Recombinant Human Transmembrane protein 107 (TMEM107)

What is TMEM107 and what is its role in cellular function?

TMEM107 is a transmembrane protein that plays a crucial role in the formation and function of primary cilia. It is particularly important in the transition zone (TZ) ciliary subcompartment, which is thought to control cilium composition and signaling by facilitating a protein diffusion barrier at the ciliary base . TMEM107 functions as a key recruiter of ciliopathy proteins to specific subdomains of the ciliary transition zone, organizing an intermediate layer that connects different structural components of the ciliary base . This organization is essential for proper cilia formation and subsequent signaling functions, particularly in developmental contexts involving the Sonic hedgehog (Shh) pathway .

What developmental processes are affected by TMEM107?

TMEM107 influences multiple critical developmental processes, particularly those dependent on primary cilia function. Studies have demonstrated that TMEM107 is essential for:

• Early vertebrate eye development and retinal ciliogenesis

• Neural tube patterning through regulation of Sonic hedgehog signaling

• Limb development, specifically in digit number determination

• Embryonic patterning across multiple tissues

The absence of TMEM107 results in characteristic developmental abnormalities including eye defects, neural tube patterning disruptions, and preaxial polydactyly (extra digits) . Interestingly, unlike some other ciliopathy proteins, TMEM107 mutations display tissue-specific effects with spatial restriction of phenotypes, suggesting differential requirements for this protein in cilia formation across distinct tissues .

How is TMEM107 related to ciliopathies?

Defects in the transition zone where TMEM107 functions are associated with several human ciliopathies, including Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) . TMEM107 plays a role in recruiting and organizing other ciliopathy-associated proteins to the transition zone, creating an architectural framework essential for cilia function . The partial retention of both Gli activator and repressor functions in TMEM107 mutants (unlike in complete cilia loss) provides important insight into how specific ciliary factors might differentially affect signaling pathways across tissues, potentially explaining the variety of clinical presentations in ciliopathy patients .

What is the molecular architecture of TMEM107 within the ciliary transition zone?

TMEM107 occupies an intermediate position in the hierarchical organization of the ciliary transition zone. Research using C. elegans models indicates that TMEM107 is situated between Layer 2 and Layer 3 proteins, functioning as a connector that recruits specific MKS module proteins including TMEM-17, TMEM-231, JBTS-14, and MKS-1 .

The localization dependency network reveals that:

• TMEM107 localization depends on Layer 1, Layer 2, and MKS-1 proteins

• TMEM107 is not localization-dependent on Layer 3 proteins

• TMEM107 and NPHP module proteins are not localization interdependent

Importantly, the TZ recruitment function of TMEM107 appears to be independent of its short cytosolic N- and C-termini, suggesting this organizational role is orchestrated by the transmembrane helices or interhelical linkers . This architectural understanding provides critical insight into how the protein functions within the transition zone complex.

How does TMEM107 influence the Sonic hedgehog signaling pathway?

TMEM107 plays a nuanced role in Shh signaling that differs from complete cilia loss. In TMEM107 mutants:

• Neural tube patterning is disrupted, particularly affecting ventral and intermediate neuronal cell types

• TMEM107 acts in combination with Gli2 and Gli3 transcription factors to pattern these neural cell populations

• Unlike complete cilia loss mutants, TMEM107 mutants retain some function of both Gli activator and repressor forms

• In limb development, TMEM107 acts in the Shh pathway to determine digit number but not digit identity by regulating only a subset of Shh target genes

This selective effect on Shh signaling outputs suggests TMEM107 may play a specialized role in modulating specific aspects of ciliary signal transduction, providing important insights into how ciliary factors affect Shh pathway functionality in distinct tissues .

What is the relationship between TMEM107 and primary cilia formation in retinal development?

TMEM107 is essential for proper ciliogenesis during retinal development. Research using both knockout and knockdown approaches has demonstrated that:

• TMEM107 absence leads to failure of primary cilia formation on early-stage organoids

• In late-stage retinal organoids, outer segments (specialized ciliary structures) fail to form without TMEM107

• Loss of TMEM107 results in down-regulation of retina-specific genes

• Neural retina structures and cell types fail to generate properly in human retinal organoid models lacking TMEM107

• TMEM107-deficient organoids develop cysts containing lipids, suggesting disrupted cell organization

Comparative analysis of primary cilia in retinal pigment epithelium (RPE) between wild-type and TMEM107-/- models shows significant differences:

These findings demonstrate that TMEM107 is critical for both the initiation and maintenance of cilia in the developing retina .

What approaches have been used to generate TMEM107-deficient models?

Researchers have utilized multiple complementary approaches to generate TMEM107-deficient models:

• CRISPR/Cas9 Gene Editing:

  • Targeting the start codon in the first exon of TMEM107, which is shared by all human TMEM107 isoforms

  • Using NcoI restriction site screening to rapidly identify knockout cell lines

  • Confirmation of editing through next-generation sequencing analysis

• shRNA-Mediated Knockdown:

  • Generation of lentiviral particles containing mCherry reporter and doxycycline-inducible expression of shRNA

  • Transduction followed by puromycin selection and FACS sorting of human induced pluripotent stem cells (hiPSCs)

  • Achieved approximately 50% down-regulation of TMEM107 gene expression as determined by RT-qPCR

• Forward Genetics Approach:

  • Isolation of the novel mutant allele "schlei" of the mouse Tmem107 gene

  • Characterization of phenotypes including preaxial polydactyly, exencephaly, and disrupted ventral neural tube patterning

Each approach offers distinct advantages for studying different aspects of TMEM107 function, from complete loss-of-function to partial knockdown, and from cellular to organismal levels.

How can TMEM107 protein-protein interactions be investigated?

Investigation of TMEM107's interactions with other transition zone proteins has been achieved through multiple complementary techniques:

• Co-immunoprecipitation (coIP) Assays:

  • Expression of GFP-tagged TMEM107 with FLAG-tagged partners (TMEM216, TMEM231, TMEM17, TMEM237) or myc-tagged MKS1

  • Use of binding conditions optimized specifically for membrane proteins

  • Assessment of biochemical associations in IMCD3 cells

• Localization Dependency Mapping:

  • Systematic analysis of protein localization in wild-type versus mutant backgrounds

  • Generation of fluorescently tagged proteins to visualize localization patterns

  • Creation of dependency maps showing hierarchical relationships between transition zone proteins

• Functional Complementation Assays:

  • Testing of mutated TMEM107 constructs to identify domains required for protein interactions

  • Demonstration that TZ recruitment function is independent of cytosolic N- and C-termini

These approaches collectively provide robust methods for dissecting the molecular interactions that underpin TMEM107's role in organizing the ciliary transition zone.

What methods are effective for analyzing TMEM107's role in retinal development?

Researchers have employed several specialized techniques to investigate TMEM107's function in retinal development:

• Retinal Organoid Models:

  • Generation of 3D retinal organoids from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs)

  • Inducible knockdown or knockout of TMEM107 during differentiation

  • Assessment of organoid structure, cell type specification, and cilia formation

• Immunofluorescence Analysis of Primary Cilia:

  • Quantification of cilia number and length in retinal pigment epithelium (RPE)

  • Comparison between wild-type and TMEM107-deficient models across developmental timepoints

  • Statistical analysis of changes in ciliogenesis (E10.5, E11.5, E12.5)

• Gene Expression Analysis:

  • RT-qPCR to quantify changes in retina-specific gene expression

  • Assessment of down-regulation effects following TMEM107 depletion

  • Correlation of gene expression changes with structural phenotypes

These methodologies provide complementary approaches to examining TMEM107's role in the complex process of retinal development and ciliogenesis.

How should researchers interpret phenotypic differences between various TMEM107-deficient models?

The interpretation of phenotypic differences across TMEM107-deficient models requires careful consideration of several factors:

• Residual Protein Function: The "schlei" mutant allele of TMEM107 retains some function of both Gli activator and repressor forms, unlike complete cilia loss mutants . This partial functionality explains the distinctive pattern of phenotypes observed.

• Tissue-Specific Requirements: TMEM107 shows differential requirements across tissues. The spatial restriction of phenotypes and lack of left-right patterning defects in TMEM107 mutants (unlike other cilia mutants) suggest tissue-specific roles for this protein in cilia formation .

• Temporal Dynamics: The effects of TMEM107 loss may vary depending on developmental timing, with critical periods for different tissues. For example, in retinal development, TMEM107 deficiency affects primary cilia at multiple developmental stages (E10.5, E11.5, E12.5) with varying severity .

• Knockdown versus Knockout Effects: Complete elimination of TMEM107 (knockout) may produce different phenotypes than partial reduction (knockdown). The ~50% down-regulation achieved through shRNA produces specific ciliary defects including extremely elongated or very short cilia with expanded bulges at their tips .

Researchers should carefully document the nature and extent of TMEM107 deficiency in their specific model system and consider these factors when comparing results across studies.

What are the critical variables in designing experiments to study TMEM107's role in ciliogenesis?

When designing experiments to investigate TMEM107's function in ciliogenesis, researchers should control for several critical variables:

• Cell Type Selection: Given the differential requirements for TMEM107 across tissues, the choice of cell type is crucial. Experiments should be conducted in relevant cell types where TMEM107 function has been established, such as retinal cells or neural tube progenitors .

• Developmental Timing: The timing of TMEM107 manipulation must be carefully controlled, particularly in developmental studies. For example, in retinal organoid models, doxycycline was applied from day 2 of the differentiation process with analysis at day 25 .

• Cilia Visualization Methods: Proper visualization and quantification of primary cilia require specific markers and imaging techniques. Studies have employed immunostaining for cilia markers combined with high-resolution microscopy to accurately assess cilia number, length, and morphology .

• Gene Dosage Effects: The level of TMEM107 reduction should be precisely quantified. RT-qPCR confirmation of knockdown efficiency (as in the ~50% reduction achieved in some studies) is essential for interpreting phenotypic outcomes .

• Genetic Background: In animal models, the genetic background can influence the penetrance and expressivity of TMEM107-related phenotypes. This variable should be controlled through appropriate backcrossing or the use of isogenic cell lines .

Careful attention to these variables will enhance experimental reproducibility and facilitate meaningful comparisons across studies.

What are promising therapeutic approaches targeting TMEM107-related ciliopathies?

While direct therapeutic applications based on TMEM107 are still in early stages, several promising research directions emerge from current understanding:

• Gene Therapy Approaches: The tissue-specific requirements for TMEM107 suggest that targeted gene therapy approaches might be feasible for treating specific aspects of TMEM107-related ciliopathies without needing system-wide correction.

• Small Molecule Modulators: Identification of small molecules that can modulate the recruitment function of TMEM107 might provide therapeutic options for ciliopathies where transition zone organization is disrupted.

• Pathway-Specific Interventions: Given TMEM107's selective effects on Shh signaling outputs, targeting specific downstream pathways (such as Gli activator/repressor balance) might provide more precise therapeutic strategies than attempting to fully restore cilia formation.

• Organoid-Based Drug Screening: The established retinal organoid models with TMEM107 deficiency provide an excellent platform for screening potential therapeutic compounds that might restore aspects of cilia formation or function .

• Combinatorial Approaches: The synthetic genetic interactions observed between TMEM107 and other ciliary genes (such as NPHP-4) suggest that combination therapies targeting multiple transition zone components might be more effective than single-target approaches .

These therapeutic directions will require further research into the precise molecular mechanisms by which TMEM107 orchestrates transition zone organization and influences developmental signaling pathways.

What are the key unanswered questions about TMEM107 function?

Despite significant advances in understanding TMEM107, several critical questions remain unanswered:

• Structural Determinants: How do the transmembrane helices or interhelical linkers of TMEM107 mediate its function in recruiting other transition zone proteins? Structural studies are needed to resolve this mechanism .

• Tissue Specificity: What molecular mechanisms explain the differential requirements for TMEM107 across tissues? Why do certain tissues display pronounced phenotypes while others are relatively spared in TMEM107-deficient models ?

• Signaling Specificity: How does TMEM107 selectively influence certain aspects of Shh signaling while sparing others? What is the molecular basis for its effect on digit number but not identity in limb development ?

• Human Disease Relevance: What is the full spectrum of human diseases associated with TMEM107 dysfunction? Are there undiagnosed or misclassified ciliopathies that might be attributed to TMEM107 variants?

• Regulatory Mechanisms: How is TMEM107 expression and function regulated during development and in adult tissues? Are there tissue-specific regulatory mechanisms that explain the differential requirements?

Addressing these questions will require integrated approaches combining structural biology, developmental genetics, cell biology, and clinical genetics to fully elucidate TMEM107's functions and disease relevance.