefit?

  • How can the "Goldilocks effect" be managed to achieve optimal MAPRE2 function?

• Human disease associations:

  • Beyond Brugada syndrome and CSC-KT, what other human conditions might involve MAPRE2 dysfunction?

  • How do common genetic variants in MAPRE2 influence disease susceptibility?