Recombinant Mouse Transmembrane protein 107 (Tmem107)

What is Tmem107 and where is it localized in cells?

Tmem107 is a four-pass transmembrane protein that localizes to the transition zone of primary cilia. The protein contains four transmembrane domains, with the fourth domain containing a highly conserved glutamic acid residue (E125) that, when mutated to glycine in the schlei mouse model, causes significant developmental abnormalities . Tmem107 is highly conserved within vertebrates and has orthologs in C. elegans, Nematostella, and Chlamydomonas, suggesting an ancient evolutionary role in cilia regulation .

At the subcellular level, Tmem107 functions at the ciliary transition zone, where it recruits other ciliopathy-associated proteins including MKS-1, TMEM-231 (JBTS20), and JBTS-14 (TMEM237) to this domain . This localization is critical for its function in regulating cilia formation and maintaining ciliary compartmentalization.

What phenotypes are associated with Tmem107 deficiency in mouse models?

Tmem107-deficient mouse models display a constellation of developmental abnormalities that are characteristic of ciliopathies. The key phenotypes include:

In schlei mutant mice (carrying E125G mutation in Tmem107), embryos exhibit shortened snouts, expanded facial midlines, cleft palates, and extensive exencephaly . Interestingly, despite these pronounced developmental defects, schlei mutants do not display left-right patterning abnormalities or kidney/liver cysts that are commonly observed in other ciliopathy models, suggesting tissue-specific requirements for Tmem107 .

How does Tmem107 affect ciliogenesis in different tissues?

Tmem107 exhibits tissue-specific effects on ciliogenesis, with differential requirements across embryonic tissues. In schlei mutants, cilia formation is compromised in several developing tissues, but notably, nodal cilia appear normal, explaining the absence of left-right patterning defects .

In the neural retina, Tmem107 is particularly enriched during development, and its loss leads to severe defects in cilia formation in this tissue . Similarly, in TMEM107-deficient retinal organoids, there is complete loss of primary cilia, down-regulation of retina-specific genes, and cyst formation .

When TMEM107 is knocked out in human ARPE-19 cells (retinal pigment epithelial cells), primary cilia formation is prevented entirely . This tissue specificity in cilia formation requirements may explain the distinctive pattern of developmental abnormalities observed in different ciliopathy syndromes.

How does Tmem107 regulate Sonic hedgehog (Shh) signaling in neural tube development?

Tmem107 plays a complex dual role in Shh signaling within the neural tube, functioning both as a positive and negative regulator depending on context. Mechanistically:

• Ventral neural tube patterning: Tmem107 acts synergistically with Gli2 as a positive mediator of Shh signaling to specify ventral neuronal cell types .

• Intermediate zone patterning: Tmem107 functions in combination with Gli3 as a negative regulator to constrain the dorsal expansion of intermediate-level neuronal cells .

• Regional specificity: In schlei mutants, Shh expression in the floorplate is lost specifically in cervical and forelimb regions but remains induced in the trunk, indicating regional differences in Tmem107 requirements .

The effects of Tmem107 on Shh signaling appear to be mediated through its role in cilia formation and function. Unlike mutants with complete loss of cilia, schlei mutants retain some function of both Gli activator and repressor forms, indicating that Tmem107 modulates rather than abolishes ciliary Shh signaling .

What experimental approaches are most effective for studying Tmem107 function in ciliary signaling?

Several complementary experimental approaches have proven effective for investigating Tmem107 function:

1. Mouse genetic models:

• Forward genetics screens identified the schlei mutation (E125G) in Tmem107

• Targeted null alleles (Tmem107 tm1Lex) provide complementary models

• Complementation tests between different alleles help confirm causality of mutations

2. Retinal organoid models:

• TMEM107-deficient human retinal organoids allow study of its role in retinal development without influence from surrounding tissues

• These models enable direct observation of primary cilia formation and signaling defects

3. Cell culture systems:

• ARPE-19 cell lines with TMEM107 knockout for studying cilia formation

• Smoothened agonist treatment assays to assess Shh pathway responsiveness

• Immunofluorescence for ciliary markers to quantify ciliogenesis defects

4. Developmental analysis techniques:

• In situ hybridization to assess spatial patterns of gene expression changes

• Immunostaining for cell-type specific markers to evaluate patterning defects

• Scanning electron microscopy to examine cilia morphology and abundance

What is the relationship between Tmem107 and other transition zone proteins in cilia assembly?

Tmem107 functions within a complex network of transition zone proteins that collectively regulate cilia assembly and compartmentalization. Key interactions include:

• Protein recruitment: Tmem107 is required for recruiting several ciliopathy-associated proteins to the transition zone, including MKS-1, TMEM-231 (JBTS20), and JBTS-14 (TMEM237) .

• Functional parallels: The tissue-specific requirements for Tmem107 in ciliogenesis parallel those of other transition zone proteins like Tctn1, which also shows differential requirements across tissues .

• Potential mechanisms: Based on its transmembrane structure, Tmem107 may function within the ciliary membrane itself (similar to Tmem237), at the transition zone (like Tmem216), or potentially in both locations (like Tmem67) .

The precise molecular mechanisms by which Tmem107 influences cilia formation likely involve regulation of protein trafficking through the transition zone, control of ciliary membrane composition, or modulation of signaling receptor localization within cilia.

How does Tmem107 deficiency affect eye development at the molecular and cellular levels?

Tmem107 plays a crucial role in eye development through its effects on primary cilia and associated signaling pathways:

• Expression pattern: Tmem107 is specifically enriched in the developing neural retina during mouse embryogenesis, with enhanced expression in differentiating retina and optic stalk .

• Molecular consequences: TMEM107 deficiency in retinal organoids leads to:

  • Complete loss of primary cilia

  • Down-regulation of retina-specific genes

  • Formation of cystic structures rather than organized retinal layers

• Signaling disruption: In TMEM107-knockout ARPE-19 cells, there is:

  • Prevention of primary cilia formation

  • Impaired response to Smoothened agonist treatment

  • Ectopic activation of the Shh pathway, suggesting Tmem107 normally constrains pathway activation in specific contexts

• Phenotypic spectrum: Eye defects in Tmem107-deficient mice range from complete anophthalmia (33.3% of mutants) to microphthalmia (47% of mutants), with additional anomalies including aphakia (lens absence) and optic nerve hypoplasia .

These findings collectively demonstrate that Tmem107 regulates retinal development through its essential role in ciliogenesis and proper Shh pathway regulation in ocular tissues.

What technical considerations are important when working with recombinant Tmem107 protein?

When working with recombinant Tmem107 protein for research purposes, several technical considerations are critical:

• Expression systems: As a multi-pass transmembrane protein, Tmem107 requires eukaryotic expression systems (mammalian or insect cells) that can properly insert the protein into membranes and perform post-translational modifications.

• Protein solubilization: Extraction requires careful optimization of detergent conditions to maintain native structure while solubilizing the protein from membranes.

• Functional assays: Since Tmem107 functions at the ciliary transition zone, assays should be designed to test:

  • Protein-protein interactions with known transition zone partners (MKS-1, TMEM-231, JBTS-14)

  • Localization to the transition zone in complementation experiments

  • Ability to rescue ciliogenesis in Tmem107-deficient cells

• Structure-function analysis: The critical E125 residue in the fourth transmembrane domain represents an important site for mutagenesis studies, as the E125G mutation in schlei mice causes significant developmental abnormalities .

• Species considerations: While Tmem107 is highly conserved within vertebrates, there are important differences across species that may affect cross-reactivity of antibodies and interaction partners .

What genetic tools are available for studying Tmem107 function in mice?

Researchers have developed several genetic tools for investigating Tmem107 function:

These models provide complementary approaches for dissecting Tmem107 function. The schlei mouse model was identified through a forward genetics screen and retains some Tmem107 function, while the targeted null allele allows assessment of complete loss of function . Notably, complementation tests between these alleles confirmed that the schlei phenotype results from the Tmem107 mutation .

How can researchers effectively visualize and quantify ciliary defects in Tmem107-deficient models?

Multiple imaging and quantification approaches have proven effective for assessing ciliary defects in Tmem107-deficient models:

• Immunofluorescence microscopy:

  • Primary antibodies: Acetylated α-tubulin or ARL13B for ciliary axoneme

  • Transition zone markers: RPGRIP1L, MKS1, NPHP4

  • Basal body markers: γ-tubulin or pericentrin

  • Counterstaining: DAPI for nuclei

• Scanning electron microscopy (SEM):

  • Provides high-resolution visualization of cilia morphology

  • Allows examination of cilia density, length, and structural abnormalities

• Quantification parameters:

  • Percentage of ciliated cells

  • Cilia length measurements

  • Localization of ciliary proteins (transition zone, axoneme)

  • Co-localization coefficients for protein interactions

• Tissue-specific analyses:

  • Neural tube sections at specific axial levels

  • Developing limb buds at key developmental stages

  • Eye primordia and developing retina

  • Nodal cilia in early embryos

What experimental paradigms can assess Shh pathway activity in the context of Tmem107 research?

To evaluate Shh pathway activity in Tmem107 research contexts, several experimental approaches have proven informative:

• Gene expression analysis:

  • qRT-PCR for Shh target genes (Ptch1, Gli1, Hhip)

  • In situ hybridization to assess spatial patterns of Shh target activation

  • RNA-seq for genome-wide transcriptional changes

• Protein analyses:

  • Western blotting to assess Gli processing (full-length Gli vs. repressor forms)

  • Immunostaining for Shh pathway components in tissue sections

  • Co-immunoprecipitation to detect protein interactions

• Functional assays:

  • Smoothened agonist (SAG) treatment of cultured cells

  • Gli reporter assays in Tmem107-deficient cells

  • Shh-responsiveness of neural progenitors derived from Tmem107 mutants

• Genetic interaction studies:

  • Generate compound mutants with Shh pathway components (Gli2, Gli3)

  • Assess genetic rescue with constitutively active Shh pathway components

In Tmem107 research, these approaches have revealed that the protein differentially regulates distinct Shh target genes in the limb to control digit number but not identity , and that TMEM107 deficiency leads to ectopic activation of the Shh pathway in cultured cells .

What are the unresolved questions regarding Tmem107 function in development and disease?

Despite significant advances, several important questions about Tmem107 remain unanswered:

• Biochemical mechanism: How does Tmem107, as a transmembrane protein, mechanistically regulate the assembly and function of the ciliary transition zone?

• Tissue specificity: What molecular factors account for the differential requirements for Tmem107 across tissues, particularly the lack of requirement in nodal cilia?

• Protein interactions: What is the complete interactome of Tmem107 at the ciliary transition zone, and how do these interactions change during development or in disease states?

• Postnatal functions: What roles does Tmem107 play in postnatal tissues and adult homeostasis, given its continued expression beyond embryonic development?

• Therapeutic potential: Could modulation of Tmem107 or its downstream effectors provide therapeutic benefit in ciliopathies or related disorders?

How might emerging technologies advance our understanding of Tmem107 biology?

Emerging technologies hold significant promise for elucidating Tmem107 biology:

• Cryo-electron microscopy: Could reveal the structure of Tmem107 within the transition zone complex and provide insights into its functional domains.

• Proximity labeling proteomics (BioID, APEX): These approaches could identify the complete protein neighborhood of Tmem107 at the transition zone under different conditions.

• Single-cell transcriptomics: Would enable precise characterization of cell type-specific responses to Tmem107 deficiency across tissues.

• CRISPR-based screening: Systematic genetic interaction screens could identify suppressors and enhancers of Tmem107 phenotypes.

• Advanced organoid models: Development of complex multi-tissue organoids could help understand how Tmem107 influences tissue interactions during organogenesis.

• In vivo imaging: Live imaging of ciliary dynamics in Tmem107 mutants could provide insights into the temporal aspects of cilia formation and function.