Recombinant Danio rerio Receptor expression-enhancing protein 2 (reep2)

What is the primary cellular function of REEP2 in Danio rerio?

REEP2 in Danio rerio, like its mammalian counterparts, belongs to the DP1/Yop1p family of endoplasmic reticulum (ER)-shaping proteins. Its primary function involves modulating membrane curvature through insertion of hydrophobic hairpin domains into the ER membrane . This protein plays a crucial role in:

• Shaping and organizing the endoplasmic reticulum architecture

• Potentially facilitating interactions between the ER and microtubule cytoskeleton

• Contributing to proper protein trafficking through the secretory pathway

• Maintaining cellular homeostasis, particularly in neurons where REEP2 is highly expressed

Experimental evidence from studies with REEP family proteins demonstrates that loss of these proteins affects ER organization, as observed in mouse models with REEP1 deficiency . The highly conserved nature of REEP proteins across species suggests similar fundamental functions in zebrafish.

What expression patterns does REEP2 exhibit in Danio rerio tissues?

While the search results don't provide comprehensive tissue-specific expression data for Danio rerio REEP2, studies of REEP2 in other species indicate it is highly expressed in the brain and testis . This suggests a potentially similar expression pattern in zebrafish, though direct experimental validation is needed for confirmation.

The restricted expression pattern in specific tissues implies specialized functions in neuronal and reproductive systems. Unlike ubiquitously expressed proteins, this tissue-specific expression indicates REEP2 may have specialized roles in neural development, function, or maintenance in zebrafish. Future research using techniques such as in situ hybridization or tissue-specific RNA-seq would be valuable to precisely map REEP2 expression patterns throughout zebrafish development and in adult tissues.

What are the optimal conditions for expressing and purifying recombinant Danio rerio REEP2 protein?

Based on available data, the following protocol represents the optimal approach for expression and purification of recombinant Danio rerio REEP2:

Expression System:

• Host: E. coli expression system

• Vector considerations: Vectors similar to those used for other membrane-associated proteins would be appropriate

• Tags: N-terminal His-tag for efficient purification

Expression Conditions:
For membrane proteins like REEP2, careful control of expression is critical to prevent toxicity to host cells. Drawing from experience with other zebrafish proteins:

• Consider using KRX cells (a derivative of E. coli K12) for expression, as they provide precise control of gene expression and minimize leaky expression that could be toxic

• Induce expression with appropriate inducer (e.g., rhamnose if using the rhamnose promoter system)

• Optimal temperature for expression would likely be 18-25°C to facilitate proper folding

Purification Protocol:

• Cell lysis in an appropriate buffer containing detergents to solubilize membrane proteins

• Purification using Ni²⁺-NTA resin for His-tagged protein

• Storage as lyophilized powder or in buffer containing 50% glycerol at -20°C/-80°C

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

Storage Buffer Components:

• Tris/PBS-based buffer

• 6% Trehalose

• pH 8.0

For experimental applications requiring high purity, additional purification steps such as size exclusion chromatography may be necessary to achieve >90% purity as determined by SDS-PAGE .

How can researchers effectively assess REEP2 membrane binding capacity in functional assays?

Membrane binding is a critical functional property of REEP2, and mutations affecting this capacity have been linked to pathological conditions in humans, including hereditary spastic paraplegias . To assess REEP2 membrane binding, researchers can employ several complementary approaches:

Subcellular Fractionation Assay:

• Express tagged REEP2 (wild-type or mutant variants) in an appropriate cell line

• Perform subcellular fractionation to separate membrane-bound from soluble proteins

• Analyze distribution using Western blotting with anti-tag antibodies

• Compare the ratio of membrane-bound to soluble REEP2 across different variants

Liposome Flotation Assay:
This in vitro approach directly measures membrane binding capacity:

• Express and purify recombinant REEP2 protein

• Prepare liposomes with composition mimicking ER membranes

• Incubate protein with liposomes, then perform density gradient centrifugation

• Collect fractions and analyze protein distribution by Western blotting

Fluorescence Microscopy Approach:

• Express fluorescently-tagged REEP2 variants in cells

• Co-stain with ER markers (e.g., calnexin or PDI)

• Analyze colocalization using confocal microscopy

• Quantify the degree of membrane association through colocalization analysis

When conducting these assays, it's critical to include appropriate controls such as known membrane-binding deficient variants (e.g., the p.Val36Glu variant which has been shown to impair membrane binding) .

What methodological approaches can be used to study REEP2-microtubule interactions in Danio rerio models?

REEP2, like other REEP family proteins, potentially interacts with microtubules, which may be important for its function in organizing the ER network. Based on information about related REEP proteins, the following methodological approaches would be effective:

Co-immunoprecipitation of REEP2 with Tubulin:

• Prepare zebrafish tissue or cell lysates (brain tissue would be optimal given REEP2's expression pattern)

• Perform immunoprecipitation using anti-REEP2 antibodies

• Detect co-precipitated tubulin by Western blotting with anti-tubulin antibodies

• Include appropriate controls (non-specific IgG, tubulin-binding deficient mutants)

Immunofluorescence Colocalization Studies:

• Express fluorescently-tagged REEP2 in cell models

• Stain microtubules with appropriate antibodies

• Analyze colocalization using high-resolution confocal or super-resolution microscopy

• Quantify colocalization coefficients and perform statistical analysis

Microtubule Cosedimentation Assay:

• Purify recombinant REEP2 protein

• Prepare stabilized microtubules in vitro

• Incubate REEP2 with microtubules

• Perform centrifugation to pellet microtubules and associated proteins

• Analyze pellet and supernatant fractions by SDS-PAGE and Western blotting

In vivo Analysis using CRISPR/Cas9 Models:

• Generate zebrafish lines with fluorescently-tagged REEP2 using CRISPR/Cas9 genome editing

• Perform live imaging of tagged REEP2 in relation to microtubule dynamics

• Compare wild-type with mutant variants having altered microtubule binding domains

Previous studies have demonstrated that both wild-type REEP2 and the p.Val36Glu variant interact with microtubules , suggesting that this function may be preserved even when membrane binding is compromised.

How can Danio rerio REEP2 be used to model human diseases associated with REEP2 mutations?

Zebrafish (Danio rerio) represents an excellent model organism for studying REEP2-associated diseases due to its genetic tractability, transparent embryos for imaging, and the conservation of REEP2 function across species. Mutations in human REEP2 are associated with hereditary spastic paraplegias (HSPs), a group of neurological conditions characterized by progressive spasticity and weakness of the lower limbs . To develop zebrafish models:

CRISPR/Cas9 Gene Editing Approach:

• Design guide RNAs targeting the zebrafish reep2 gene

• Introduce specific mutations corresponding to human disease-causing variants:

  • p.Val36Glu variant for dominant HSP models

  • p.Phe72Tyr variant for recessive HSP models

• Confirm mutations through sequencing

• Establish stable transgenic lines

Phenotypic Analysis:

• Motor behavior assessment using standardized swimming assays

• Neuroanatomical analysis focusing on motor neuron development and axonal integrity

• Electrophysiological recordings to assess neural function

• ER morphology studies in affected neurons using transmission electron microscopy

Rescue Experiments:

• Attempt rescue of phenotypes by introducing wild-type human REEP2

• Compare efficacy of rescue between different mutant forms

• Test potential therapeutic compounds that might compensate for REEP2 dysfunction

The advantage of the zebrafish model is that it enables investigation of both dominant and recessive inheritance patterns associated with different REEP2 mutations, as observed in human families . Molecular phenotyping can help explain how different mutations cause disease through distinct mechanisms (dominant-negative effect versus loss-of-function).

What are the key experimental considerations when investigating REEP2's role in endoplasmic reticulum shaping?

REEP2's function in ER shaping requires specialized experimental approaches to visualize and quantify changes in ER morphology. Key considerations include:

Visualization Techniques:

• High-resolution imaging using techniques such as:

  • Confocal microscopy with ER-specific markers

  • Super-resolution microscopy (STED, PALM, or STORM) for detailed ER structure

  • Transmission electron microscopy for ultrastructural analysis

• Live cell imaging to capture dynamic changes in ER morphology

Quantitative Analysis Methods:

• Morphometric analysis of ER structure:

  • Tubule length and branching frequency

  • Sheet-to-tubule ratio

  • Three-dimensional reconstruction of ER networks

• Statistical approaches to quantify structural differences between wild-type and mutant conditions

Experimental Controls and Variables:

• Include both positive controls (known ER-shaping protein mutants) and negative controls

• Consider cell-type specific effects, as REEP2 function may vary between different cell types

• Evaluate temporal aspects of ER remodeling during development or under stress conditions

• Compare effects of different REEP2 mutations:

  • The p.Val36Glu variant which affects membrane binding

  • The p.Phe72Tyr variant which decreases affinity for membranes

Functional Assays:

• Assess ER stress responses using appropriate markers (e.g., XBP1 splicing, ATF6 activation)

• Evaluate calcium homeostasis, as ER shape affects calcium signaling

• Measure effects on protein trafficking and secretion

• Investigate interactions with other ER-shaping proteins through proximity labeling techniques

The evidence from human studies suggests that loss of association of REEP2 with membranes leads to pathological conditions , highlighting the importance of proper experimental design to capture subtle changes in ER morphology that may have significant functional consequences.

How does Danio rerio REEP2 structurally and functionally compare to other REEP family proteins?

Danio rerio REEP2 belongs to the larger REEP/DP1/Yop1p family of proteins, which can be divided into two subfamilies based on sequence homology and function. A comprehensive comparison reveals:

Structural Comparison:

• N-terminal Domain:

  • REEP2 contains two highly conserved hydrophobic domains at its N-terminus, a feature shared with REEP1, REEP3, and REEP4

  • These domains form hairpins that insert into membranes and promote oligomerization

  • The p.Phe72Tyr mutation site in REEP2 is conserved across all REEP family members up to zebrafish

• C-terminal Domain:

  • In REEP1, the C-terminus mediates interaction with microtubules

  • In REEP3 and REEP4, basic residues between the two hydrophobic domains facilitate microtubule binding

  • The specific microtubule-binding mechanism of REEP2 appears to differ from that of REEP1

Functional Comparison:

• Membrane Binding:

  • All REEP family proteins interact with membranes

  • The p.Val36Glu mutation in REEP2 disrupts membrane binding in a dominant-negative manner

  • Different REEP proteins may preferentially associate with different membrane curvatures

• Expression Patterns:

  • REEP2 is highly expressed in brain and testis

  • REEP1 and REEP2 show preferential expression in certain tissues (based on the title of search result )

  • This differential expression suggests specialized functions in specific cell types

• Disease Associations:

  • Mutations in both REEP1 and REEP2 are associated with hereditary spastic paraplegias

  • This suggests functional conservation and importance in neuronal maintenance

Evolutionary Conservation:
The conservation of key amino acids like Phe72 in REEP2 from zebrafish to humans highlights the evolutionary importance of these residues for proper protein function . This conservation provides a strong rationale for using zebrafish as a model organism to study REEP2 function and related diseases.

What are the key differences in optimal experimental conditions between Danio rerio REEP2 and mammalian REEP2 proteins?

When working with Danio rerio REEP2 versus mammalian REEP2 proteins, researchers should consider several important differences in experimental approaches:

Expression Systems:

• For zebrafish REEP2:

  • E. coli-based expression systems have been successfully used

  • Consider using KRX cells to minimize leaky expression, as demonstrated for other zebrafish proteins

  • Optimal induction conditions may differ from mammalian proteins

• For mammalian REEP2:

  • Mammalian expression systems may be preferred to ensure proper post-translational modifications

  • Insect cell systems might provide better yield for structural studies

Buffer Conditions:
Drawing from experiences with other zebrafish proteins compared to mammalian counterparts:

• Salt Sensitivity:

  • Optimal salt concentrations may differ significantly

  • For instance, zebrafish DNA polymerase β shows maximum activity at 10 mM KCl and 50 mM NaCl

  • Testing a range of ionic strengths is advisable for novel zebrafish proteins

• Divalent Cation Requirements:

  • Zebrafish proteins often show different optimal Mg²⁺ and Mn²⁺ concentrations compared to mammalian orthologs

  • For example, zebrafish DNA polymerase β shows a sharp activity peak at 1 mM Mg²⁺ versus 5 mM for rat enzyme

Functional Assays:

• Temperature Considerations:

  • Zebrafish proteins have evolved to function at lower temperatures (optimal temperature ~28°C)

  • Experimental assays should account for temperature differences when comparing across species

• pH Optima:

  • Optimal pH conditions may differ between zebrafish and mammalian proteins

  • Systematic testing of buffer conditions is recommended

Storage Stability:
For zebrafish REEP2, recommended storage conditions include:

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use

• Avoid repeated freeze-thaw cycles

• Consider storage in buffer containing 50% glycerol

These differences highlight the importance of optimizing experimental conditions specifically for Danio rerio REEP2 rather than assuming identical conditions to mammalian orthologs will be optimal.

How can researchers overcome challenges in generating functional antibodies against Danio rerio REEP2?

Generating specific antibodies against Danio rerio REEP2 presents several challenges due to its membrane-associated nature and potential conservation with other REEP family members. The following approaches can help overcome these challenges:

Antigen Design Strategies:

• Peptide-based approach:

  • Select unique epitopes not conserved in other REEP family proteins

  • Focus on hydrophilic regions of the protein that are likely accessible

  • Avoid the highly conserved hydrophobic domains to prevent cross-reactivity

• Recombinant protein fragments:

  • Express soluble fragments of REEP2 (particularly the C-terminal region)

  • Use His-tagged constructs for efficient purification

  • Verify proper folding of the antigen before immunization

Immunization Protocols:

• Use of multiple species:

  • Generate antibodies in rabbits for polyclonal antibodies

  • Consider hamster or guinea pig for additional specificity, as they may recognize different epitopes than rabbit antibodies

• Adjuvant selection:

  • Choose adjuvants that enhance immune response without causing protein denaturation

  • Consider gentle adjuvants for membrane proteins

Purification and Validation Strategies:

• Affinity purification:

  • Purify antibodies using immobilized antigen

  • Perform negative selection against related REEP proteins to remove cross-reactive antibodies

• Comprehensive validation:

  • Test antibody specificity in REEP2 knockout/knockdown models

  • Verify recognition of both denatured (Western blot) and native (immunoprecipitation) forms

  • Perform preabsorption tests with immunizing antigen

• Cross-reactivity testing:

  • Test against other REEP family members expressed in the same tissues

  • Evaluate in multiple assays (immunoblotting, immunohistochemistry, etc.)

Alternative Approaches:
If generating specific antibodies proves challenging, consider:

• Epitope tagging approaches:

  • Generate transgenic zebrafish lines expressing tagged versions of REEP2

  • Use commercial antibodies against the tag for detection

• Proximity labeling methods:

  • Use BioID or APEX2 fusion proteins to identify REEP2-proximal proteins

  • This avoids the need for direct REEP2 antibodies

What are the critical parameters for successful mutagenesis studies of Danio rerio REEP2?

Mutagenesis studies of REEP2 in zebrafish require careful consideration of several critical parameters to ensure successful outcomes:

Target Selection:

• Disease-relevant mutations:

  • Focus on conserved residues known to cause human disease

  • The p.Val36Glu mutation causes dominant HSP

  • The p.Phe72Tyr mutation is associated with recessive HSP

• Functional domain targeting:

  • Target the N-terminal hydrophobic domains involved in membrane interaction

  • Consider residues at the interface between REEP2 and potential binding partners

Mutagenesis Strategies:

• CRISPR/Cas9 approach:

  • Design specific guide RNAs with minimal off-target effects

  • Consider using base editors for precise point mutations

  • Include PAM site mutations that don't affect protein function to prevent re-cutting

• Homology-directed repair:

  • Provide donor templates containing desired mutations

  • Include silent mutations to facilitate genotyping

  • Consider using long homology arms to increase efficiency

Genotyping Considerations:

• Screening strategy:

  • Design primers for efficient screening of founders

  • Establish restriction enzyme digestion assays when possible

  • Consider high-resolution melting analysis for point mutations

• Verification methods:

  • Confirm mutations by sequencing

  • Verify germline transmission

  • Check for potential off-target effects in critical genes

Phenotypic Analysis Parameters:

• Developmental timing:

  • Assess phenotypes at multiple developmental stages

  • Consider potential compensatory mechanisms in early development

• Tissue-specific effects:

  • Focus analysis on tissues with high REEP2 expression (brain)

  • Perform cell-type specific analyses within these tissues

• Functional readouts:

  • ER morphology assessment

  • Motor behavior analysis

  • Electrophysiological measurements in relevant neuronal populations

• Molecular phenotyping:

  • Analyze interactions with membranes for p.Val36Glu-equivalent mutations

  • Assess protein stability and localization for p.Phe72Tyr-equivalent mutations

Controls and Validation:

• Include appropriate controls:

  • Wild-type siblings

  • Different allelic variants (null versus point mutations)

  • Rescue experiments with wild-type human REEP2

The successful implementation of these parameters will enable meaningful mutagenesis studies that provide insight into REEP2 function in zebrafish and its relevance to human disease.

What are the most promising avenues for future research on Danio rerio REEP2?

Based on current knowledge and gaps in understanding, several promising research directions emerge:

• Comprehensive Expression Mapping:

  • Detailed characterization of REEP2 expression patterns throughout zebrafish development

  • Cell-type specific expression analysis in the brain

  • Regulatory mechanisms controlling REEP2 expression

• High-Resolution Structural Studies:

  • Cryo-EM or X-ray crystallography studies of REEP2 interaction with membranes

  • Structural analysis of REEP2 oligomers and their membrane-shaping properties

  • Conformational changes associated with disease-causing mutations

• In vivo Functional Studies:

  • Generation of zebrafish models with mutations equivalent to human disease variants

  • Live imaging of ER dynamics in REEP2 mutant neurons

  • Rescue experiments with structure-guided engineered variants

• Interactome Analysis:

  • Comprehensive identification of REEP2 binding partners

  • Comparison with other REEP family members to identify unique interactions

  • Determination of tissue-specific interaction networks

• Therapeutic Development:

  • Screens for compounds that can rescue REEP2 mutant phenotypes

  • Development of strategies to enhance membrane binding of defective REEP2 variants

  • Gene therapy approaches for REEP2-associated diseases

The zebrafish model offers unique advantages for these studies due to its genetic tractability, transparent embryos for imaging, and conservation of REEP2 function . The reference cross DNA panel for zebrafish, consisting of 520 F2 progeny, provides a valuable resource for genetic mapping studies that may further elucidate REEP2 function and regulation .

How might integrative approaches combining structural biology, genetics, and cell biology advance our understanding of REEP2 function?

An integrative approach combining multiple disciplines represents the most promising strategy to fully elucidate REEP2 function:

Structural Biology Contributions:

• Determination of REEP2 structure in membrane-bound state

• Analysis of conformational changes associated with disease-causing mutations

• Structural basis for REEP2 oligomerization and membrane curvature induction

Genetic Approaches:

• CRISPR/Cas9-based generation of zebrafish models with specific REEP2 mutations

• Forward genetic screens to identify genetic modifiers of REEP2 function

• Utilization of the reference cross DNA panel for fine mapping of genetic interactions

Cell Biology Techniques:

• Advanced imaging of ER dynamics in living cells

• Analysis of REEP2's role in ER-microtubule contact sites

• Investigation of REEP2's impact on secretory pathway function

Integration Methods:

• Combined structural-functional studies:

  • Structure-guided mutagenesis to test specific hypotheses

  • Correlation of structural features with functional outcomes in vivo

• Multi-omics integration:

  • Correlation of transcriptomic, proteomic, and lipidomic data

  • Network analysis to position REEP2 in cellular pathways

• Cross-species comparative approaches:

  • Parallel studies in zebrafish and mammalian models

  • Evolutionary analysis of REEP protein function

• Translation to disease mechanisms:

  • Direct testing of patient-derived mutations in zebrafish

  • Development of zebrafish-based platforms for therapeutic screening