Recombinant Rat Transmembrane protein 231 (Tmem231)

What is the basic structure of rat Tmem231 and how does it differ from other species?

Rat Tmem231 is a transmembrane protein composed of four transmembrane domains. Based on computational analysis, Tmem231 is predicted to have N- and C-terminal cytoplasmic domains, with the N-terminus typically being shorter than the C-terminus . The protein structure includes:

• Four alpha-helical transmembrane domains of approximately 20-23 amino acids each

• Two intracellular loops (with the first loop typically longer than the second)

• A short extracellular loop of approximately 8 amino acids

• A C-terminal domain of approximately 71 amino acids

Rat Tmem231 shares high homology with mouse Tmem231 (83% identity and 91% similarity) but has lower homology with human TMEM231 (43% identity and 64% similarity) . The protein contains several highly conserved regions across species, particularly in the C-terminus, which includes the amino acid blocks PRSIV and VTWAL that are preserved across multiple mammals .

What is the primary function of Tmem231 in cellular processes?

Tmem231 is a component of the B9 complex that functions at the transition zone (TZ) of primary cilia, located between the basal body and axoneme . Its primary functions include:

• Formation of the diffusion barrier between cilia and plasma membrane

• Regulation of ciliary membrane protein composition

• Control of protein entry into cilia (permitting or preventing specific proteins)

• Organization of the Meckel syndrome (MKS) complex at the transition zone

• Supporting proper ciliary signaling, particularly Hedgehog signaling

Knockout studies in mice have shown that Tmem231 is essential for the localization of several proteins to the ciliary transition zone, including B9d1, Mks1, and Tmem67 .

What expression systems are most effective for producing recombinant rat Tmem231?

Based on the research data, mammalian expression systems are generally preferred for recombinant rat Tmem231 production due to their capacity for proper post-translational modifications and membrane protein folding . The following considerations are important:

• Mammalian cells: HeLa cells have been successfully used for expressing GFP-tagged Tmem231 constructs for localization studies .

• Vectors: pEGFP-C1 vectors have been used for creating fusion proteins with GFP tags for visualization studies .

• Tags: His-tags are commonly used for purification purposes , while GFP tags are useful for localization studies.

For successful expression:

• Clone the full-length or partial Tmem231 coding sequence into an appropriate expression vector

• Include appropriate tags (His, GFP, or FLAG) depending on downstream applications

• Transfect mammalian cells using reagents like Superfect

• Allow 24-48 hours for expression before harvesting

What purification methods yield the highest purity and functional activity for recombinant rat Tmem231?

For optimal purification of recombinant rat Tmem231:

• Affinity chromatography: For His-tagged constructs, use nickel or cobalt affinity columns .

• Buffer composition: PBS-based buffers are typically used for storage of purified protein .

• Preservation methods:

  • Lyophilization provides longer shelf life (up to 12 months at -20°C/-80°C)

  • Addition of 5-50% glycerol helps maintain protein stability

  • Reconstitution at 0.1-1.0 mg/mL concentration is recommended

As Tmem231 is a membrane protein, it may be necessary to include mild detergents during purification to maintain protein solubility and stability.

What assays can be used to verify the functional activity of recombinant rat Tmem231?

Several experimental approaches can verify functional activity of recombinant rat Tmem231:

• Protein-protein interaction assays:

  • Coimmunoprecipitation (Co-IP) with known binding partners like B9d1 and Mks1

  • Mass spectrometric identification of binding partners using LAP-tagged Tmem231

• Localization studies:

  • Immunofluorescence microscopy to verify localization to the transition zone of cilia

  • The protein should show a distinct pattern around the nuclear membrane and in the cytoplasm, with concentration at the ciliary transition zone

• Functional complementation:

  • Rescue experiments in Tmem231-knockout cells to restore B9d1 localization at the transition zone

  • Restoration of Arl13b and Inpp5e localization to cilia in knockout models

• Validation of membrane topology:

  • N- and C-terminal domains should be oriented as predicted by computational models

  • Deletion constructs (e.g., N-terminal 35 amino acid deletion) can verify domain importance

How can recombinant rat Tmem231 be used to study ciliary function in vitro?

Recombinant rat Tmem231 can be used to study ciliary function through:

• Ciliary composition analysis:

  • Overexpression of wild-type or mutant Tmem231 to observe effects on cilia formation

  • Immunostaining for ciliary markers like ARL13B to assess cilia integrity

• Transition zone assembly studies:

  • Expression of tagged Tmem231 to track transition zone component assembly

  • FRAP (Fluorescence Recovery After Photobleaching) analysis to study dynamics

• Protein trafficking analysis:

  • Monitoring movement of fluorescently-tagged ciliary proteins in the presence of wild-type versus mutant Tmem231

  • Quantification of ciliary versus non-ciliary localization of membrane proteins

• Hedgehog signaling assays:

  • Luciferase reporter assays for Hedgehog pathway activity

  • Quantification of Gli transcription factor localization and processing

How should researchers design experiments to study the effect of Tmem231 mutations on ciliary function?

When designing experiments to study Tmem231 mutations:

• Cell model selection:

  • Choose appropriate cell types that form primary cilia (e.g., MEFs, IMCD3, or RPE1 cells)

  • Consider tissue-specific cell types relevant to MKS/JBTS phenotypes (renal, neural, etc.)

• Mutation design strategy:

  • Incorporate known disease-causing mutations (e.g., p.Asn90Ile, p.Pro125Ala, p.Leu81Phe, p.Ala216Pro)

  • Create deletion constructs targeting specific domains (e.g., N-terminal transmembrane domain)

  • Design splice site mutations to study effects on mRNA processing

• Control selection:

  • Include wild-type Tmem231 as positive control

  • Use empty vector transfection as negative control

  • Include known functional mutants as reference points

• Readout parameters:

  • Primary: Localization of B9d1 and other TZ components

  • Secondary: Cilia formation, length, and markers (ARL13B staining)

  • Tertiary: Downstream signaling effects (Hedgehog pathway activation)

Example experimental workflow:

• Transfect cells with wild-type or mutant Tmem231 constructs

• Induce ciliogenesis through serum starvation (24-48 hours)

• Perform immunofluorescence for ciliary and TZ markers

• Quantify cilia formation, protein localization, and signaling readouts

What are the critical controls needed when working with recombinant rat Tmem231 in different experimental contexts?

Critical controls for Tmem231 experiments include:

• For localization studies:

  • Wild-type Tmem231-GFP fusion protein

  • GFP-only vector control

  • Nuclear staining (DAPI) to define cellular compartments

  • Additional markers for ciliary structures (acetylated tubulin for axoneme)

• For protein-protein interaction studies:

  • Empty vector as negative control

  • Known interactors (B9d1, Mks1) as positive controls

  • Non-interacting transmembrane protein as specificity control

  • Input samples to verify expression levels

• For functional rescue experiments:

  • Untransfected Tmem231-knockout cells

  • Wild-type Tmem231 for complete rescue

  • Non-functional mutant (e.g., p.Asn90Ile) that fails to rescue

  • Dose-response with varying levels of expression

• For mRNA expression analysis:

  • Housekeeping gene controls (β-actin) for normalization

  • Tissue-matched controls from unaffected samples

  • Controls for splice variant detection

How can recombinant rat Tmem231 be used to study disease mechanisms of ciliopathies?

Recombinant rat Tmem231 provides valuable tools for studying ciliopathy mechanisms:

• Disease mutation modeling:

  • Create disease-relevant mutations (MKS, JBTS, OFD3) in recombinant Tmem231

  • Assess functional consequences on protein localization, stability, and interaction

  • Compare effects of different mutations on ciliary composition and signaling

• Pathway analysis:

  • Study impact on Hedgehog signaling components

  • Investigate effects on other ciliary signaling pathways (Wnt, PDGF)

  • Identify differential effects of mutations on specific pathways

• Structure-function analysis:

  • Correlate disease mutations with protein domains

  • Map interaction interfaces disrupted by specific mutations

  • Identify critical residues for transition zone assembly

• Therapeutic strategy testing:

  • Test small molecules that might stabilize mutant Tmem231

  • Evaluate compounds that bypass Tmem231 function by activating downstream pathways

  • Screen for molecules that enhance remaining function of hypomorphic mutants

Experiments have shown that disease-associated mutations like p.Asn90Ile and p.Pro125Ala compromise Tmem231's ability to support MKS complex organization at the transition zone, providing a mechanistic basis for ciliopathy development .

What are the key differences between Tmem231 mutations that cause Joubert syndrome versus Meckel syndrome?

The genotype-phenotype correlation for Tmem231 mutations reveals important differences:

Research indicates that gene conversion events affecting TMEM231, leading to loss of exon 4, when combined with certain missense mutations (c.712G>A) cause Joubert syndrome, while different combinations (with c.334T>G) cause Meckel-Gruber syndrome .

The severity spectrum appears related to the remaining functional capacity of Tmem231 at the transition zone, with complete loss of function being associated with the more severe MKS phenotype .

How does rat Tmem231 compare functionally with human and mouse orthologs in experimental systems?

Comparative analysis of Tmem231 across species reveals important similarities and differences:

All three orthologs appear to function in ciliary transition zone organization, but with some experimental considerations:

• Antibody cross-reactivity: Some antibodies against human TMEM231 may not recognize rat or mouse orthologs due to sequence differences, necessitating species-specific antibodies .

• Expression systems: Rat and mouse Tmem231 are frequently expressed in rodent cell lines for better compatibility, while human TMEM231 may be studied in human cell lines.

• Functional conservation: The core function in transition zone organization appears conserved across species, as evidenced by similar phenotypes in knockout models and conservation of key interaction partners .

The high conservation between rat and mouse Tmem231 makes rat models particularly valuable for studying mouse ciliopathy models, while specific human disease mutations may need to be studied in human cell systems for the most accurate translation to human disease.

What insights about evolutionary conservation of the transition zone can be gained from studying Tmem231 across species?

Studying Tmem231 across species provides valuable evolutionary insights:

• Functional conservation: The role of Tmem231 in transition zone organization is conserved from nematodes (C. elegans TMEM-231) to mammals, indicating fundamental importance in ciliary biology .

• Structural elements:

  • The four transmembrane domain topology is preserved across species

  • Conserved C-terminal motifs (PRSIV, VTWAL) suggest critical functional elements

  • Alignment analysis reveals six highly conserved serine residues and seven conserved leucine residues across species

• Interaction networks:

  • The B9 complex components and their interactions are conserved

  • In C. elegans, TMEM-231 works with MKS module components MKSR-1 (B9d1), MKSR-2 (B9d2), and MKS-6 (Cc2d2a) to control protein entry into cilia

  • Similar phenotypes (e.g., aberrant ciliary protein composition) result from Tmem231 mutations across species

• Specific adaptations:

  • Minor variations in sequence and expression patterns may reflect species-specific adaptations

  • Differential susceptibility to certain mutations suggests evolutionary pressures

Research on TMEM-231 in C. elegans has shown that, like in mammals, it localizes to the transition zone and controls ciliary composition, suggesting that this function evolved early and has been maintained throughout evolution .

What are common challenges in expressing and purifying functional recombinant rat Tmem231, and how can they be addressed?

Common challenges with recombinant rat Tmem231 expression and purification include:

• Low expression levels:

  • Problem: Transmembrane proteins often express poorly

  • Solution: Optimize codon usage for expression system; use strong promoters (e.g., EF1α); lower expression temperature (30°C instead of 37°C); consider using fusion partners to enhance solubility

• Protein aggregation:

  • Problem: Membrane proteins may aggregate during expression/purification

  • Solution: Include appropriate detergents (0.5% Triton X-100 has been used successfully for permeabilization ); consider fusion with solubility-enhancing tags; purify under mild conditions

• Improper folding:

  • Problem: Loss of native conformation affecting function

  • Solution: Express in mammalian cells rather than bacterial systems; include molecular chaperones; optimize buffer conditions

• Degradation during purification:

  • Problem: Proteolytic cleavage of target protein

  • Solution: Include protease inhibitors; maintain samples at 4°C; minimize purification time; consider adding stabilizing agents (glycerol 5-50%)

• Verification of functionality:

  • Problem: Difficult to assess if purified protein maintains native activity

  • Solution: Perform binding assays with known partners (B9d1, Mks1); verify correct subcellular localization when expressed in cells; compare activity to positive controls

For storage and stability:

• Reconstitute lyophilized protein at 0.1-1.0 mg/mL

• Add 5-50% glycerol as a stabilizing agent

• Store at -20°C/-80°C for long-term preservation

How can researchers troubleshoot localization issues when studying Tmem231 in cellular systems?

When troubleshooting Tmem231 localization issues:

• Absence of ciliary/transition zone localization:

  • Potential causes: Improper fusion protein design; overexpression artifacts; cell type not forming primary cilia; insufficient ciliogenesis induction

  • Solutions:

    • Verify ciliation with acetylated tubulin staining

    • Ensure serum starvation protocol is effective (24-48 hours is typical)

    • Try both N- and C-terminal tags to determine optimal configuration

    • Validate antibody specificity with known controls

• Diffuse cytoplasmic localization:

  • Potential causes: Protein overexpression; N-terminal domain deletion (known to affect localization ); improper fixation

  • Solutions:

    • Titrate expression levels by adjusting transfection conditions

    • Compare with endogenous protein localization

    • Optimize fixation protocol (4% paraformaldehyde for 20 minutes works for HeLa cells )

• Aggregation artifacts:

  • Potential causes: Protein misfolding; excessive expression; improper detergent treatment

  • Solutions:

    • Reduce expression levels

    • Optimize permeabilization conditions (0.5% Triton X-100 for 5 minutes is effective )

    • Consider alternative cell types

• No co-localization with transition zone markers:

  • Potential causes: Primary antibody incompatibility; epitope masking; timing issues

  • Solutions:

    • Use multiple transition zone markers (B9d1, Mks1) for validation

    • Try different fixation and permeabilization protocols

    • Ensure cells are examined at appropriate time points post-transfection (48 hours optimal)

For optimal visualization results in HeLa cells, researchers have successfully used the following protocol: fixation in 3% paraformaldehyde for 20 minutes, permeabilization with 0.5% Triton X-100 for 5 minutes, and nuclear staining with DAPI (2.5 μg/ml) .

How can CRISPR-Cas9 genome editing be used to study Tmem231 function in rat models?

CRISPR-Cas9 offers powerful approaches for studying Tmem231:

• Knockout model generation:

  • Design guide RNAs targeting early exons of rat Tmem231

  • Create complete knockout to study loss-of-function phenotypes similar to mouse Tmem231-/- models (polydactyly, kidney cysts, impaired Hedgehog signaling)

  • Generate tissue-specific knockouts using Cre-loxP systems

• Knock-in strategies:

  • Introduce disease-specific mutations (e.g., p.Asn90Ile, p.Pro125Ala)

  • Create fluorescent protein fusions for live imaging of Tmem231

  • Generate epitope-tagged versions for biochemical studies

• Domain analysis:

  • Introduce precise deletions of specific domains (transmembrane domains, N-terminus, C-terminus)

  • Create chimeric proteins to determine domain-specific functions

• Transcriptional regulation:

  • Target CRISPRa/CRISPRi to Tmem231 regulatory regions to modulate expression

  • Study dosage effects on ciliary transition zone organization

• Experimental design considerations:

  • Off-target effects: Use multiple guide RNAs and verify editing specificity

  • Phenotypic validation: Compare to established knockout models

  • Controls: Include wild-type and heterozygous animals for comparison

For validating genomic modifications, researchers should verify editing at the DNA level (sequencing), mRNA level (RT-PCR), and protein level (Western blot, immunofluorescence), with particular attention to potential splice variants that may arise from certain modifications .

What advanced microscopy techniques can provide deeper insights into Tmem231 dynamics at the ciliary transition zone?

Advanced microscopy approaches for studying Tmem231 dynamics include:

• Super-resolution microscopy:

  • STED (Stimulated Emission Depletion) microscopy to resolve transition zone subdomains

  • STORM/PALM to map precise localization of Tmem231 relative to other TZ components

  • SIM (Structured Illumination Microscopy) for improved resolution of ciliary structures

• Live-cell imaging techniques:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure Tmem231 turnover at the TZ

  • Photoactivatable/photoconvertible fluorescent protein fusions to track protein movement

  • FRET analysis to study real-time interactions with binding partners

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence localization with ultrastructural analysis

  • Study Tmem231 localization relative to TZ ultrastructure

  • Map protein distribution at nanometer resolution

• Expansion microscopy:

  • Physical expansion of specimens to increase effective resolution

  • Particularly valuable for resolving the compact ciliary transition zone

• Light sheet microscopy:

  • Reduced phototoxicity for extended live imaging

  • Especially useful for developmental studies in embryos with ciliopathy phenotypes

For optimal results, researchers should consider:

• Using minimally invasive tags (small epitope tags or fluorescent proteins)

• Validating expression levels close to endogenous conditions

• Combining multiple techniques for complementary information

• Including appropriate markers for ciliary subdomains (basal body, transition zone, axoneme)