Recombinant Danio rerio Transmembrane protein 186 (tmem186)

How does the structure of recombinant tmem186 compare to other transmembrane proteins in zebrafish?

The structure of recombinant tmem186 shares several characteristics with other zebrafish transmembrane proteins but has unique features. Unlike Transmembrane protein 216 (tmem216), which consists of 160 amino acids , tmem186 has 228 amino acids . Both proteins feature hydrophobic segments that span the lipid bilayer, but their amino acid compositions differ significantly. The MADVCLRLQYACPWQSNMAAHG sequence in tmem216 contrasts with the MDMMMMSTRLIHLSRHFPQYGF sequence in tmem186 . Similar to other transmembrane proteins in Danio rerio, tmem186 likely contains cytoplasmic domains that may interact with intracellular signaling molecules, though the specific binding partners are yet to be fully elucidated compared to better-characterized proteins like Tmem184a, which has established roles in angiogenesis .

What expression systems are recommended for producing recombinant tmem186?

For recombinant tmem186 expression, E. coli-based systems are commonly employed, similar to the approaches used for other Danio rerio transmembrane proteins. Based on experiences with related proteins, KRX cells are often preferred over BL21(DE3) or BL21(DE3)pLysS strains due to reduced leaky expression . For optimal expression:

• Clone the full tmem186 coding sequence (1-228) into an expression vector with a purification tag (His-tag is commonly used)

• Transform into KRX cells rather than standard BL21 strains to minimize protein degradation

• Induce expression under controlled conditions to prevent inclusion body formation

• Purify using affinity chromatography followed by size exclusion to ensure homogeneity

In vitro expression systems may result in proteolysis of zebrafish transmembrane proteins, as observed with other Danio rerio proteins, making bacterial expression generally more reliable for obtaining intact protein .

How can knockout/knockdown approaches be utilized to study tmem186 function in zebrafish?

Knockout/knockdown studies of tmem186 in zebrafish can be designed using several approaches, informed by methodologies employed for related transmembrane proteins:

• Morpholino-based knockdown: Design morpholinos targeting the tmem186 translation start site or splice junctions. Inject into one-cell stage embryos (1-2 nl at 0.2-0.4 mM concentration) and validate knockdown efficiency via western blotting .

• CRISPR/Cas9 knockout: Design guide RNAs targeting conserved regions of tmem186, particularly within the transmembrane domains. After generating F0 mosaic animals, screen for germline transmission and establish stable knockout lines.

• Dominant-negative approach: Similar to studies with Tmem184a, express truncated versions of tmem186 lacking functional domains to disrupt endogenous protein function .

For phenotypic analysis, researchers should examine both embryonic development and adult physiology, with particular attention to systems where transmembrane proteins typically function (vascular system, neural development, epithelial integrity). Complementation studies using recombinant protein can help confirm specificity of observed phenotypes. When analyzing results, it's essential to monitor for off-target effects by including appropriate controls and rescue experiments.

What are the critical parameters for biochemical characterization of recombinant tmem186?

Biochemical characterization of recombinant tmem186 requires attention to several critical parameters:

During biochemical characterization, compare kinetic parameters (Km, kcat, catalytic efficiency) with orthologs from other species to identify conserved properties versus zebrafish-specific features, similar to approaches used for other Danio rerio proteins . For storage stability assessment, monitor protein integrity after freeze-thaw cycles using both functional and structural assays, as repeated freezing and thawing is not recommended for transmembrane proteins .

What are the optimal conditions for storage and handling of recombinant tmem186?

Optimal storage and handling of recombinant tmem186 should follow established protocols for transmembrane proteins:

• Short-term storage: Store working aliquots at 4°C for up to one week to minimize freeze-thaw cycles .

• Long-term storage: Store at -20°C, or preferably -80°C for extended periods . Prepare small aliquots to avoid multiple freeze-thaw cycles.

• Buffer composition: Use Tris-based buffer with 50% glycerol for stability . For functional studies, optimize buffer conditions including pH, salt concentration, and potential stabilizing additives.

• Reconstitution: For lyophilized protein, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Consider adding 5-50% glycerol as a cryoprotectant (with 50% being a standard final concentration) .

• Sample preparation: Briefly centrifuge vials before opening to bring contents to the bottom, particularly for lyophilized preparations .

These conditions are based on established protocols for related Danio rerio transmembrane proteins and should be optimized specifically for tmem186 through stability trials monitoring activity and structural integrity over time under different storage conditions.

What experimental controls are essential when studying tmem186 function in zebrafish models?

When studying tmem186 function in zebrafish models, several essential controls must be implemented:

• Knockdown/knockout validation:

  • Western blotting to confirm protein reduction

  • qRT-PCR to assess transcript levels

  • Include non-targeting morpholinos or guide RNAs as negative controls

• Phenotypic analysis controls:

  • Wild-type siblings from the same clutch

  • Rescue experiments with wild-type tmem186 mRNA to confirm specificity

  • Dose-response studies to assess morpholino or CRISPR specificity

• Protein expression controls:

  • Loading controls (tubulin, as used for VE-cadherin detection )

  • Subcellular fraction controls to confirm membrane localization

  • Tagged versus untagged protein comparisons to assess tag interference

• Functional assays:

  • Positive controls using proteins with known activity

  • Temperature controls for zebrafish development

  • Vehicle controls for any treatments or interventions

These controls ensure that observed phenotypes are specific to tmem186 disruption rather than off-target effects or experimental artifacts, following established practices in zebrafish protein research .

How can researchers effectively validate antibodies for detecting native tmem186 in zebrafish samples?

Antibody validation for detecting native tmem186 in zebrafish samples requires a systematic approach:

• Specificity testing:

  • Compare staining patterns in wild-type versus tmem186 knockout/knockdown samples

  • Perform peptide competition assays to confirm binding to the intended epitope

  • Test across multiple tissues and developmental stages to ensure consistent detection

• Cross-reactivity assessment:

  • Evaluate antibody performance across species if using commercial antibodies

  • Test against recombinant tmem186 and related family members to confirm specificity

  • Perform western blots under reducing and non-reducing conditions to identify potential cross-reactivity

• Optimization protocols:

  • For western blotting: Test different sample preparation methods, particularly for membrane proteins (similar to methods used for VE-cadherin detection )

  • For immunohistochemistry: Compare fixation protocols, antigen retrieval methods, and blocking conditions

  • For flow cytometry: Optimize permeabilization conditions for intracellular domains

• Validation methods matrix:

Consider generating zebrafish-specific antibodies if commercial options lack specificity, similar to the approach used for VE-cadherin detection where a polyclonal antibody was generated against amino acids 186-372 .

What are common challenges in expressing and purifying recombinant tmem186, and how can they be addressed?

Common challenges in expressing and purifying recombinant tmem186 and strategies to address them include:

• Protein degradation:

  • Challenge: Proteolysis during expression, as observed with other Danio rerio proteins expressed in vitro

  • Solution: Use KRX cells instead of BL21(DE3) strains to reduce leaky expression , add protease inhibitors during all purification steps, and optimize induction temperature (typically lower temperatures reduce proteolysis)

• Low solubility:

  • Challenge: Transmembrane proteins often form inclusion bodies in bacterial systems

  • Solution: Express as fusion proteins with solubility tags (MBP, SUMO), use specialized E. coli strains designed for membrane proteins, or employ insect cell expression systems for improved folding

• Purification difficulties:

  • Challenge: Obtaining homogeneous preparations of membrane proteins

  • Solution: Implement two-step purification protocols combining affinity chromatography with size exclusion, carefully select detergents compatible with downstream applications, and consider nanodiscs for maintaining native-like lipid environments

• Protein instability:

  • Challenge: Loss of activity during storage

  • Solution: Add stabilizing agents (glycerol, specific lipids), avoid repeated freeze-thaw cycles by storing small aliquots , and determine optimal buffer conditions through thermal shift assays

• Functional validation:

  • Challenge: Confirming proper folding and activity

  • Solution: Develop activity assays based on predicted function, compare structural characteristics with well-characterized homologs, and validate through complementation studies in knockout models

These approaches are based on methods successfully applied to other transmembrane proteins from Danio rerio and should be optimized specifically for tmem186 .

How can researchers distinguish between direct and indirect effects when analyzing tmem186 knockdown phenotypes?

Distinguishing between direct and indirect effects in tmem186 knockdown studies requires multiple complementary approaches:

• Temporal analysis:

  • Track phenotype progression over time to identify primary versus secondary effects

  • Use inducible knockdown systems to initiate tmem186 depletion at different developmental stages

  • Compare with the temporal progression of phenotypes in other zebrafish transmembrane protein studies

• Tissue-specific knockdown:

  • Use tissue-specific promoters to restrict tmem186 knockdown to specific cell types

  • Compare phenotypes between global and tissue-restricted knockdown to identify cell-autonomous effects

  • Employ mosaic analysis through cell transplantation between wild-type and knockdown embryos

• Molecular pathway analysis:

  • Examine changes in known signaling pathways (similar to Vegfr2 pathway analysis in Tmem184a studies )

  • Perform transcriptomic analysis at early timepoints after knockdown

  • Use phosphoproteomic approaches to identify rapidly altered signaling events

• Rescue experiments:

  • Full-length tmem186 rescue to confirm knockdown specificity

  • Domain-specific mutants to identify critical functional regions (similar to the heparin-binding domain analysis in Tmem184a )

  • Rescue with downstream effectors to establish pathway relationships

• Cross-comparison with related phenotypes:

  • Compare with phenotypes of interacting proteins or pathway components

  • Analyze double knockdowns to identify genetic interactions, similar to the Tmem184a and Vegfr2b synergy observed in previous studies

What advanced analytical techniques are most informative for studying tmem186 structure-function relationships?

Several advanced analytical techniques are particularly informative for studying tmem186 structure-function relationships:

• Structural analysis techniques:

  • Cryo-electron microscopy: For determining three-dimensional structure of the intact protein in a membrane environment

  • NMR spectroscopy: Particularly useful for analyzing dynamic regions and ligand interactions

  • X-ray crystallography: If stable, well-diffracting crystals can be obtained

  • Hydrogen-deuterium exchange mass spectrometry: To identify conformational changes upon binding partners

• Functional mapping approaches:

  • Alanine scanning mutagenesis: Systematically replace conserved residues to identify critical functional sites

  • Domain swapping: Exchange domains with related transmembrane proteins to determine specificity determinants

  • Chimeric proteins: Create fusions with domains from related proteins (similar to analysis of the heparin binding domain in Tmem184a )

• Interaction studies:

  • Proximity labeling (BioID, APEX): To identify neighboring proteins in cellular contexts

  • Crosslinking mass spectrometry: To capture direct interaction interfaces

  • Förster resonance energy transfer (FRET): For analyzing dynamic interactions in living cells

• In silico approaches:

  • Molecular dynamics simulations: To study protein behavior in membrane environments

  • Homology modeling: Based on related transmembrane proteins with known structures

  • Evolutionary coupling analysis: To identify co-evolving residues likely to be functionally important

When implementing these techniques, researchers should consider the specialized challenges of membrane proteins, including the need for appropriate detergents or lipid environments to maintain native-like conformations. Integration of multiple complementary techniques typically provides the most comprehensive understanding of structure-function relationships.

What are the most promising future research directions for understanding tmem186 function in zebrafish?

The most promising future research directions for understanding tmem186 function in zebrafish include:

• Comprehensive phenotypic characterization: Systematic analysis of tmem186 knockout zebrafish across developmental stages and tissue types, with particular attention to systems where related transmembrane proteins function (vascular development, neural patterning, epithelial morphogenesis).

• Interactome mapping: Application of proximity labeling and affinity purification approaches to identify the protein interaction network of tmem186, potentially revealing pathway connections similar to the heparan sulfate and VEGF signaling links discovered for Tmem184a .

• Evolutionary analysis: Comparative studies of tmem186 across vertebrate species to identify conserved domains and species-specific adaptations, providing insights into fundamental versus specialized functions.

• Integration with omics data: Correlation of tmem186 expression patterns with transcriptomic, proteomic, and metabolomic datasets to place the protein in broader cellular networks and identify potential functional contexts.

• Therapeutic implications: Investigation of whether tmem186 function in zebrafish has parallels to human health conditions, potentially establishing new zebrafish models for studying human diseases related to transmembrane protein dysfunction.