Recombinant Pongo abelii Microtubule-associated protein RP/EB family member 2 (MAPRE2)

Basic Research Questions

• What is MAPRE2 and what are its primary functions in cellular processes?

MAPRE2 (Microtubule Associated Protein RP/EB Family Member 2) belongs to the evolutionary conserved microtubule plus-end tracking proteins, specifically the end-binding (EB) family. Unlike its paralogs MAPRE1 and MAPRE3, MAPRE2 is not required for the persistent growth and stabilization of microtubules . Instead, it plays crucial roles in:

• Reorganization of microtubules during early apical-basal differentiation in epithelia

• Regulation of cell adhesion and focal adhesion turnover

• Mitotic progression and genome stability

• Neural crest cell migration, particularly cranial neural crest cells

• Cardiac electrophysiology and ventricular conduction

The protein contains a highly conserved calponin-homology (CH) domain that is responsible for tracking and interacting with the growing plus-end tips of microtubules . MAPRE2 acts as an adaptor to recruit signaling molecules to microtubule ends and interacts with proteins such as MAP4K4 and HAX1 in the focal adhesion turnover complex .

• How do MAPRE2 mutations affect neural crest cell migration and what methodologies are used to study this?

MAPRE2 mutations significantly alter neural crest cell migration, which has been demonstrated through multiple methodological approaches:

In vitro methodologies:

• Scratch assay combined with live imaging allows tracking of single neural crest cells as they migrate into an open area

• Nuclei labeling with H2B-RFP for easy tracking of individual cells

• Quantitative assessment of migration speed and directionality

Key findings:

• Loss-of-function mutations (Q152* heterozygous or homozygous) result in decreased migration speed of cranial neural crest cells

• The N68S/N68S mutation, in contrast, increases migration speed compared to isogenic controls

• Haploinsufficiency is sufficient to cause migration phenotypes, as seen in patients with these mutations

In vivo approaches:

• Xenotransplantation of human-derived MAPRE2 neural crest progenitors into developing chicken embryos

• Zebrafish models showing that knockdown of MAPRE2 results in craniofacial defects and aberrant branchial arch formation

• What is the relationship between MAPRE2 and focal adhesion dynamics?

MAPRE2 plays a critical role in focal adhesion (FA) dynamics through the following mechanisms:

Molecular interactions:

• MAPRE2 interacts with MAP4K4 and HAX1 in the focal adhesion turnover complex

• It influences wound healing and cell motility through these interactions

Experimental observations:

• Microtubules specifically grow toward focal adhesions and promote FA turnover rate at the cell periphery

• MAPRE2 appears to serve as an adaptor to recruit signaling molecules to microtubule ends

Quantitative findings:

• MAPRE2 Q152* knockdown lines (both heterozygous and homozygous) show significantly larger vinculin-positive focal adhesion spots compared to wild-type controls

• This can be visualized using immunohistochemistry and confocal microscopy of iPSC-derived cranial neural crest cells plated on fibronectin-coated plates

The altered focal adhesion dynamics likely contribute to the abnormal cell motility observed in MAPRE2 mutant cranial neural crest cells, providing a mechanism for the craniofacial phenotypes seen in patients.

• How does MAPRE2 differ from other members of the EB protein family?

The end-binding (EB) protein family has three main members (EB1, EB2, and EB3), with MAPRE2 encoding EB2. Key differences include:

These differences indicate that despite sharing structural similarities, MAPRE2 has evolved distinct functions from its paralogous proteins, particularly in specialized cellular contexts.

Advanced Research Questions

• What are the best approaches for generating and validating MAPRE2 knockout/knockdown models?

Creating reliable MAPRE2 loss-of-function models requires careful consideration of several factors:

CRISPR/Cas9 system for germline knockout:

• Target the calponin homology domain for functional disruption

• Generate isogenic control lines simultaneously for proper comparison

• Validate knockout efficiency through both Western blotting and RT-qPCR

• Assess both mRNA levels (using primers in different exons) and protein expression

siRNA for transient knockdown:

• Design and validate multiple siRNAs targeting different regions of MAPRE2

• Confirm knockdown efficiency by Western blot in established cell lines (e.g., HeLa)

• Verify knockdown in target cells (e.g., iPSC-CMs) by immunofluorescence

Validation considerations:

• For Q152* mutation models, expect approximately 95% reduction in mRNA and 96% reduction in protein for homozygous knockouts

• For heterozygous models, expect approximately 45% reduction in mRNA and 55% reduction in protein

• For N68S mutations, protein levels may be reduced despite normal mRNA levels due to protein instability (observed 70% reduction)

Rescue experiments:

• Include rescue controls to confirm phenotype specificity

• The "Goldilocks effect" should be considered - both overexpression and insufficient protein can lead to similar phenotypes

• How can I investigate MAPRE2's role in cardiac electrophysiology and sodium channel function?

MAPRE2 has recently been implicated in Brugada syndrome through genome-wide association studies. To investigate its role in cardiac electrophysiology:

Zebrafish model approaches:

• Generate mapre2 knockout zebrafish using CRISPR/Cas9

• Perform voltage mapping of larval hearts at 5 days post-fertilization

• Measure ventricular maximum upstroke velocity of action potentials and conduction velocity

• Conduct ECG on adult fish hearts to detect QRS prolongation

• Use patch clamping to measure sodium current density in ventricular myocytes

iPSC-CM approaches:

• Create MAPRE2 knockdown or knockout in human induced pluripotent stem cell-derived cardiomyocytes

• Perform patch clamping to detect arrhythmias

• Use immunocytochemistry to examine adherens junction organization

• Assess N-cadherin localization and detyrosinated tubulin levels

Key findings to validate:

• MAPRE2 loss typically leads to decreased ventricular maximum upstroke velocity and conduction velocity

• QRS prolongation is observed in ECGs of knockout models

• Sodium current density is decreased in knockout ventricular myocytes

• Adherens junctions appear disorganized with mislocalization of mature N-cadherin

• Detyrosinated tubulin levels are decreased

• What techniques can be used to study MAPRE2's impact on microtubule dynamics?

MAPRE2 affects microtubule dynamics in several ways that can be studied using these approaches:

Microtubule plus-end tracking:

• Transfect cells with EB3-GFP to visualize microtubule plus-ends

• Perform live-cell imaging to track individual microtubules

• Generate kymographs for quantitative analysis

• Calculate growth velocity and distance parameters

Expected results in MAPRE2 knockdown:

• Increased microtubule growth velocity (1.11-1.26 fold increase)

• Increased microtubule growth distance (1.22-1.34 fold increase)

Tubulin post-translational modification analysis:

• Use Western blotting with antibodies specific to detyrosinated α-tubulin

• Calculate the ratio of detyrosinated to total α-tubulin

• Perform immunofluorescence to visualize tubulin modifications in situ

Microtubule architecture assessment:

• Immunofluorescence with antibodies against α-tubulin

• Confocal microscopy to assess microtubule bundling and straightness

• Computational analysis of microtubule organization and dynamics

Rescue strategies:

• Knockdown of tubulin tyrosine ligase (TTL) in MAPRE2 knockout models can rescue tubulin detyrosination and associated phenotypes

• How should I design experiments to investigate the structural instability of MAPRE2 mutations?

The structural analysis of MAPRE2 mutations, particularly the N68S mutation, requires multifaceted approaches:

Computational structure analysis:

• Use homology modeling based on MAPRE1 structures (e.g., 1VKA template)

• Analyze conservation scores across multiple species - residue 68 shows 100% conservation, suggesting structural/functional importance

• Calculate protein stability changes induced by mutations using tools like DUET, FoldX, or Rosetta

• Assess aggregation propensity changes using tools like TANGO

Expression and stability analysis:

• Compare mRNA and protein levels between mutant and isogenic control lines

• For N68S mutation, note that despite normal mRNA levels, protein expression may be reduced by about 70%

• Assess protein half-life using cyclohexamide chase assays

Structural impact predictions:

• N68S mutation increases aggregation propensity of the APR from 41.8 to 44.6

• Although this increase alone might not significantly affect aggregation tendency, the mutation is destabilizing

• The destabilization can hamper native folding by requiring higher energy states, indirectly increasing aggregation

Microtubule binding assays:

• In vitro microtubule-polymerization assays can detect changes in binding affinity

• Previous studies with missense mutations showed increased affinity and dwell time at microtubules, indicating gain-of-function effects

• What are the contradictions and knowledge gaps in the current understanding of MAPRE2 function?

Several contradictions and knowledge gaps exist in the current MAPRE2 literature:

Contradictory findings on tubulin detyrosination:

Mutation effects - Loss vs. Gain of function:

• Some MAPRE2 mutations (Q152*) act through loss-of-function mechanisms

• Other mutations (missense mutations in the CH domain) show increased microtubule binding, suggesting gain-of-function effects

• The "Goldilocks effect" hypothesis suggests that both overactive and insufficient protein can lead to similar clinical pathologies

Tissue-specific effects:

• How MAPRE2 functions differently in diverse tissues (cardiac, neural crest, epithelial) remains unclear

• The 43 amino acid segment unique to MAPRE2 (not present in EB1 or EB3) has an unknown function

Research gaps:

• Detailed 3D structure of MAPRE2 remains unknown

• Complete interactome of MAPRE2 across different cell types is not established

• Mechanistic understanding of how MAPRE2 variants lead to skin creases phenotype is incomplete

• The connection between MAPRE2, microtubule dynamics, and sodium channel function needs further clarification

• What methods should be used to study the neural crest differentiation protocol for MAPRE2 research?

To effectively study MAPRE2 in neural crest development, optimized differentiation protocols are essential:

Neural crest differentiation protocol:

• Start with induced pluripotent stem cells (iPSCs)

• Adapt protocols from previous studies (e.g., references 26,27 mentioned in source material)

• Validate cranial neural crest identity through gene expression analysis of specific markers:

  • TFAP2A, p75, SOX10, and ETS1 (cranial neural crest markers) should increase

  • OCT4, SOX2, Nanog, and PHOX2B (pluripotency genes) should decrease

Quality control metrics:

• Flow cytometry for surface markers (p75, HNK1)

• Immunofluorescence for neural crest transcription factors

• RT-qPCR for quantitative assessment of marker expression

• Single-cell RNA sequencing to confirm population homogeneity

Experimental considerations:

• Generate isogenic control lines using CRISPR/Cas9 technology

• Create both heterozygous and homozygous mutant lines

• Ensure cranial identity of neural crest cells to study craniofacial development accurately

• Consider timing of differentiation and maturation for phenotypic analysis

Migration assays for functional assessment:

• Scratch assay combined with live-cell imaging

• Nuclear labeling with H2B-RFP for cell tracking

• Quantification of migration speed, directionality, and persistence

• Supplementation with focal adhesion visualization using vinculin staining