Recombinant Mouse Coiled-coil domain-containing protein 151 (Ccdc151)

What is Ccdc151 and what is its primary function in biological systems?

Ccdc151 (Coiled-coil domain-containing protein 151) is an evolutionarily conserved axonemal protein present in motile ciliated species . It encodes a coiled-coil protein that plays a critical role in ensuring the correct attachment of outer dynein arm (ODA) complexes to microtubules in motile cilia . The proper arrangement of these dynein arm complexes provides the mechanical force necessary for cilia beat .

Functionally, Ccdc151 shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas , indicating its highly conserved role across species. In humans, the CCDC151 gene is encoded by accession number NM_145045.4 , and loss-of-function mutations are associated with Primary Ciliary Dyskinesia (PCD), a disorder characterized by respiratory distress and defective left-right body asymmetry .

How can Ccdc151 expression patterns be effectively studied across different tissues?

Multiple complementary techniques can be employed to comprehensively study Ccdc151 expression:

• Reporter gene expression analysis: Microcomputed tomography (microCT) X-ray imaging of Ccdc151–β-galactosidase reporter expression in whole-mount tissues provides three-dimensional visualization of expression patterns. This approach has successfully revealed Ccdc151 expression in ependymal cells lining the ventricular brain system in mice .

• In situ hybridization techniques: Whole-mount in situ hybridization (WISH) has proven effective for detecting Ccdc151 expression in tissues with motile cilia, particularly in zebrafish models . This method involves:

  • Synthesizing digoxigenin-labeled RNA probes from linearized plasmid templates

  • Fixing staged embryos overnight at 4°C in 4% paraformaldehyde

  • Performing hybridization according to standard protocols

• Transcript analysis: For investigating specific transcript variants, methods include:

  • Nested PCR amplification from cDNA using KOD polymerase

  • Recombination with Gateway vectors via BP Clonase II reaction

  • Subcloning into epitope-tagged destination vectors via LR Clonase reaction

• Protein localization: Immunofluorescence techniques using antibodies against Ccdc151 or epitope-tagged versions can visualize protein distribution in ciliated cells .

What phenotypes are associated with Ccdc151 mutations in various model organisms?

Ccdc151 mutations produce distinct but overlapping phenotypes across species that highlight its critical role in ciliary function:

These phenotypic patterns demonstrate that while the fundamental role of Ccdc151 in ciliary function is conserved across species, its importance in specific physiological contexts varies, likely reflecting the differential reliance on ciliary function across organisms.

How does Ccdc151 contribute to the assembly of outer dynein arms in motile cilia?

Ccdc151 serves as a crucial component in the molecular pathway for outer dynein arm (ODA) assembly in motile cilia:

Mechanistically, Ccdc151 functions as part of the ODA docking complex, which is essential for proper attachment of dynein arms to the axonemal microtubules . Research demonstrates that CCDC151 deficiency disrupts not only the assembly of the ODAs themselves but also the incorporation of ODA targeting and docking components CCDC114 and ARMC4 into axonemes .

The assembly pathway appears to involve a hierarchical series of interactions where CCDC151 is required for the subsequent recruitment of other ODA complex components. Studies in human cells show that mutations in CCDC151 abolish its assembly into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components .

Experimental evidence supporting this mechanism includes:

• Analysis of CCDC151-deficient human respiratory cells showing complete loss of ODAs and severely impaired ciliary beating

• Studies in mice demonstrating that SNAP-tagged Dnah5 (a major ODA component) can be used as a reporter for functional rescue in PCD mouse models exhibiting loss of these complexes from cilia

• Findings that CCDC151 expression occurs in vertebrate left-right organizers, consistent with the laterality defects observed in affected individuals

This evidence collectively establishes Ccdc151 as a key upstream mediator in the ODA assembly and docking pathway.

What is the global prevalence of Ccdc151 mutations, and how do they contribute to Primary Ciliary Dyskinesia?

CCDC151 mutations represent a subset of the genetic causes of Primary Ciliary Dyskinesia (PCD), which has a higher global prevalence than previously recognized:

Based on allele frequency calculations from large genetic databases, the global prevalence of PCD (including cases caused by CCDC151 mutations) is at least 1 in 7,554, which is higher than previous estimates of approximately 1 in 11,000 to 1 in 16,566 in various populations . This calculation excludes many disease-causing variants currently classified as variants of uncertain significance (VUS), suggesting the true prevalence may be even higher.

CCDC151 mutations specifically lead to PCD through disruption of the outer dynein arm assembly pathway. In individuals with CCDC151 mutations, respiratory cilia show a complete loss of ODAs and severely impaired beating . Molecular analysis has identified specific mutations such as the nonsense mutation c.925G>T:p.(E309*) in CCDC151 .

The mechanistic pathway involves:

• Loss of functional CCDC151 protein in the axoneme

• Failure of ODA targeting and docking components (CCDC114, ARMC4) to assemble

• Subsequent failure of ODA components like DNAH5 to incorporate

• Complete loss of ciliary motility and resulting clinical manifestations

This understanding has important clinical implications, suggesting that PCD due to CCDC151 mutations may be underdiagnosed in the general population. In a study of bronchiectasis patients from the UK 100,000 Genomes Project, PCD was found to be significantly underdiagnosed, with less than 2.5% of severe cases receiving appropriate testing .

How can CRISPR/Cas9 technology be optimized for generating Ccdc151 knockout models?

CRISPR/Cas9 editing represents a powerful approach for generating Ccdc151 mutant models, with several methodological considerations for optimization:

Design principles for sgRNAs:

• Optimize for on-target ranking, minimal predicted off-target sites, and appropriate distance to the target site

• Select sgRNAs that target early exons or functionally crucial domains of Ccdc151

Procedure for generating knockout mice:

• sgRNA production: Synthesize using MEGAshortscript™ T7 Transcription Kit or similar systems

• Injection preparation: Co-inject sgRNA (25ng/μL), ssODN template (if using homology-directed repair), and Cas9 mRNA into zygotes

• Embryo transfer: Transfer injected zygotes into oviducts of pseudopregnant females

• Founder identification: Screen progeny for the desired modifications using PCR and Sanger sequencing

• Strain establishment: Backcross founders carrying the mutation into appropriate strain backgrounds (typically requiring at least 1-2 generations)

Efficiency considerations:

• As noted in search result , projects may seek to "improve the efficiency of CRISPR/Cas9 induced gene correction, via a novel linkage method"

• This suggests exploring methods beyond standard approaches, such as using modified Cas9 variants or optimized delivery systems

Validation approaches:

• Confirm gene disruption at the DNA level (sequencing)

• Verify absence of Ccdc151 protein expression (Western blot, immunofluorescence)

• Assess functional consequences through phenotypic characterization of motile cilia (e.g., using HSVM)

This methodological framework provides a comprehensive approach for generating and validating Ccdc151 knockout models for further experimental studies.

What imaging techniques are most effective for assessing ciliary defects in Ccdc151 mutant models?

A multi-modal imaging approach provides the most comprehensive assessment of ciliary defects in Ccdc151 mutant models:

Ultrastructural analysis:

• Transmission electron microscopy (TEM): Essential for examining axonemal ultrastructure, particularly the absence of outer dynein arms characteristic of Ccdc151 mutations . Sample preparation typically involves fixation in glutaraldehyde, post-fixation in osmium tetroxide, and embedding in resin before ultrathin sectioning.

Functional analysis:

• High-speed video microscopy (HSVM): Critical for assessing ciliary beat patterns and frequency . This technique captures digital videos at frame rates exceeding 200 frames per second, enabling detailed analysis of the ciliary waveform and beat coordination.

Expression and localization analysis:

• Microcomputed tomography (microCT): Particularly valuable for whole-mount tissues when using reporter gene systems like Ccdc151–β-galactosidase .

• Immunofluorescence: Essential for protein localization studies, using antibodies against Ccdc151 or epitope-tagged versions. As demonstrated in mouse studies, using tags like SNAP can enable visualization of protein dynamics in live cells .

Molecular interaction analysis:

• Pulldown experiments and co-immunoprecipitation (CoIP): Important for studying the association of CCDC151 with other proteins in the dynein arm assembly pathway .

In vivo expression analysis:

• Whole-mount in situ hybridization (WISH): Effective for determining spatial expression patterns in embryonic tissues, particularly in zebrafish models .

Integration of these techniques provides complementary data on structure, function, expression patterns, and molecular interactions, enabling comprehensive characterization of phenotypes resulting from Ccdc151 mutations.

How does Ccdc151 dysfunction specifically impact male fertility, and what experimental approaches can assess this?

Ccdc151 dysfunction significantly impacts male fertility through effects on sperm flagellar function, which can be studied through several experimental approaches:

Mechanistic basis:
The impact on fertility is primarily attributed to immotility of sperm, as demonstrated in studies of Ccdc151-null animals and in related coiled-coil domain-containing protein mutations (e.g., CCDC40) . This occurs because:

• Sperm flagella share structural and functional similarities with motile cilia

• Proper outer dynein arm assembly is essential for flagellar motility

• Ccdc151 is required for this assembly process

Experimental approaches:

• Conditional gene deletion in adult males: As performed with Ccdc151, this approach isolates fertility effects from developmental impacts, showing that "conditional deletion of the Ccdc151 gene in adult males causes low sperm counts and defective sperm motility" .

• Sperm analysis techniques:

  • Computer-assisted sperm analysis (CASA) for quantitative motility assessment

  • Transmission electron microscopy (TEM) to assess flagellar ultrastructure

  • Hypo-osmotic swelling test (HOST) to assess membrane integrity and select viable sperm

• Molecular analysis:

  • Targeted next-generation sequencing to identify specific mutations

  • Transcript analysis via PCR and sequencing

  • Protein localization studies in sperm flagella

• Fertility rescue approaches:

  • Intracytoplasmic sperm injection (ICSI) using sperm selected by HOST

  • Potential gene therapy approaches using CRISPR/Cas9-mediated gene correction

These methodologies provide a comprehensive framework for investigating the reproductive consequences of Ccdc151 dysfunction and developing potential interventions for male infertility associated with ciliopathies.

What are the molecular signaling pathways associated with Ccdc151 in ciliary development and function?

The molecular signaling networks involving Ccdc151 extend beyond its structural role in cilia, with evidence for both upstream regulatory pathways and downstream functional interactions:

Transcriptional regulation:
Research suggests that Ccdc151 expression is likely regulated as part of the motile ciliogenesis program, potentially downstream of master regulators such as MCIDAS (Multiciliate Differentiation And DNA Synthesis Associated Cell Cycle Protein) , which is listed among the genes mentioned in search result .

Protein interaction networks:

• Dynein arm assembly pathway: Ccdc151 functions within a hierarchical assembly pathway that includes CCDC114 and ARMC4, which fail to localize to axonemes in CCDC151-deficient cells .

• RNA regulatory interactions: Studies with SNAP-DNAH5 mice have shown that "SNAP-DNAH5 might interact with RNA regulatory proteins in maturing motile ciliated cells suggestive of translational regulation" , indicating potential post-transcriptional regulation mechanisms.

Signaling pathways beyond motility:
The search results indicate that CCDC151 has acquired additional functions beyond ciliary motility, particularly in vertebrates:

• In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and "genetically interacts with prickle1 in this process" , suggesting a connection to planar cell polarity signaling.

• In mammalian cells, "CCDC151 is implicated in the regulation of primary cilium length" , indicating a role in primary (non-motile) cilia that may involve distinct signaling mechanisms.

These multiple signaling contexts demonstrate that Ccdc151 functions within a complex molecular network that extends beyond its structural role in dynein arm assembly, with species-specific adaptations that have evolved additional signaling functions.

How do mouse models of Ccdc151 deficiency contribute to understanding human ciliopathies?

Mouse models of Ccdc151 deficiency provide invaluable insights into human ciliopathies through several research applications:

Disease modeling:
The Ccdc151 knockout mouse recapitulates key features of human Primary Ciliary Dyskinesia (PCD), including laterality defects and male infertility . Additionally, these mice develop congenital hydrocephalus, providing a model system to study this serious complication of ciliopathy that can occur in human patients .

Reporter systems for therapeutic development:
The SNAP-Dnah5 mouse model (relevant for studying Ccdc151 function) serves as "a reporter for functional rescue in PCD mouse models which exhibit loss of these complexes from the cilia" . This enables quantitative assessment of therapeutic efficacy, as "the effectiveness of the therapy could then be graded on the restoration of SNAP-DNAH5 fluorescence in the motile cilia" .

Mechanistic insights:
Ccdc151 mouse models have revealed important aspects of ciliary biology, including:

• The finding that "there is a very low level of ODA turnover in mature cilia"

• Evidence that "the Dnah5 transcript localises to large apical clusters in ciliated tracheal epithelial cells"

• Demonstration that Ccdc151 is expressed in ependymal cells lining the ventricular brain system

Therapeutic development platform:
Mouse models provide a platform for testing therapeutic approaches, including "CRISPR/Cas9 induced gene correction, via a novel linkage method, to develop a genome editing therapy for PCD" .

Developmental timing studies:
Conditional Ccdc151 gene deletion in adult males has demonstrated that Ccdc151 is required not only during development but also for ongoing ciliary function in adults, causing "low sperm counts and defective sperm motility" when deleted in adulthood.

These applications illustrate how mouse models of Ccdc151 deficiency contribute to a deeper understanding of human ciliopathies and facilitate the development of potential therapeutic interventions.