CCDC86 Antibody

What is CCDC86 and what are its key cellular functions?

CCDC86 (Coiled-coil domain-containing protein 86), also known as Cyclon, is a nucleolar protein that was initially identified as an immediate-early cytokine-responsive gene induced by interleukin 3 (IL-3) in hematopoietic cell lines . More recent research has established CCDC86 as a novel component of the perichromosomal layer, which is a network of proteins and RNAs coating the outer surface of mitotic chromosomes .

CCDC86 functions include:

• Regulation of chromosome segregation during mitosis

• Contribution to proper spindle regulation

• Modulation of kinetochore-microtubule attachments

• Regulation of T-cell apoptosis upon activation (activation-induced cellular death)

• Potential role in cancer progression as a MYCN-regulated gene

The protein contains a coiled-coil domain in its C-terminus and three conserved AT-hook-like motifs that are important for its localization to chromosomes .

What are the structural characteristics and molecular properties of CCDC86?

CCDC86 has several important structural features:

The AT-hook-like motifs found in CCDC86 are typically present in DNA-binding and DNA-remodeling proteins, including high-mobility group (HMG) proteins and the BRG1 protein, with a preference for AT-rich regions . These motifs are crucial for CCDC86's localization to chromosomes during mitosis.

How does CCDC86 associate with other proteins during the cell cycle?

CCDC86 displays dynamic localization patterns throughout the cell cycle:

• Interphase: Localizes primarily to the nucleolus

• Early mitosis: Disperses in the cytoplasm with some enrichment at the chromosome periphery

• Anaphase: Becomes highly enriched at the chromosome periphery

• Telophase/G1: Relocates to reforming nucleoli

CCDC86 interacts with several proteins including:

• Ki-67: A key interaction partner that is required for CCDC86's localization to the chromosome periphery. Proximity ligation assays (PLA) have confirmed this interaction in vivo, with signals distributed throughout the nuclear space and enrichment at the nucleolar periphery .

• Nucleolin: Co-localizes with CCDC86 at the chromosome periphery and in nucleolar-derived foci (NDFs) .

• Other nucleolar proteins: Including fibrillarin, NOP56, and multiple histones .

What are the optimal conditions for using CCDC86 antibodies in different experimental applications?

Based on validated protocols, here are the recommended conditions for CCDC86 antibody applications:

For Western blot analysis of immunoprecipitates, it's recommended to use Goat anti-Rabbit Light Chain HRP Conjugate with 5% Normal Pig Serum added to the blocking buffer .

What approaches are most effective for studying CCDC86 localization and dynamics during mitosis?

For investigating CCDC86 localization during mitosis, researchers have successfully employed several approaches:

• Fixed-cell immunofluorescence:

  • Co-stain with Ki-67 to establish mitotic stage and assess co-localization

  • Include DNA stain (DAPI) to visualize chromosomes

  • Use lamin A/C co-staining to precisely score cells at telophase/mitotic exit

• Live-cell imaging with fluorescently tagged proteins:

  • GFP-tagged CCDC86 constructs (both N- and C-terminally tagged) have been successfully used

  • Time-lapse imaging at 30-minute intervals for up to 25 hours has revealed dynamics during mitotic progression

  • This approach allows assessment of cell division outcomes and survival of daughter cells

• Proximity ligation assays (PLA):

  • Effective for confirming protein-protein interactions in situ

  • Has been successfully used to demonstrate CCDC86 interaction with Ki-67

  • Provides spatial information about where interactions occur within the cell

When analyzing CCDC86 localization during mitosis, it's important to clearly define and identify different mitotic stages using appropriate markers and morphological criteria.

How can researchers effectively deplete CCDC86 to study its functional role?

RNA interference (RNAi) has been successfully used to deplete CCDC86 and study resulting phenotypes:

• siRNA approach:

  • At least two independent siRNA oligonucleotides targeting CCDC86 should be used to control for off-target effects

  • Effective knockdown has been achieved by 48 hours post-transfection

  • Protein depletion should be confirmed by Western blot

• Phenotype analysis methods:

  • Flow cytometry: For cell cycle profile analysis (G1, S, G2/M, sub-G1 populations)

  • Immunofluorescence: To analyze chromosome alignment, spindle morphology, and perichromosomal protein localization

  • Live-cell imaging: To assess mitotic timing and cell fate after division

• Rescue experiments:

  • Expression of siRNA-resistant CCDC86 cDNA can confirm phenotype specificity

  • This approach successfully rescued the chromosome misalignment phenotype in published studies

When conducting CCDC86 depletion experiments, researchers should be aware that resulting phenotypes include:

• Chromosome alignment defects

• Altered spindle length

• Abnormal cytoplasmic aggregates containing Ki-67 and nucleolin

• Increased apoptosis and mitotic defects

How do the AT-hook domains of CCDC86 contribute to its function and chromosomal localization?

CCDC86 contains three conserved AT-hook-like motifs that play crucial roles in its localization and function:

• Domain structure and conservation:

  • AT-hook motifs are found in various DNA-binding and DNA-remodeling proteins

  • They have preference for AT-rich DNA regions

  • In CCDC86, three AT-hook-like motifs are present and highly conserved

• Functional analysis through domain deletion:
  Studies using truncated CCDC86 proteins revealed:

  • Deletion of the first AT-hook domain (GFP-CCDC86 Δ63) prevented localization to chromosomes during metaphase and anaphase

  • Proteins lacking this domain only accumulated on chromosomes after nuclear envelope reformation

  • Deletion of first and second domains (GFP-CCDC86 Δ121) or all three domains (GFP-CCDC86 Δ197) similarly failed to localize to chromosomes

• Interaction with Ki-67:

  • Interestingly, the AT-hook domains appear dispensable for Ki-67 interaction

  • Proximity ligation assays showed that both wild-type CCDC86 and the Δ63 mutant interact with Ki-67

  • This suggests separate domains mediate protein-protein interactions versus chromosomal localization

These findings indicate that the AT-hook domains, particularly the first one, are essential for CCDC86's proper recruitment to chromosomes during mitosis, while other domains likely mediate protein-protein interactions.

What is the relationship between CCDC86 and the MYCN oncogene, and its relevance to neuroblastoma?

CCDC86 has been identified as a MYCN-regulated gene with significant implications for neuroblastoma:

• Transcriptional regulation:

  • CCDC86 expression is regulated by the MYCN oncogene

  • This places CCDC86 within oncogenic signaling networks

• Prognostic value:

  • CCDC86 expression levels represent a "powerful marker for prognostic outcomes in neuroblastoma"

  • High expression levels correlate with poorer patient outcomes

• Mechanistic implications:

  • The role of CCDC86 in chromosome segregation and mitotic progression may explain its contribution to oncogenesis

  • Chromosome segregation errors can lead to genomic instability, a hallmark of cancer

  • CCDC86's function in regulating apoptosis of activated T-cells may also contribute to immune evasion mechanisms

This connection between CCDC86, MYCN, and neuroblastoma suggests that CCDC86 may serve not only as a biomarker but potentially as a therapeutic target in MYCN-amplified neuroblastomas.

How does CCDC86 depletion affect the organization of the perichromosomal layer and mitotic progression?

CCDC86 depletion has complex effects on both chromosome organization and mitotic progression:

These findings suggest CCDC86 is essential for maintaining proper chromosome organization and segregation during mitosis, with its depletion leading to serious defects that ultimately trigger cell death.

How can researchers address discrepancies in CCDC86 molecular weight observed in experimental settings?

The calculated molecular weight of CCDC86 is approximately 40 kDa, but the observed molecular weight in some experimental systems is around 65 kDa . This discrepancy requires careful consideration:

• Possible explanations:

  • Post-translational modifications (phosphorylation, ubiquitination, SUMOylation)

  • Alternative splicing variants

  • Protein-protein interactions resistant to SDS-PAGE separation

  • Anomalous migration due to charged residues or structural features

• Validation approaches:

  • Use multiple antibodies targeting different epitopes

  • Perform CCDC86 knockdown/knockout to confirm band specificity

  • Include positive controls with known CCDC86 expression

  • Consider mass spectrometry analysis to confirm protein identity and modifications

• Technical considerations:

  • Optimize sample preparation (different lysis buffers, denaturing conditions)

  • Run gradient gels for better resolution

  • Consider using different protein standards alongside your samples

  • Pretreat samples with phosphatases or other enzymes to identify potential modifications

When reporting CCDC86 detection in publications, clearly specify the observed molecular weight and antibody used to facilitate comparison across studies.

What controls are essential when studying CCDC86 interactions with Ki-67 and other perichromosomal proteins?

When investigating CCDC86 interactions, several critical controls should be included:

• For co-immunoprecipitation experiments:

  • IgG control: Use matched isotype control antibodies

  • Reciprocal IP: Pull down with anti-Ki-67 and blot for CCDC86, and vice versa

  • Input controls: Analyze a portion of pre-IP lysate

  • Knockout/knockdown controls: Perform parallel experiments in cells depleted of the target protein

• For proximity ligation assays (PLA):

  • Single primary antibody controls: Omit one antibody to establish background

  • Irrelevant protein controls: Use antibodies against proteins not expected to interact

  • Subcellular localization controls: Include markers for different cellular compartments

  • GFP-only control when using GFP-tagged proteins (as done in the CCDC86-Ki-67 PLA studies)

• For colocalization studies:

  • Quantitative metrics: Use Pearson's or Mander's coefficients

  • Cell cycle phase controls: Ensure precise identification of mitotic stages

  • Resolution considerations: Be aware of the limits of optical resolution (typically ~200nm)

  • Z-stack analysis: Examine colocalization in 3D rather than single optical sections

• For functional studies:

  • Rescue experiments: Express siRNA-resistant wild-type protein

  • Domain deletion/mutation controls: As demonstrated with the AT-hook domain studies

  • Cell cycle synchronization: To enrich for specific mitotic phases

These controls help ensure that observed interactions are specific and biologically relevant rather than technical artifacts.

How might CCDC86 serve as a potential therapeutic target or biomarker in cancer research?

The identification of CCDC86 as a MYCN-regulated gene with prognostic value in neuroblastoma opens several research avenues:

• Biomarker development:

  • Evaluate CCDC86 expression across different cancer types

  • Correlate expression levels with clinical outcomes and treatment responses

  • Develop standardized assays for CCDC86 detection in patient samples

  • Investigate whether CCDC86 expression can stratify patients for specific therapeutic approaches

• Therapeutic targeting possibilities:

  • Design inhibitors targeting CCDC86-specific functions

  • Explore synthetic lethality approaches in MYCN-amplified cancers

  • Investigate whether CCDC86 inhibition could sensitize cancer cells to conventional therapies

  • Develop strategies to disrupt specific protein-protein interactions (e.g., CCDC86-Ki-67)

• Mechanistic investigations needed:

  • Determine precise downstream effects of CCDC86 in cancer progression

  • Identify cancer-specific functions versus normal physiological roles

  • Explore potential immune modulatory functions given CCDC86's role in T-cell biology

  • Characterize tissue-specific expression patterns and functions

The connection between CCDC86, chromosome segregation, and cancer progression makes this protein a promising subject for translational cancer research.

What methodological advances would enhance our understanding of CCDC86's role in the perichromosomal layer?

Several technological and methodological approaches could advance CCDC86 research:

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM, PALM, SIM) to resolve fine structures at the chromosome periphery

  • Correlative light and electron microscopy (CLEM) to connect fluorescent signals with ultrastructural features

  • FRET/FLIM approaches to measure direct protein-protein interactions in live cells

  • Lattice light-sheet microscopy for high-speed, low-phototoxicity imaging of mitotic dynamics

• Proteomic approaches:

  • BioID or APEX proximity labeling to identify the complete interactome of CCDC86

  • Chromatin immunoprecipitation sequencing (ChIP-seq) to identify potential DNA binding sites of CCDC86 via its AT-hook domains

  • Phosphoproteomics to identify cell cycle-dependent modifications of CCDC86

  • Cross-linking mass spectrometry to map interaction interfaces

• Genomic technologies:

  • CRISPR-Cas9 genome editing for complete knockout and endogenous tagging

  • Conditional/inducible knockout systems to study acute loss of CCDC86

  • Single-cell transcriptomics to assess cell cycle-dependent expression

  • Domain-specific mutations introduced at the endogenous locus

These methodological advances would provide more precise insights into CCDC86's molecular functions and regulatory mechanisms at the chromosome periphery.