DEUP1 Antibody

What is DEUP1 and what cellular functions does it regulate?

DEUP1 (deuterosome assembly protein 1), also known as coiled-coil domain-containing protein 67 (CCDC67) or deuterosome protein 1, is a cytoplasmic protein involved in multiciliated epithelial cell differentiation. As a member of the CEP63 protein family, DEUP1 is a key component of deuterosomes, which are electron-dense structures involved in centriole amplification . Interestingly, recent studies have revealed that while DEUP1 was traditionally thought to be essential for centriole amplification, loss-of-function experiments demonstrated that cells lacking deuterosomes due to DEUP1 gene ablation can still produce basal bodies and functional multicilia . This suggests that DEUP1's role is more complex than initially thought, possibly involving the regulation of centriole production timing rather than being absolutely required for amplification.

What are the key structural and molecular characteristics of DEUP1?

In humans, the canonical DEUP1 protein consists of 604 amino acid residues with a molecular mass of approximately 71 kDa . Up to two different isoforms have been reported for this protein, and its subcellular localization is primarily cytoplasmic. DEUP1 is evolutionarily conserved, with orthologs identified in multiple species including mouse, rat, bovine, frog, chimpanzee, and chicken . As a paralog of Cep63, DEUP1 serves as a key component of deuterosomes, which are specialized structures involved in massive centriole amplification during multiciliated cell differentiation . The protein's ability to self-organize into larger aggregates called deuterosome clusters (DCs) appears to have functional significance in regulating centriole number and multiciliated cell development .

What are the common applications for DEUP1 antibodies in research?

DEUP1 antibodies are versatile tools in multiciliogenesis research, with several validated applications:

Researchers should note that DEUP1 antibodies from different sources may vary in their specific applications, so validation for particular experimental contexts is recommended .

What are the optimal fixation and staining protocols for DEUP1 immunodetection?

For optimal DEUP1 immunodetection, researchers should consider the following protocol based on successful studies:

• Fixation: Fix cells with 4% paraformaldehyde (PFA) and 2% sucrose in phosphate-buffered saline (PBS) for 15 minutes on ice .

• Washing: Wash three times with PBS for 5 minutes each .

• Blocking: Block with 3% bovine serum albumin (BSA), 5% horse serum, and 0.1% Triton X-100 in PBS for 30 minutes at room temperature .

• Primary antibody incubation: Dilute DEUP1 antibody in 3% BSA in PBS and incubate overnight at 4°C .

• Secondary antibody treatment: After washing, incubate with species-specific Alexa Fluor conjugated secondary antibodies for 2 hours at room temperature .

• Mounting: Mount using an anti-fade mounting medium such as VECTASHIELD HardSet .

For co-staining with other centriolar markers, consider using antibodies against Centrin, SAS6, CP110, or CEP164 to differentiate between various centriolar structures .

How can researchers validate the specificity of DEUP1 antibodies?

To ensure experimental rigor, researchers should validate DEUP1 antibody specificity through multiple approaches:

• Knockout/knockdown controls: Compare staining in wild-type cells versus DEUP1 knockout or knockdown cells to confirm signal specificity .

• Multiple antibody validation: Use antibodies targeting different epitopes of DEUP1 to confirm consistent localization patterns .

• Recombinant protein competition: Pre-incubate the antibody with recombinant DEUP1 protein to demonstrate competitive blocking of specific binding.

• Co-localization studies: Confirm that DEUP1 antibody staining co-localizes with known deuterosome markers and shows expected distribution patterns during multiciliated cell differentiation .

• Western blot analysis: Verify that the antibody detects a band of the expected molecular weight (approximately 71 kDa) in human samples .

What control samples should be included in DEUP1 antibody experiments?

Robust experimental design for DEUP1 studies should include the following controls:

For in vivo studies, appropriate controls might include in utero electroporation with membrane-bound GFP (mGFP) constructs for comparison with GFP-DEUP1 constructs .

How can DEUP1 antibodies be used to study centriole amplification in multiciliated cells?

DEUP1 antibodies provide powerful tools for investigating the complex process of centriole amplification:

• Time-lapse imaging: By combining GFP-tagged DEUP1 constructs with antibodies against centriolar components, researchers can perform live imaging to track deuterosome dynamics during multiciliogenesis .

• Quantitative analysis of deuterosome fate: DEUP1 antibodies enable researchers to distinguish between scattered deuterosomes and deuterosome clusters (DCs), allowing analysis of their differential roles in centriole production .

• Co-immunoprecipitation studies: DEUP1 antibodies can be used to pull down protein complexes, helping identify interaction partners involved in centriole amplification.

• Super-resolution microscopy: Using DEUP1 antibodies with techniques like STORM or STED microscopy can reveal ultrastructural details of deuterosome organization beyond conventional microscopy limits.

• Correlative light and electron microscopy (CLEM): DEUP1 antibodies can help correlate fluorescence signals with electron microscopy ultrastructure, providing comprehensive views of deuterosome morphology and function .

What insights have gain-of-function and loss-of-function studies revealed about DEUP1's role?

Contrasting findings from DEUP1 gain-of-function and loss-of-function studies have revealed nuanced aspects of its biological role:

These seemingly contradictory results suggest that DEUP1 plays a regulatory role in centriole amplification rather than being absolutely required for the process, with precise expression levels being critical for normal multiciliated cell development .

How can researchers investigate deuterosome cluster formation using DEUP1 antibodies?

To study deuterosome cluster (DC) formation, researchers can employ several approaches using DEUP1 antibodies:

• Morphological characterization: Use DEUP1 antibodies in combination with super-resolution microscopy to measure deuterosome size (average diameter of 300 nm for individual deuterosomes) and track their aggregation into larger DCs .

• Composition analysis: Co-stain with antibodies against centriolar components (Centrin, SAS6, CP110) to determine how these proteins are incorporated into DCs versus scattered deuterosomes .

• Temporal dynamics: Conduct time-course experiments using DEUP1 antibodies to track deuterosome behavior during differentiation, noting that scattered deuterosomes gradually disappear as cells become multiciliated, while DCs self-organize into larger aggregates .

• Functional correlation: Combine DEUP1 staining with markers for multiciliation (GT335) and cell boundaries (ZO-1) to correlate DC presence with cell fate outcomes (primary ciliated versus multiciliated cells) .

• In vivo validation: Use in utero electroporation of GFP-DEUP1 constructs followed by tissue immunostaining to confirm DC formation in developing ependymal tissue .

What are common challenges in DEUP1 antibody experiments and how can they be addressed?

Researchers may encounter several technical challenges when working with DEUP1 antibodies:

For optimal results, researchers should conduct preliminary titration experiments and include appropriate controls to establish optimal conditions for their specific experimental system .

How should researchers interpret conflicting data in DEUP1 expression studies?

When confronted with seemingly contradictory results in DEUP1 research, consider these interpretative frameworks:

• Developmental timing effects: DEUP1's role may vary across different stages of multiciliated cell differentiation. DC-positive cells are more prevalent at early postnatal stages but become scarce later, suggesting stage-specific functions .

• Cell-type specificity: Different multiciliated cell types (ependymal cells versus airway epithelial cells) may exhibit distinct DEUP1 dependencies or expression patterns .

• Dose-dependent effects: While complete loss of DEUP1 may be compensated by alternative pathways, overexpression can disrupt normal development, indicating the importance of precise expression levels .

• Methodological differences: Variations in experimental approaches (in vivo versus in vitro, fixed versus live imaging) may yield apparently conflicting results due to different temporal or spatial resolution .

• Functional redundancy: Other proteins or pathways may compensate for DEUP1 loss in knockout models but become disrupted with DEUP1 overexpression .

Research suggests that while DEUP1 is not absolutely required for centriole amplification, its dysregulation can significantly impact multiciliated cell development and function .

What factors might affect the reproducibility of DEUP1 antibody experiments?

Several factors can influence the reproducibility of DEUP1 antibody-based experiments:

• Antibody source and lot-to-lot variation: Different suppliers produce antibodies with varying specificity and sensitivity. Even within the same product, lot-to-lot variations can occur .

• Cell culture conditions: For in vitro ependymal cell studies, differentiation induction methods (such as switching from 10% FBS to 0% FBS media) can affect DEUP1 expression patterns and deuterosome formation .

• Fixation and permeabilization protocols: Different fixation methods can affect epitope accessibility and antibody binding efficiency .

• Developmental stage heterogeneity: When working with developing tissues or differentiating cells, population heterogeneity can lead to variable results if not properly controlled for developmental stage .

• Imaging parameters and quantification methods: Differences in microscopy settings, image processing, and quantitative analysis approaches can lead to different interpretations of similar biological phenomena .

To enhance reproducibility, researchers should maintain detailed protocols, validate antibodies in their specific experimental systems, and clearly report all methodological details in publications .

What are emerging applications for DEUP1 antibodies in multiciliated cell research?

DEUP1 antibodies are poised to contribute to several cutting-edge research directions:

• Single-cell proteomics: Combining DEUP1 immunostaining with single-cell mass spectrometry could reveal protein interaction networks during different phases of deuterosome formation and function.

• Spatial transcriptomics: DEUP1 antibodies used in conjunction with RNA sequencing technologies could map gene expression patterns around deuterosomes and deuterosome clusters.

• In vivo deuterosome dynamics: Advanced intravital imaging using fluorescently tagged DEUP1 antibodies or constructs could reveal real-time deuterosome behavior in living tissues .

• Developmental timing mechanisms: DEUP1 antibodies could help identify "DC antagonists" that appear in late postnatal stages and potentially regulate deuterosome cluster formation .

• Comparative evolutionary studies: Using DEUP1 antibodies across different species could illuminate the evolutionary conservation and divergence of multiciliogenesis mechanisms .

How might DEUP1 research contribute to understanding ciliopathies and related diseases?

DEUP1 research has potential implications for understanding and addressing ciliary disorders:

• Hydrocephalus mechanisms: Since ectopic DEUP1 expression impairs cerebrospinal fluid flow, understanding its regulation may provide insights into conditions involving dysfunctional ependymal cilia, such as hydrocephalus .

• Respiratory ciliopathies: As DEUP1 marks Airway Deuterosomal Cells, further research could illuminate pathological mechanisms in primary ciliary dyskinesia and other respiratory conditions affecting mucociliary clearance .

• Developmental disorders: The inhibitory effect of DEUP1 overexpression on multiciliated cell differentiation suggests that its dysregulation could contribute to developmental abnormalities involving ciliated tissues .

• Therapeutic targeting: Modulating DEUP1 expression or function might represent a novel approach to addressing certain ciliopathies by influencing centriole amplification and multiciliated cell development .

• Diagnostic applications: DEUP1 antibodies could potentially serve as diagnostic tools for identifying abnormalities in deuterosome formation or centriole amplification in patient samples .