Recombinant Mouse Centrobin (Cntrob), partial

What is Centrobin (Cntrob) and what are its primary functions in cellular biology?

Centrobin (Centrosomal BRCA2-interacting protein, also known as Cntrob or LYST-interacting protein 8) is a centrosomal protein that plays crucial roles in centriole duplication and microtubule stability. It primarily localizes to daughter centrioles under normal conditions but can associate with mother centrioles upon serum starvation . Recent studies have revealed that centrobin functions as a positive regulator of vertebrate ciliogenesis by contributing to the removal of CP110 (a key negative regulator of ciliogenesis) from the mother centriole and stabilizing microtubules during axonemal extension . The protein has been implicated in several cellular processes including cell division, centrosome maturation, and primary cilium formation. Functional studies using knockout models have demonstrated that centrobin loss abrogates primary ciliation upon serum starvation, with ultrastructural analysis revealing defective axonemal extension after mother centriole docking .

How does the structure of Mouse Centrobin relate to its function?

Mouse Centrobin contains functional domains that mediate its interactions with other proteins and cellular structures. Particularly significant is the C-terminal region (amino acids 365-903) that interacts with both CP110 and tubulin . This C-terminal portion is essential for ciliogenesis, as demonstrated through rescue experiments. The protein structure can be divided into at least two functional regions:

• N-terminal region (amino acids 1-364): Not directly involved in CP110 interaction or ciliogenesis

• C-terminal region (amino acids 365-903): Critical for CP110 interaction and proper ciliogenesis

Further subdivision of the C-terminal region has identified that amino acids 452-903 constitute a ciliation-permissive fragment that can rescue ciliogenesis defects when expressed in centrobin-null cells . This structural organization allows centrobin to simultaneously perform multiple functions, including microtubule stabilization and CP110 regulation, both of which are required for normal ciliogenesis.

What expression systems are available for producing Recombinant Mouse Centrobin?

Multiple expression systems have been successfully employed to produce Recombinant Mouse Centrobin with varying attributes:

What are the optimal conditions for reconstituting lyophilized Recombinant Mouse Centrobin?

For optimal reconstitution of lyophilized Recombinant Mouse Centrobin, follow this methodological approach:

• Centrifuge the vial briefly prior to opening to bring all material to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for long-term storage stability

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store aliquots at -20°C to -80°C for maximum retention of activity

The addition of glycerol serves as a cryoprotectant, preventing protein denaturation during freeze-thaw cycles. The standard final concentration of glycerol used is 50%, but this can be adjusted based on downstream applications . For applications requiring higher protein concentrations, gradual reconstitution with gentle mixing is recommended to avoid protein aggregation. If precipitation occurs during reconstitution, try using a buffer system with optimized pH (typically 7.2-7.4) rather than pure water.

How can researchers effectively detect and localize Centrobin in cellular studies?

Detection and localization of Centrobin in cellular studies requires careful consideration of methodology:

Immunofluorescence Microscopy:

• Fix cells with 4% paraformaldehyde for 10 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 for 5 minutes

• Block with 3% BSA in PBS for 1 hour

• Incubate with anti-Centrobin primary antibody (1:200-1:500 dilution)

• Detect using fluorophore-conjugated secondary antibodies

• Co-stain with centrosomal markers (e.g., γ-tubulin, pericentrin) for colocalization studies

Important considerations:

• During serum starvation experiments, centrobin relocalization from daughter to mother centrioles can be observed, requiring time-course imaging

• For high-resolution studies, super-resolution microscopy techniques can reveal distinct localization patterns at different cell cycle stages

• When comparing centrobin levels between experimental conditions, quantitative image analysis should measure fluorescence intensity at individual centrioles rather than whole-cell measurements

For biochemical detection, Western blotting using denaturing conditions (SDS-PAGE) can identify the approximately 110 kDa centrobin protein . In co-immunoprecipitation experiments, gentler lysis conditions using non-ionic detergents are preferable to preserve protein-protein interactions, such as the centrobin-CP110 interaction .

What methods can be used to study Centrobin's protein-protein interactions?

Several complementary approaches have been successfully employed to study Centrobin's interactions with its binding partners:

Co-immunoprecipitation (Co-IP):

• Prepare cell lysates using mild lysis buffers (e.g., containing 0.5% NP-40 or 1% Triton X-100)

• Incubate lysates with anti-Centrobin antibody coupled to protein A/G beads

• Wash complexes thoroughly and elute with SDS sample buffer

• Analyze by Western blot for potential interacting partners such as CP110, tubulin, or BRCA2

This approach has successfully demonstrated Centrobin's interaction with CP110, particularly involving the C-terminal region (amino acids 365-903) of Centrobin .

Far Western Blot Analysis:

• Separate purified recombinant Centrobin and potential binding partners by SDS-PAGE

• Transfer proteins to membrane and renature

• Incubate membrane with purified potential binding partner

• Detect interaction using antibodies against the binding partner

This technique has been used to characterize Centrobin's interaction with Keratin 5 .

Yeast Two-Hybrid Screening:
For discovering novel interactions, yeast two-hybrid screening using centrobin fragments as bait against cDNA libraries can identify potential binding partners. This approach can be followed by validation using Co-IP or Far Western analysis.

Proximity Labeling Methods:
Newer methods like BioID or APEX2 proximity labeling, where Centrobin is fused to a biotin ligase or peroxidase, can identify proteins in close proximity to Centrobin in living cells, revealing both stable and transient interactions in their native cellular context.

How can genome editing be applied to study Centrobin function in cellular models?

Genome editing, particularly CRISPR-Cas9 technology, has revolutionized the study of Centrobin function:

CRISPR-Cas9 Knockout Strategy:

• Design sgRNAs targeting early exons of the CNTROB gene (multiple guides recommended)

• Transfect cells with Cas9 and sgRNA expression constructs

• Screen clones for complete ablation of Centrobin expression using Western blot

• Verify genomic modifications by sequencing

• Perform phenotypic characterization focusing on centriole duplication, ciliogenesis, and cell division

This approach has been successfully implemented in hTERT-RPE1 cells, revealing Centrobin's essential role in primary cilium formation . CNTROB null cells exhibited an increased frequency of monocentriolar and acentriolar cells, demonstrating Centrobin's importance in centriole duplication.

Rescue Experiments:
To confirm specificity of observed phenotypes and study structure-function relationships:

• Generate expression constructs for full-length or truncated Centrobin variants

• Introduce these constructs into CNTROB null cells

• Assess restoration of normal function

These experiments have determined that the C-terminal region of Centrobin (particularly amino acids 452-903) is sufficient to rescue ciliogenesis defects .

Domain-Specific Modifications:
Using CRISPR-Cas9 with homology-directed repair, specific functional domains can be modified rather than completely ablated:

• Design repair templates containing desired mutations

• Co-transfect with CRISPR-Cas9 components

• Screen for precise editing events

This approach allows for more subtle investigation of structure-function relationships, such as identifying specific residues involved in CP110 or tubulin binding.

What strategies can be employed to study the dynamics of Centrobin during the cell cycle?

Understanding Centrobin dynamics throughout the cell cycle requires specialized approaches:

Live-Cell Imaging:

• Generate stable cell lines expressing fluorescently-tagged Centrobin (e.g., GFP-Centrobin)

• Ensure expression levels approximate endogenous protein to avoid artifacts

• Perform time-lapse confocal microscopy throughout cell cycle progression

• Co-express markers for different cell cycle phases or centrosomal structures

For quantitative analysis, fluorescence recovery after photobleaching (FRAP) can measure Centrobin turnover rates at centrioles during different cell cycle stages.

Cell Synchronization Protocols:
To enrich populations at specific cell cycle stages:

• G1/S arrest: Double thymidine block

• S phase: Single thymidine block

• G2/M arrest: RO-3306 (CDK1 inhibitor)

• Mitotic arrest: Nocodazole or Taxol treatment

After synchronization, cells can be released and sampled at defined time points to create a temporal map of Centrobin localization and interaction patterns.

Centrobin Redistribution Study:
To specifically investigate the redistribution of Centrobin from daughter to mother centrioles during serum starvation:

• Culture cells in complete medium

• Transfer to serum-free medium for various durations (0-48 hours)

• Fix and immunostain for Centrobin and markers distinguishing mother from daughter centrioles (e.g., Centrin, Cep164)

• Quantify Centrobin levels at each centriole type over time

Research has shown that serum starvation induces Centrobin association with mother centrioles, coinciding with the initiation of ciliogenesis .

How can researchers investigate the role of Centrobin in ciliogenesis and associated pathologies?

Investigating Centrobin's role in ciliogenesis requires multifaceted approaches:

Ultrastructural Analysis:

• Generate CNTROB knockout or knockdown cells

• Induce ciliogenesis through serum starvation

• Process for scanning electron microscopy (SEM) or transmission electron microscopy (TEM)

• Examine axonemal extension and basal body docking

This approach has revealed that Centrobin loss leads to defective axonemal extension after mother centriole docking to the plasma membrane .

Functional Ciliary Assays:

• Sonic Hedgehog (Shh) pathway activation: Measure Gli1 transcriptional response

• Ciliary trafficking: Track movement of IFT proteins using FRAP or photoactivation

• Mechanosensation: Measure calcium influx in response to flow

Animal Model Studies:
For organismal relevance, zebrafish models provide valuable insights:

• Design morpholinos targeting zebrafish centrobin mRNA

• Inject morpholinos into zebrafish embryos at the 1-2 cell stage

• Analyze phenotypes at various developmental stages

• Perform rescue experiments with wild-type or mutant centrobin mRNA

Centrobin-depleted zebrafish embryos exhibit microcephaly, curved and shorter bodies, and defects in laterality control – all features indicative of ciliary dysfunction . This model system allows for assessment of Centrobin's role in ciliopathy-like conditions.

CP110 Relationship Analysis:
To examine the regulatory relationship between Centrobin and CP110:

• Perform CP110 knockdown in CNTROB null cells

• Assess rescue of ciliogenesis defects

• Analyze CP110 localization in wild-type versus CNTROB null cells before and after serum starvation

These experiments have shown that CP110 knockdown in CNTROB nulls partially rescues ciliogenesis, indicating that both microtubule stabilization and CP110 regulation by Centrobin are required for ciliogenesis .

How should researchers quantify and interpret changes in Centrobin localization between experimental conditions?

Quantitative analysis of Centrobin localization requires rigorous methodological approaches:

Intensity Measurement Protocol:

• Acquire z-stack images of cells immunostained for Centrobin and centriole markers

• Generate maximum intensity projections

• Define regions of interest (ROIs) around individual centrioles

• Measure integrated fluorescence intensity within each ROI

• Subtract local background signal

• Normalize to the intensity of a centriole marker (e.g., γ-tubulin) if comparing across different samples

Comparative Analysis Framework:
When analyzing Centrobin redistribution between mother and daughter centrioles, such as during serum starvation experiments:

Research has demonstrated that mother centrioles in cells lacking daughter centrioles (1:1 cells) contained nearly twice as much centrobin compared to normal cells, suggesting redistribution due to higher affinity for daughter centrioles . When interpreting such data, consider that protein relocalization may reflect changes in binding affinity, competition between binding sites, or post-translational modifications rather than simply changes in expression levels.

What are common challenges in working with Recombinant Centrobin and how can they be addressed?

Researchers frequently encounter several challenges when working with Recombinant Centrobin:

Solubility Issues:

• Challenge: Recombinant Centrobin, particularly full-length protein, may show limited solubility due to its size (~110 kDa) and structural properties.

• Solution: Express as fusion protein with solubility-enhancing tags (MBP, SUMO); optimize buffer conditions with increased salt (150-300 mM NaCl) and mild detergents (0.1% Triton X-100); consider expressing functional domains separately.

Protein Stability:

• Challenge: Purified Centrobin may exhibit degradation during storage.

• Solution: Add protease inhibitors during purification; store with 50% glycerol at -80°C; prepare fresh working aliquots; avoid repeated freeze-thaw cycles.

Functional Activity Assessment:

• Challenge: Confirming that recombinant protein retains native activities.

• Solution: Include positive controls in interaction assays; compare activity of protein from different expression systems; validate using cellular assays.

Antibody Cross-Reactivity:

• Challenge: Antibodies may cross-react with related proteins, especially in complex samples.

• Solution: Validate antibody specificity using CNTROB knockout cells; perform competitive blocking with recombinant protein; use multiple antibodies targeting different epitopes.

Quantification Variability:

• Challenge: Variability in immunofluorescence quantification between experiments.

• Solution: Include internal standards in each experiment; normalize to stable reference proteins; perform technical replicates; use automated image analysis pipelines to reduce subjective biases.

How can researchers reconcile contradictory findings regarding Centrobin function across different model systems?

When faced with apparently contradictory findings regarding Centrobin function, consider these methodological approaches:

Systematic Comparison Framework:

• Catalog specific differences in experimental systems:

  • Species differences (mouse vs. human vs. zebrafish)

  • Cell type variations (embryonic vs. differentiated cells)

  • Acute vs. chronic depletion methods

  • Complete knockout vs. partial knockdown

• Directly compare phenotypes using standardized assays:

  • Apply identical protocols across cell lines

  • Use the same antibodies and detection methods

  • Quantify results using consistent metrics

• Consider context-dependent functions:

  • Test for cell-cycle dependency

  • Examine tissue-specific roles

  • Investigate compensatory mechanisms in different systems

Specific Reconciliation Examples:
The dual role of Centrobin in centriole duplication and ciliogenesis might appear contradictory, but can be reconciled by understanding its dynamic localization patterns. During the cell cycle, Centrobin primarily associates with daughter centrioles and promotes duplication. Upon serum starvation, it relocates to mother centrioles where it facilitates CP110 removal and ciliogenesis . This demonstrates how temporal and spatial regulation can allow the same protein to perform distinct functions in different contexts.

Similarly, while studies in RPE1 cells show complete abrogation of ciliogenesis upon Centrobin loss , other cell types might show less severe phenotypes due to compensatory mechanisms or different dependencies on specific ciliogenesis pathways. When analyzing such differences, consider whether they represent truly contradictory functions or context-dependent manifestations of the same molecular activities.

How conserved is Centrobin structure and function across vertebrate species?

Centrobin shows significant evolutionary conservation across vertebrate species, with important implications for research:

Sequence Conservation Analysis:
Comparison of Centrobin orthologs reveals:

• High conservation of C-terminal domains involved in CP110 and tubulin binding

• More variable N-terminal regions

• Particularly strong conservation of the ciliation-permissive fragment (amino acids 452-903)

The zebrafish centrobin gene encodes a predicted 2,610-bp full-length cDNA, which has been deposited in GenBank under accession number MF461638 . Sequence alignment between mouse and zebrafish Centrobin shows conservation of key functional domains, supporting the use of zebrafish as a model organism for studying Centrobin function.

Functional Conservation Evidence:
The following experimental evidence supports functional conservation:

• Morpholino-mediated knockdown of centrobin in zebrafish results in phenotypes consistent with ciliary dysfunction (microcephaly, curved bodies, laterality defects)

• These phenotypes mirror cellular defects observed in mammalian cell culture models

• Rescue experiments with mammalian Centrobin in zebrafish embryos can partially restore normal development

This cross-species functional conservation validates translational approaches and suggests that mechanisms of Centrobin action in ciliogenesis are fundamental across vertebrates.

What are the advantages and limitations of different model systems for studying Centrobin function?

Each model system offers distinct advantages and limitations for Centrobin research:

Cell Culture Models:

Animal Models:

Experimental Approaches by System:
For zebrafish studies, morpholino-mediated knockdown provides a rapid assessment method, but CRISPR-Cas9 genome editing offers more specific gene targeting. The zebrafish model is particularly valuable for studying ciliopathy-related phenotypes, as demonstrated by the observation of microcephaly and body curvature in centrobin-depleted embryos .

For cellular models, hTERT-RPE1 cells have proven particularly valuable for dissecting the molecular mechanisms of Centrobin function in ciliogenesis, allowing detailed ultrastructural analysis and protein interaction studies .

How can researchers effectively translate findings about Centrobin from model systems to human disease relevance?

Translating Centrobin research to human disease contexts requires strategic approaches:

Disease-Relevant Phenotypic Analysis:

• Identify human diseases with phenotypes matching centrobin dysfunction (e.g., ciliopathies with microcephaly)

• Analyze patient samples for CNTROB mutations or expression changes

• Create cellular disease models using patient-derived cells or engineered mutations

• Test whether restoring Centrobin function can rescue disease phenotypes

Functional Genomic Screening:
For discovering disease-relevant interactions:

• Perform synthetic lethality screens with CNTROB in disease-relevant cell types

• Identify genetic modifiers that enhance or suppress Centrobin loss phenotypes

• Map these modifiers to human disease genes and pathways

Therapeutic Target Validation:
To assess Centrobin as a potential therapeutic target:

• Develop small molecules or peptides targeting specific Centrobin interactions (e.g., Centrobin-CP110)

• Test these agents in cellular and animal models

• Assess restoration of ciliary function in disease models

Integration with Human Genetics:
Current evidence from animal models suggests that Centrobin deficiency leads to phenotypes resembling human ciliopathies, including microcephaly and laterality defects . These findings highlight the potential relevance of CNTROB as a candidate gene for human ciliopathies, particularly those affecting brain development. Researchers should consider screening CNTROB in patients with unexplained ciliopathy-like syndromes, especially those with primary microcephaly.