Recombinant Danio rerio Transmembrane protein 18 (tmem18)

What is the molecular structure of Danio rerio tmem18?

Danio rerio tmem18 is a small protein of 152 amino acids that belongs to the transmembrane protein 18 family (IPR026721). Unlike previous models suggesting three transmembrane domains, recent structural analyses indicate tmem18 likely contains four transmembrane domains . The protein is predominantly alpha-helical in structure. In zebrafish, tmem18 is encoded by a protein-coding gene located on chromosome 23 . The protein contains a positively charged C-terminus that includes a nuclear localization signal, which is critical for its DNA-binding functionality .

What are the primary cellular functions of tmem18 in Danio rerio?

Tmem18 in zebrafish is predicted to enable DNA binding activity and plays a role in fat cell differentiation pathways . The protein primarily localizes to the nuclear membrane where it can bind to DNA in a sequence-specific manner . This binding activity brings chromatin very close to the nuclear membrane, potentially repressing transcription of target genes . Additionally, tmem18 has been found to physically interact with key components of the nuclear pore complex, suggesting a role in nucleocytoplasmic transport or chromatin organization .

In which tissues is tmem18 primarily expressed in zebrafish?

In Danio rerio, tmem18 expression has been documented in multiple tissues, including:

• Gill

• Heart

• Liver

• Nervous system

• Pleuroperitoneal region

This expression pattern suggests tmem18 may have tissue-specific functions across different physiological systems in zebrafish.

What expression systems are most effective for producing recombinant Danio rerio tmem18?

For producing recombinant Danio rerio tmem18, researchers should consider several expression systems based on the protein's characteristics:

Bacterial Expression System (E. coli):

• For DNA-binding domain studies, a C-terminal fragment containing the positively charged region can be expressed as a fusion protein with tags like 6xHis or GST

• Culture conditions: LB medium supplemented with appropriate antibiotics

• Induction: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Temperature: Lower temperature (16-25°C) during induction to enhance proper folding

• Challenge: Full-length tmem18 with multiple transmembrane domains may form inclusion bodies

Eukaryotic Expression Systems:

• Insect cells (Sf9, Sf21) using baculovirus expression system

• Mammalian cells (HEK293, CHO) for studies requiring mammalian post-translational modifications

• Yeast (Pichia pastoris) for membrane protein expression

The choice of expression system should be guided by the specific research questions and downstream applications.

What purification strategies are most effective for recombinant Danio rerio tmem18?

Purification Protocol for Recombinant tmem18:

• Cell Lysis and Membrane Fraction Isolation:

  • For full-length protein: Gentle lysis methods to preserve membrane integrity

  • Buffer composition: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail

  • Ultracentrifugation at 100,000 × g to isolate membrane fractions

• Membrane Protein Solubilization:

  • Detergents: n-dodecyl-β-D-maltoside (DDM), digitonin, or CHAPSO at 1-2% w/v

  • Incubation: 1-2 hours at 4°C with gentle rotation

• Affinity Chromatography:

  • For His-tagged proteins: Ni-NTA resin

  • For GST-fusion proteins: Glutathione Sepharose

  • Imidazole gradient elution (20-250 mM) for His-tagged proteins

• Size Exclusion Chromatography:

  • Buffer: 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05-0.1% detergent

  • Columns: Superdex 75 or 200 depending on construct size

For the DNA-binding domain alone (C-terminus), standard protein purification methods may be suitable without requiring detergent solubilization.

How can researchers characterize the DNA-binding specificity of Danio rerio tmem18?

To characterize the DNA-binding specificity of recombinant Danio rerio tmem18, researchers should employ multiple complementary approaches:

Electrophoretic Mobility Shift Assay (EMSA):

• Generate purified recombinant tmem18 or its C-terminal DNA-binding domain

• Design oligonucleotide probes based on predicted binding sequences

• Incubate protein with labeled DNA probes

• Analyze mobility shifts on non-denaturing polyacrylamide gels

• Perform competition assays with unlabeled DNA to confirm specificity

Chromatin Immunoprecipitation (ChIP):

• Express epitope-tagged tmem18 in zebrafish cells or embryos

• Cross-link protein-DNA complexes with formaldehyde

• Immunoprecipitate with antibodies against the epitope tag

• Identify bound DNA sequences through sequencing (ChIP-seq)

Systematic Evolution of Ligands by Exponential Enrichment (SELEX):

• Incubate recombinant tmem18 with a random oligonucleotide library

• Isolate protein-DNA complexes

• Amplify bound DNA by PCR

• Repeat selection processes for multiple rounds

• Sequence enriched DNA to identify consensus binding motifs

Reporter Gene Assays:

• Clone potential tmem18 binding sequences upstream of a reporter gene

• Co-transfect with tmem18 expression vector

• Measure reporter gene activity to assess transcriptional effects

Previous research has demonstrated that TMEM18 can suppress expression from a reporter vector containing its target sequence , suggesting that similar approaches could be effective for the zebrafish ortholog.

What methods can be used to study tmem18 interaction with the nuclear pore complex?

To investigate the interaction between tmem18 and the nuclear pore complex (NPC) in Danio rerio, researchers can employ several techniques:

Co-immunoprecipitation (Co-IP):

• Generate antibodies against tmem18 or use epitope-tagged recombinant protein

• Prepare nuclear membrane fractions from zebrafish tissues or cells

• Immunoprecipitate tmem18 and identify interacting NPC components by mass spectrometry

• Validate interactions by reciprocal Co-IP with antibodies against NPC components

Proximity Ligation Assay (PLA):

• Fix zebrafish cells or tissue sections

• Incubate with primary antibodies against tmem18 and specific NPC proteins

• Use secondary antibodies conjugated with DNA oligonucleotides

• Perform ligation and rolling circle amplification

• Visualize interaction signals by fluorescence microscopy

Fluorescence Resonance Energy Transfer (FRET):

• Generate fusion constructs of tmem18 and NPC components with appropriate fluorophores

• Express in zebrafish cells or embryos

• Measure energy transfer to detect protein-protein proximity

BioID or APEX Proximity Labeling:

• Generate fusion constructs of tmem18 with BioID or APEX2

• Express in zebrafish cells

• Activate biotinylation of proteins in proximity to tmem18

• Purify biotinylated proteins and identify by mass spectrometry

How can researchers investigate the role of tmem18 in zebrafish adipogenesis and metabolism?

To study tmem18's role in adipogenesis and metabolism in zebrafish, researchers can implement the following experimental approaches:

Genetic Manipulation:

• CRISPR-Cas9 Gene Editing:

  • Design sgRNAs targeting zebrafish tmem18

  • Generate knockout or knockin lines

  • Assess phenotypes related to adiposity and lipid metabolism

• Morpholino Knockdown:

  • Design splice-blocking or translation-blocking morpholinos

  • Inject into one-cell stage embryos

  • Analyze early developmental effects on adipose tissue formation

• Transgenic Overexpression:

  • Generate tissue-specific promoter-driven tmem18 constructs

  • Create stable transgenic lines with adipose tissue or brain-specific expression

  • Assess effects on adipogenesis and whole-body metabolism

Phenotypic Analysis:

• Adipose Tissue Visualization:

  • Nile Red or Oil Red O staining to visualize neutral lipids

  • Fluorescent lipid analogs for in vivo imaging

  • Transgenic lines with fluorescently labeled adipocytes

• Metabolic Parameters:

  • Whole-body triglyceride and cholesterol quantification

  • Glucose tolerance tests

  • Oxygen consumption and activity measurements

• Gene Expression Analysis:

  • qPCR for adipogenesis markers (pparγ, cebpα, fabp11a)

  • RNA-seq to identify genome-wide expression changes

  • In situ hybridization to localize expression in tissues

Dietary Interventions:

• High-Fat Diet Challenge:

  • Feed wild-type and tmem18-modified fish with high-fat diets

  • Compare weight gain, adipose tissue expansion, and metabolic parameters

  • Analyze gene expression changes in response to diet

Based on mouse studies, researchers should pay particular attention to food intake and energy expenditure, as loss of Tmem18 in mice results in increased body weight (exacerbated by high-fat diet) due to increased food intake, while overexpression reduces food intake and increases energy expenditure .

What experimental designs are appropriate for studying tmem18 function in the zebrafish nervous system?

Given tmem18's expression in the nervous system and its potential role in energy balance regulation through central mechanisms , researchers can employ these approaches:

Neuroanatomical Studies:

• Expression Mapping:

  • In situ hybridization to precisely localize tmem18 expression in brain regions

  • Immunohistochemistry to detect protein distribution

  • Single-cell RNA-seq to identify neuron populations expressing tmem18

• Neuronal Circuit Analysis:

  • Transgenic reporter lines to visualize tmem18-expressing neurons

  • Anterograde and retrograde tracing to map connections

  • Calcium imaging to assess neuronal activity

Functional Studies:

• Neuron-Specific Manipulation:

  • Conditional knockdown or overexpression using neuron-specific promoters

  • Optogenetic or chemogenetic activation/inhibition of tmem18-expressing neurons

  • Focal brain region injection of viral vectors for localized manipulation

• Behavioral Assays:

  • Feeding behavior quantification

  • Activity and energy expenditure measurements

  • Responses to metabolic challenges (fasting, refeeding)

Molecular Mechanisms:

• Transcriptional Profiling:

  • RNA-seq of specific brain regions after tmem18 manipulation

  • ChIP-seq to identify direct target genes in neurons

  • Analysis of neuropeptide expression changes

• Signaling Pathway Analysis:

  • Investigation of interaction with known energy balance pathways

  • Protein-protein interaction studies in neuronal contexts

  • Phosphorylation state analysis following nutritional challenges

How can researchers resolve contradictions in the literature regarding tmem18 transmembrane topology?

The literature presents contradictions regarding tmem18's transmembrane topology, with earlier work suggesting three transmembrane domains while more recent evidence indicates four transmembrane domains . To resolve this contradiction, researchers should:

Computational Prediction Refinement:

• Apply multiple prediction algorithms (TMHMM, Phobius, HMMTOP)

• Compare results across algorithms and across species orthologs

• Generate consensus topology models

Experimental Validation Approaches:

• Substituted Cysteine Accessibility Method (SCAM):

  • Generate cysteine mutants throughout the protein sequence

  • Express in membrane systems

  • Probe accessibility with membrane-permeable and impermeable reagents

  • Map topology based on reactivity patterns

• Protease Protection Assays:

  • Express epitope-tagged versions of tmem18 in cell systems

  • Prepare microsomes or membrane fractions

  • Treat with proteases with/without membrane permeabilization

  • Detect protected fragments by Western blotting

• Glycosylation Mapping:

  • Insert glycosylation sites at various positions

  • Express in eukaryotic systems

  • Assess glycosylation status to determine luminal exposure

• Cryo-EM or X-ray Crystallography:

  • Purify sufficient quantities of stable, homogeneous protein

  • Determine high-resolution structure to definitively resolve topology

Validation in Zebrafish System:

• Generate fusion constructs with reporters positioned at different segments

• Express in zebrafish cells or tissues

• Determine subcellular localization and membrane topology in vivo

What approaches can identify genome-wide binding sites and transcriptional targets of tmem18 in zebrafish?

To comprehensively identify genome-wide binding sites and transcriptional targets of tmem18 in zebrafish, researchers should implement an integrated approach:

ChIP-seq Analysis:

• Generate epitope-tagged tmem18 constructs (HA, FLAG, or V5)

• Express in zebrafish embryos or cell lines

• Perform chromatin immunoprecipitation followed by high-throughput sequencing

• Analyze binding sites with peak calling algorithms

• Identify enriched sequence motifs and genomic features

CUT&RUN or CUT&Tag:

• Use antibodies against tmem18 or epitope tags

• Perform targeted DNA cleavage around binding sites

• Sequence released fragments

• Compare with ChIP-seq results for validation

ATAC-seq Combined with tmem18 Manipulation:

• Generate tmem18 knockdown, knockout, or overexpression models

• Perform ATAC-seq to identify changes in chromatin accessibility

• Correlate changes with potential tmem18 binding sites

RNA-seq for Transcriptional Effects:

• Manipulate tmem18 levels (knockdown, knockout, overexpression)

• Perform RNA-seq to identify differentially expressed genes

• Compare with binding site data to distinguish direct from indirect targets

• Validate key targets with qPCR and reporter assays

Integrated Data Analysis:

• Correlate binding sites with gene expression changes

• Perform pathway and Gene Ontology enrichment analyses

• Construct regulatory networks centered on tmem18

• Compare with known obesity and metabolism-related pathways

This integrated approach would provide comprehensive insights into the regulatory functions of tmem18 in zebrafish and could reveal mechanisms by which TMEM18 genetic variation contributes to obesity risk in humans .

How conserved is tmem18 function between Danio rerio and mammalian models?

Studies indicate significant conservation of tmem18 structure and function across vertebrates, including between zebrafish and mammals:

Sequence and Structural Conservation:

Functional Conservation:

• Nuclear Membrane Localization: Both zebrafish and mammalian tmem18 localize to the nuclear membrane

• DNA Binding Activity: The DNA-binding capability appears conserved, mediated by the positively charged C-terminus

• Metabolic Regulation: In mice, Tmem18 influences body weight, food intake, and energy expenditure , which likely extends to zebrafish given the conservation of adipogenic pathways

• Expression Patterns: Both zebrafish and mammalian tmem18 are expressed in the nervous system and metabolically active tissues

Model System Complementarity:

• Zebrafish offers advantages for high-throughput screening, developmental studies, and in vivo imaging

• Mouse models provide closer physiological relevance to human metabolism

• Cell culture systems from both species can interrogate molecular mechanisms

Researchers should consider the strengths of each model system when designing experiments to study tmem18 function, with zebrafish particularly valuable for early developmental processes and genetic screens.

How can zebrafish tmem18 research inform human obesity genetics studies?

Zebrafish tmem18 research can provide valuable insights into human obesity genetics through several approaches:

Functional Validation of Human Variants:

• Identify human GWAS variants near TMEM18 associated with obesity

• Generate corresponding mutations in zebrafish tmem18

• Assess phenotypic consequences on adiposity and metabolism

• Test whether human variants can rescue zebrafish tmem18 mutant phenotypes

Regulatory Network Mapping:

• Identify downstream targets of tmem18 in zebrafish

• Determine conservation of these pathways in humans

• Assess whether these target genes contain obesity-associated variants

• Construct network models connecting TMEM18 to broader metabolic regulation

Drug Discovery Applications:

• Develop zebrafish-based screening platforms for tmem18 modulators

• Test compounds affecting tmem18 function or expression

• Validate hits in mammalian systems

• Identify potential therapeutic targets for obesity

Developmental Origins:

• Track tmem18 expression during zebrafish development

• Identify critical periods for metabolic programming

• Correlate with human developmental patterns

• Explore early interventions targeting tmem18 pathways

Given the strong and reproducible association between TMEM18 genetic variants and obesity in humans , and evidence that TMEM18 itself (rather than adjacent genes) mediates effects on adiposity through central nervous system action , zebrafish models offer a valuable system for mechanistically dissecting these relationships.

What are the optimal protocols for analyzing tmem18 gene expression in zebrafish tissues?

RNA Extraction and Quality Control:

• Tissue Preparation:

  • Flash-freeze dissected tissues in liquid nitrogen

  • For whole embryos: Pool 20-30 embryos per developmental stage

  • For adult tissues: Carefully dissect specific regions (brain, liver, adipose, etc.)

• RNA Isolation:

  • TRIzol extraction followed by column purification

  • DNase treatment to remove genomic DNA contamination

  • Quality assessment by spectrophotometry (A260/A280 ratio) and gel electrophoresis

  • For small samples: Use RNA extraction kits optimized for limited material

Quantitative RT-PCR:

• Primer Design:

  • Design intron-spanning primers to avoid genomic DNA amplification

  • Optimal tmem18 primers: Forward 5'-[sequence based on zebrafish tmem18]-3', Reverse 5'-[sequence based on zebrafish tmem18]-3'

  • Amplicon size: 80-150 bp for optimal qPCR efficiency

• Reference Genes:

  • Use multiple reference genes (ef1α, rpl13a, actb1)

  • Verify stability across experimental conditions

  • Apply geometric averaging for normalization

• Reaction Conditions:

  • Two-step RT-PCR protocol

  • cDNA synthesis: 500ng total RNA per 20μl reaction

  • qPCR cycling: Initial denaturation (95°C, 3 min), followed by 40 cycles of 95°C for 15s and 60°C for 30s

In Situ Hybridization:

• Probe Design:

  • Generate antisense RNA probes (400-800 bp) targeting tmem18 mRNA

  • Include sense probes as negative controls

• Protocol Optimization:

  • Fixation: 4% paraformaldehyde, 4-16 hours depending on sample size

  • Proteinase K treatment: Titrate concentration (5-10 μg/ml) and time (5-30 minutes) based on developmental stage

  • Hybridization temperature: 65-70°C for RNA probes

  • Anti-DIG antibody concentration: 1:2000-1:5000 dilution

What challenges exist in generating antibodies against zebrafish tmem18 and how can they be overcome?

Generating effective antibodies against transmembrane proteins like zebrafish tmem18 presents several challenges:

Challenge 1: Limited Antigenic Regions

• Solution: Target the C-terminal domain (known to be exposed and functionally important)

• Design synthetic peptides corresponding to hydrophilic, exposed regions

• Use protein structure prediction to identify optimal epitopes

Challenge 2: Protein Conformation

• Solution: Express recombinant fragments rather than full-length protein

• Focus on the DNA-binding C-terminal domain for antibody generation

• Consider native conformation during immunization strategies

Challenge 3: Cross-Reactivity

• Solution: Compare sequences with other zebrafish proteins to identify unique regions

• Test antibody specificity against tmem18-knockout samples as negative controls

• Perform peptide competition assays to verify specificity

Challenge 4: Low Expression Levels

• Solution: Use signal amplification methods for detection

• Implement tyramide signal amplification for immunohistochemistry

• Consider concentrated samples (nuclear fractions) for Western blotting

Recommended Approach:

• Generate multiple antibodies against different epitopes

• Use both polyclonal and monoclonal approaches

• Validate with multiple techniques (Western blot, immunoprecipitation, immunohistochemistry)

• Consider epitope tags in recombinant studies when native antibodies are challenging

What emerging technologies could advance understanding of tmem18 function in zebrafish models?

Several cutting-edge technologies show promise for advancing tmem18 research in zebrafish:

Single-Cell Multi-Omics:

• Single-cell RNA-seq to identify cell populations expressing tmem18

• Single-cell ATAC-seq to map chromatin accessibility changes

• Spatial transcriptomics to preserve tissue context while profiling expression

• Integration of multiple data types to build comprehensive regulatory models

Advanced Genome Editing:

• Prime editing for precise modification of tmem18 sequences

• Base editing for introducing specific point mutations

• Inducible CRISPR systems for temporal control of gene modification

• Tissue-specific Cas9 expression for targeted manipulation

Live Imaging Technologies:

• Lattice light-sheet microscopy for high-resolution, low-phototoxicity imaging

• Optogenetic tools combined with calcium imaging to manipulate and monitor tmem18-expressing neurons

• FRET-based sensors to detect protein-protein interactions in vivo

• Expansion microscopy for super-resolution imaging of subcellular structures

Protein Structure and Interaction Analysis:

• AlphaFold2 and related AI methods for structure prediction

• Cryo-electron tomography for visualizing nuclear membrane complexes

• Proximity labeling methods (TurboID, APEX) for mapping protein interaction networks

• High-throughput screening of chemical modulators of tmem18 function

These technologies could help resolve outstanding questions about tmem18's precise molecular function, regulatory networks, and role in obesity pathogenesis.

How might understanding tmem18 contribute to therapeutic approaches for obesity?

Understanding the molecular mechanisms of tmem18 function could contribute to obesity therapeutics through several pathways:

Target Identification:

• Characterization of the transcriptional network regulated by tmem18

• Identification of downstream effectors mediating metabolic effects

• Discovery of proteins interacting with tmem18 at the nuclear pore complex

• Mapping of signaling pathways connecting tmem18 to appetite regulation

Therapeutic Strategies:

• Small Molecule Development:

  • Compounds modulating tmem18 DNA-binding activity

  • Molecules affecting tmem18 interaction with the nuclear pore complex

  • Agents regulating tmem18 expression in specific tissues

• Gene Therapy Approaches:

  • Targeted delivery of tmem18 to hypothalamic neurons

  • CRISPR-based modulation of tmem18 expression

  • Correction of obesity-associated regulatory variants

• Precision Medicine Applications:

  • Genetic screening for TMEM18 variants to personalize interventions

  • Biomarkers based on tmem18 pathway activity

  • Targeted interventions for specific genetic subgroups

Given that TMEM18 functions within the central nervous system to influence food intake and energy expenditure , therapeutic approaches targeting these pathways could represent a novel strategy for obesity treatment.