Recombinant Human Transmembrane protein 231 (TMEM231)

What is the basic structure and localization of TMEM231?

TMEM231 is a 36-kD two-pass transmembrane protein that localizes specifically to the transition zone (TZ) at the base of the ciliary axoneme. It contains two membrane-spanning domains with both N-terminal and C-terminal regions being essential for its function . The protein demonstrates evolutionary conservation, with orthologs identified in both vertebrates and invertebrates including Caenorhabditis elegans, suggesting fundamental importance in ciliary biology across species . Structural predictions have shown that missense variants, such as the p.R7W substitution, can significantly alter protein conformation and increase local hydrophobicity at and near the affected amino acid residue .

What protein complexes does TMEM231 participate in?

TMEM231 functions as an integral component of the Meckel syndrome (MKS) complex at the ciliary transition zone. Mass spectrometric analysis has confirmed that TMEM231 interacts with multiple proteins within this complex, including B9d1, Mks1, Tctn1, Tctn2, Tctn3, Cc2d2a (Mks6), and Tmem17 . These interactions have been validated through coimmunoprecipitation experiments with epitope-tagged versions of these proteins. Particularly significant is the relationship between TMEM231 and B9d1, as these proteins appear to be essential for each other's localization to the transition zone, creating an interdependent functional relationship critical for proper ciliary composition .

What are the primary functions of TMEM231 in cellular physiology?

TMEM231 plays a crucial role in regulating the composition of ciliary membrane proteins by functioning as a gatekeeper at the transition zone. Research has demonstrated that TMEM231, as part of the MKS complex, controls the localization of specific proteins to cilia, including Arl13b and Inpp5e . This regulatory function is essential for proper ciliary signaling pathways, particularly the Hedgehog pathway which is critical for embryonic development. TMEM231 also contributes to the structural organization of the transition zone itself, with mutations disrupting the localization of other transition zone components and compromising the ciliary diffusion barrier function .

How do mutations in TMEM231 contribute to the pathogenesis of ciliopathies like Meckel syndrome and orofaciodigital syndrome?

Mutations in TMEM231 disrupt the organization and function of the ciliary transition zone, compromising its ability to regulate the protein composition of the ciliary membrane. In mouse models, TMEM231 mutations lead to mislocalization of key ciliary proteins including Arl13b and Inpp5e, which in turn affects critical developmental signaling pathways such as Hedgehog signaling . The resulting phenotypes include polydactyly, kidney cysts, and hepatic ductal plate malformations - hallmarks of human Meckel syndrome.

In humans, TMEM231 mutations identified in orofaciodigital syndrome type 3 (OFD3) and Meckel syndrome patients have been shown to compromise transition zone function . For example, the homozygous c.19C>T (p.R7W) variant alters the protein structure, increasing local hydrophobicity, which likely impairs its ability to interact with other transition zone components . The spectrum of phenotypes observed correlates with the severity of functional disruption, with more severe mutations typically associated with Meckel syndrome and milder variants with OFD3.

What are the species-specific differences in TMEM231 function and expression?

What is the relationship between TMEM231 and ciliary signaling pathways?

TMEM231 plays a critical role in regulating ciliary signaling pathways, particularly the Hedgehog pathway, by controlling the protein composition of the ciliary membrane. Mouse Tmem231 mutants exhibit abrogated Hedgehog signaling, leading to developmental abnormalities such as dorsalization of the neural tube . This disruption occurs because TMEM231 mutations affect the localization of key ciliary membrane proteins like Arl13b and Inpp5e, which are essential for proper signal transduction .

The connection between TMEM231 and signaling extends beyond Hedgehog pathways. As a component of the transition zone, TMEM231 likely influences multiple signaling cascades that depend on proper ciliary function, including Wnt and PDGF signaling. Understanding the full spectrum of signaling pathways affected by TMEM231 dysfunction remains an active area of research with significant implications for disease mechanisms in ciliopathies.

What are the most effective approaches for expressing and purifying recombinant TMEM231 for structural and functional studies?

For successful expression and purification of recombinant TMEM231, a multi-step approach is recommended:

• Expression system selection: Mammalian expression systems (HEK293 or CHO cells) are preferable for TMEM231 as they provide appropriate post-translational modifications and membrane insertion machinery. Alternatively, insect cell systems can be used for higher yield.

• Construct design: Incorporate a localization and affinity purification (LAP) tag (as described in previous studies) to facilitate both detection and purification . Consider expressing different domains separately if the full-length protein proves challenging.

• Purification strategy: Use a combination of affinity chromatography (utilizing the LAP tag) followed by size exclusion chromatography to obtain pure protein. For membrane proteins like TMEM231, detergent selection is critical - mild non-ionic detergents such as DDM or LMNG are typically effective.

• Verification methods: Confirm protein identity and integrity using mass spectrometry and Western blotting with specific antibodies. Circular dichroism can be employed to verify proper folding.

When designing experiments, consider that TMEM231's transmembrane nature makes it challenging to work with, and strategies that have worked for other transition zone proteins may provide useful guidance.

What cellular and animal models are most suitable for studying TMEM231 function?

Several experimental models have proven valuable for TMEM231 research:

• Cellular models:

  • IMCD3 cells (mouse inner medullary collecting duct cells) provide a well-characterized system for studying ciliary biology

  • hTERT-RPE1 cells (human retinal pigmented epithelial cells) readily form primary cilia and are amenable to genetic manipulation

  • Patient-derived fibroblasts offer a physiologically relevant context for studying disease-causing mutations

• Animal models:

  • Mouse models with targeted Tmem231 mutations have successfully recapitulated ciliopathy phenotypes including polydactyly, kidney cysts, and hepatic ductal plate malformations

  • C. elegans models allow for rapid genetic manipulation and in vivo imaging of transition zone structure and function

  • Zebrafish models provide advantages for developmental studies and high-throughput screening

• Genetic approaches:

  • CRISPR/Cas9-mediated gene editing for generating precise mutations

  • Conditional knockout systems (Cre-loxP) for tissue-specific and temporal control of gene expression

  • Rescue experiments with wild-type or mutant constructs to verify functional consequences

When selecting a model system, consider the specific research question, as different models may be better suited for structural studies, developmental analyses, or therapeutic testing.

What imaging techniques are most effective for visualizing TMEM231 localization and interactions at the ciliary transition zone?

The nanoscale size of the ciliary transition zone requires specialized imaging approaches:

• Super-resolution microscopy techniques:

  • Stimulated emission depletion (STED) microscopy achieves resolution of ~50-80 nm

  • Stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) offer even higher resolution (~20 nm)

  • Structured illumination microscopy (SIM) provides ~100 nm resolution with less specialized equipment

• Immunofluorescence optimization:

  • Use epitope-tagged TMEM231 constructs (such as LAP-tagged versions) for enhanced detection sensitivity

  • Employ specific antibodies against endogenous TMEM231 along with markers for basal bodies (γ-tubulin or centrin) and axonemes (acetylated α-tubulin)

  • Consider methanol fixation which often better preserves ciliary structures compared to paraformaldehyde

• Live-cell imaging approaches:

  • Fluorescence recovery after photobleaching (FRAP) to study protein dynamics within the transition zone

  • Förster resonance energy transfer (FRET) for analyzing protein-protein interactions in living cells

• Electron microscopy:

  • Transmission electron microscopy for ultrastructural analysis of transition zone architecture

  • Immunogold labeling for precise localization of TMEM231 at the nanometer scale

Combining multiple imaging modalities provides complementary insights into TMEM231's localization, dynamics, and interactions within the complex three-dimensional structure of the transition zone.

How can researchers distinguish between direct and indirect effects of TMEM231 dysfunction in experimental systems?

Differentiating direct from indirect effects of TMEM231 dysfunction requires a systematic approach:

• Temporal analysis: Investigate the sequential development of cellular and molecular phenotypes following TMEM231 disruption. Early effects are more likely to be direct consequences, while later manifestations may represent secondary adaptations.

• Rescue experiments: Design complementation studies with wild-type TMEM231 or specific mutants to determine which phenotypes can be directly rescued. Domain-specific constructs can help map functional regions of the protein responsible for particular cellular effects .

• Interactome analysis: Compare protein interaction profiles between wild-type and mutant TMEM231 using approaches like immunoprecipitation coupled with mass spectrometry . Lost interactions in mutants may indicate direct functional relationships.

• Comparative studies: Analyze phenotypes across different TMEM231 mutation types and compare them with mutations in known interaction partners like B9d1 or Mks1. Shared phenotypes suggest common pathways, while unique effects may indicate TMEM231-specific functions .

• Epistasis experiments: Systematically combine TMEM231 mutations with mutations in other pathway components to establish hierarchical relationships and distinguish between parallel and sequential effects.

When interpreting results, consider that TMEM231's role as part of a multiprotein complex means that its dysfunction may simultaneously affect multiple cellular processes through different mechanisms.

How should researchers interpret conflicting data regarding TMEM231 function across different model systems?

When confronted with conflicting data about TMEM231 across different model systems, consider the following analytical framework:

• Genetic background effects: The same Tmem231 mutation can produce different phenotypic severity depending on genetic background, as demonstrated by the difference between pure C57BL/6 mice (embryonic lethal at E15.5) versus C57BL/6-CD1 mixed background (survival until birth) . Document and account for genetic background in all experiments.

• Species-specific differences: While core functions are conserved between species, the exact composition of transition zone complexes and their regulatory mechanisms may differ. The C. elegans ortholog of TMEM231 shares functional properties but exists in a simpler ciliary system .

• Cell type specificity: Different cell types express varying levels of TMEM231 interacting partners and may have distinct requirements for transition zone function. Compare data across multiple cell types when possible.

• Technical considerations:

  • Antibody specificity issues may lead to contradictory localization data

  • Different fixation methods significantly affect ciliary structure preservation

  • Overexpression artifacts can misrepresent normal protein behavior

• Mutational effects: Different mutations may affect distinct protein domains and functions. Classify mutations based on their molecular consequences (null, hypomorphic, dominant negative) rather than simply by the associated syndrome.

By systematically addressing these factors, researchers can reconcile apparently conflicting data and develop more comprehensive models of TMEM231 function that account for context-dependent effects.

What approaches should be used to correlate in vitro findings on TMEM231 with in vivo disease phenotypes?

Translating in vitro TMEM231 findings to in vivo disease relevance requires multifaceted approaches:

• Genotype-phenotype correlation studies:

  • Catalog specific TMEM231 mutations identified in patients with ciliopathies

  • Document associated clinical manifestations in detail

  • Compare functional consequences in cellular models with specific clinical features

• Patient-derived models:

  • Generate patient-specific induced pluripotent stem cells (iPSCs)

  • Differentiate iPSCs into relevant cell types (renal, neural, hepatic)

  • Compare cellular phenotypes with patient manifestations

• In vivo validation strategies:

  • Create knock-in mouse models with specific human mutations

  • Use tissue-specific conditional knockout approaches to isolate organ-specific effects

  • Employ rescue experiments with human TMEM231 variants in animal models

• Quantitative phenotyping:

  • Develop standardized metrics for phenotypic severity in both clinical and model systems

  • Use these metrics to create correlation matrices between molecular defects and physiological outcomes

  • Apply statistical approaches to identify the strongest predictive relationships

• Therapeutic testing pipeline:

  • Screen for compounds that correct specific cellular defects in vitro

  • Validate promising candidates in animal models

  • Track biomarkers that reflect transition zone function for translational potential

This integrated approach enables researchers to build robust connections between molecular mechanisms and disease manifestations, potentially identifying therapeutic targets and biomarkers for ciliopathies caused by TMEM231 dysfunction.