Recombinant Danio rerio Transmembrane protein 127 (tmem127)

What is TMEM127 and what is its significance in zebrafish research?

TMEM127 is a transmembrane protein identified as a tumor suppressor gene, initially characterized in human pheochromocytomas (catecholamine-secreting tumors of neural crest origin). TMEM127 functions as a negative regulator of the mTOR pathway, a central regulator of cell growth and metabolism .

Zebrafish (Danio rerio) models offer several advantages for studying tmem127:

• Transparency of embryos allowing direct visualization of developmental processes

• High fecundity and rapid development facilitating genetic studies

• Cost-efficient genetic manipulation methods

• Conservation of many cancer-related genes between humans and zebrafish

Zebrafish provides an excellent model to investigate tmem127's roles in endosomal trafficking, mTOR regulation, and tumor suppression in an in vivo context while offering unique advantages over mammalian models.

What techniques are available for studying tmem127 expression in zebrafish?

Several methodological approaches can be employed to study tmem127 expression:

• RT-qPCR analysis:

  • Extract RNA from whole embryos or dissected tissues

  • Perform reverse transcription followed by qPCR with primers specific to zebrafish tmem127

  • Normalize to appropriate reference genes (ef1α, β-actin)

• In situ hybridization:

  • Generate RNA probes complementary to tmem127 mRNA

  • Perform whole-mount in situ hybridization to visualize spatial expression patterns

  • Section samples for cellular resolution of expression

• Transgenic reporter lines:

  • Create constructs with the tmem127 promoter driving fluorescent protein expression

  • Generate stable transgenic lines using Tol2 transposition

  • Image live embryos at different developmental stages

• Protein detection methods:

  • Western blotting using antibodies against zebrafish tmem127

  • Immunohistochemistry to determine protein localization at tissue level

  • Immunofluorescence for subcellular localization studies

How can recombinant Danio rerio TMEM127 protein be produced for research applications?

Production of recombinant zebrafish tmem127 involves several methodological steps:

• Gene cloning:

  • Amplify tmem127 coding sequence from zebrafish cDNA

  • Clone into appropriate expression vector with purification tags

  • Verify sequence integrity

• Expression system selection:

  • For transmembrane proteins like tmem127, eukaryotic systems are preferred:

    • Mammalian cells (HEK293, CHO)

    • Insect cells with baculovirus

    • Yeast expression systems

  • Bacterial systems may be suitable for soluble domains only

• Protein expression optimization:

  • Test different induction conditions and expression parameters

  • For membrane proteins, consider detergent solubilization strategies

  • Optimize temperature, time, and media composition

• Purification strategy:

  • Affinity chromatography using engineered tags (His, GST, MBP)

  • Size exclusion chromatography for further purification

  • Verification of purity by SDS-PAGE and western blotting

• Functional validation:

  • Structure analysis by circular dichroism

  • Binding assays with potential interaction partners

  • Activity assays based on known functions

How does TMEM127 function in endosomal trafficking and how can this be studied in zebrafish?

TMEM127 has been shown to partially overlap with early endosomal markers Rab5 and EEA1, with approximately 68% of TMEM127-positive structures colocalizing with Rab5 and 38% with EEA1 . This suggests a significant role in endosomal trafficking processes.

Methodological approaches for studying tmem127 in endosomal trafficking:

• Colocalization studies:

  • Generate fluorescently-tagged zebrafish tmem127 constructs

  • Co-express with markers for different endosomal compartments (Rab5, EEA1, Rab7, Rab11)

  • Quantify colocalization using confocal microscopy and analysis software

• Trafficking assays:

  • Measure internalization of fluorescently-labeled cargo proteins

  • Track endosomal movement and maturation in real-time

  • Compare wild-type and tmem127-deficient cells

• Analysis of endosomal morphology:

  • Quantify size, number, and distribution of endosomal compartments

  • Assess effects of tmem127 knockdown/knockout on endosomal compartments

  • Document ultrastructural changes by electron microscopy

• RET receptor trafficking studies:

  • Since TMEM127 depletion promotes RET accumulation on the cell surface , measure:

    • Surface biotinylation to quantify surface vs. internalized RET

    • Internalization rates using antibody feeding assays

    • Recycling efficiency of internalized receptors

• Quantification methods:

How can CRISPR-Cas9 be optimized for generating zebrafish tmem127 knockout models?

CRISPR-Cas9 gene editing provides an efficient method for generating zebrafish tmem127 knockout models. The following protocol outlines the key methodological considerations:

• gRNA design optimization:

  • Target early exons to ensure functional knockout

  • Use zebrafish-specific CRISPR design tools to minimize off-targets

  • Consider targeting conserved functional domains

  • Design multiple gRNAs to increase efficiency and enable deletion strategies

• Delivery methods:

  • Microinject Cas9 protein with gRNA (RNP complex) for immediate activity

  • Alternative: inject Cas9 mRNA with gRNA for slightly delayed activity

  • Optimize injection volume (1-2 nl) and concentration

  • Target one-cell stage embryos for maximal distribution

• Mutation detection strategies:

• Establishing stable lines:

  • Raise F0 mosaic fish to adulthood

  • Screen for germline transmission by fin-clip genotyping

  • Select founders with frameshift mutations

  • Outcross to wild-type to generate F1 heterozygotes

  • Incross F1 heterozygotes to obtain homozygous knockouts in F2

• Validation approaches:

  • Verify loss of tmem127 protein by western blotting

  • Perform RT-qPCR to assess mRNA levels (potential nonsense-mediated decay)

  • Characterize phenotypes in homozygous mutants

  • Conduct rescue experiments with wild-type tmem127 to confirm specificity

CRISPR-Cas9 efficiency for zebrafish tmem127 can be optimized by targeting conserved regions and using multiple guide RNAs to create larger deletions that ensure complete loss of function .

How does tmem127 interact with the mTOR pathway and what methods can investigate this in zebrafish?

TMEM127 has been identified as a negative regulator of mTOR signaling. In human cells, it dynamically associates with the endomembrane system and colocalizes with perinuclear (activated) mTOR . TMEM127 mutations lead to hyperphosphorylation of mTOR targets, suggesting a role in constraining mTOR activity.

Methodological approaches to study tmem127-mTOR interactions in zebrafish:

• Analysis of mTOR signaling activity:

  • Western blotting for phosphorylated mTOR targets:

    • Phospho-S6K (Thr389)

    • Phospho-4E-BP1 (Thr37/46)

    • Phospho-S6 (Ser235/236)

  • Comparison between wild-type and tmem127 mutant zebrafish

• Amino acid sensing experiments:

  • Since TMEM127 influences mTOR regulation by amino acids :

    • Subject embryos to amino acid starvation/repletion

    • Monitor mTOR translocation to lysosomes

    • Assess phosphorylation of targets under different nutrient conditions

• Pharmacological manipulation:

  • Treat zebrafish with mTOR inhibitors:

    • Rapamycin (mTORC1-specific)

    • Torin1 (mTORC1/2 inhibitor)

  • Determine if inhibition rescues phenotypes in tmem127 mutants

• Subcellular localization studies:

  • Generate fluorescently tagged constructs for tmem127 and mTOR

  • Examine colocalization under different conditions

  • Track dynamic interactions using live imaging

• Quantification of mTOR pathway activation in tmem127 mutants:

• Genetic interaction studies:

  • Generate double mutants with tmem127 and mTOR pathway components

  • Assess epistatic relationships through phenotypic analysis

  • Perform rescue experiments with mTOR modulators

What is the relationship between tmem127 and RET signaling, and how can this be investigated in zebrafish?

Recent research has revealed that loss of TMEM127 causes wild-type RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive signaling and promotes proliferation . This provides a mechanistic link between TMEM127 loss and oncogenic signaling.

Methodological approaches to study tmem127-RET interactions in zebrafish:

• RET protein localization analysis:

  • Immunofluorescence to detect RET distribution in wild-type vs. tmem127 mutants

  • Surface biotinylation assays to quantify surface vs. internalized RET

  • Live imaging with fluorescently tagged RET to track trafficking dynamics

• RET internalization studies:

  • Antibody feeding assays to measure internalization rates

  • GDNF-stimulated internalization experiments (GDNF is the RET ligand)

  • Comparison between wild-type and tmem127-deficient cells

• RET signaling pathway activation:

  • Western blotting for phosphorylated RET and downstream effectors:

    • ERK1/2 (MAPK pathway)

    • AKT (PI3K pathway)

    • STAT3 (JAK/STAT pathway)

  • Transcriptional reporter assays for RET-dependent gene expression

• Functional consequences assessment:

  • Proliferation assays in RET-expressing tissues

  • EdU incorporation to measure cell proliferation in vivo

  • Long-term monitoring for spontaneous tumor development

  • Response to selective RET inhibitors (e.g., Selpercatinib at 0.1 μM)

• Quantitative comparison of RET parameters in tmem127-deficient zebrafish:

These methodological approaches would determine whether the relationship between TMEM127 and RET is conserved in zebrafish and potentially identify new therapeutic strategies for tumors with TMEM127 mutations.

What are the challenges in translating zebrafish tmem127 findings to human cancer research?

Translating findings from zebrafish tmem127 studies to human cancer research involves several methodological challenges:

• Genetic and functional considerations:

  • While many genes are conserved between species, differences in protein structure, expression patterns, and function may exist

  • Careful sequence alignment and domain analysis are required to identify conserved regions

  • Genome duplication in teleosts may result in functional redundancy not present in humans

• Physiological differences:

  • Differences in tissue architecture between fish and mammals

  • Variations in tumor microenvironment components

  • Species-specific metabolic adaptations

• Experimental validation approaches:

  • Complementary studies in human cells to validate zebrafish findings

  • Patient-derived xenograft models to bridge zebrafish and human cancer biology

  • Correlation of zebrafish phenotypes with clinical data from TMEM127-mutant patients

• Technical considerations for cross-species validation:

• Strengths of zebrafish models for translational research:

  • High-throughput screening capabilities for drug discovery

  • Ability to perform genetic interaction studies at scale

  • Real-time visualization of tumor initiation and progression

  • Conservation of core cancer pathways between zebrafish and humans

Human TMEM127 mutations are associated with pheochromocytomas and renal cell carcinomas . Zebrafish models can provide insights into the conserved mechanisms of TMEM127 function, particularly its roles in endosomal trafficking, mTOR regulation, and RET signaling, which are likely to be relevant across species.