CEP192 Antibody

What is CEP192 and what role does it play in centrosome biology?

CEP192 is a key centrosomal protein required for mitotic centrosome maturation and bipolar spindle assembly. It functions as a major regulator of pericentriolar material (PCM) recruitment, centrosome maturation, and centriole duplication . CEP192 acts as a centrosome-specific activating scaffold for AURKA (Aurora-A kinase) and PLK1 (Polo-like kinase 1) , positioning it as a critical component in the centrosomal signaling network.

The expression of CEP192 is tightly regulated during the cell cycle, with protein levels peaking during G2/M phase to facilitate its primary functions in centrosome maturation and spindle assembly . This cell cycle-dependent regulation ensures proper coordination of centrosome activities with mitotic progression.

Fluorescence recovery after photobleaching (FRAP) experiments have demonstrated that GFP-Cep192 shows hardly any recovery after 400 seconds, which is typical behavior for centrosomal core components and scaffolding proteins of the corona . This indicates that CEP192 is a stable structural element of the centrosome rather than a transient visitor.

What applications are CEP192 antibodies typically used for?

CEP192 antibodies are versatile research tools employed in multiple experimental applications:

CEP192 antibodies have proven particularly valuable for visualizing centrosome dynamics throughout the cell cycle. They can be used to track centrosome maturation, duplication, and function in both normal cellular processes and disease states . The consistent centrosomal localization of CEP192 throughout mitosis makes it an excellent marker for centrosome-focused studies .

How can I validate the specificity of a CEP192 antibody?

Validating antibody specificity is crucial for obtaining reliable research results. For CEP192 antibodies, consider these validation approaches:

• Cellular depletion: Compare antibody staining in control cells versus cells where CEP192 has been depleted through RNAi or CRISPR-Cas9 approaches. Expected outcome would be significantly reduced signal in depleted cells .

• Western blot analysis: Verify that the antibody detects a band of appropriate molecular weight (~192 kDa) in cellular extracts. Compare detection across multiple cell lines that express CEP192, such as HEK-293T, A431, and HeLa cells .

• Immunofluorescence colocalization: Confirm that the antibody signal colocalizes with other known centrosomal markers in immunofluorescence experiments, particularly during different cell cycle phases .

• Recombinant protein recognition: Test antibody recognition of recombinant CEP192 protein fragments, particularly those corresponding to the immunogenic regions .

A comprehensive validation typically combines multiple approaches to ensure antibody reliability.

How does CEP192 interact with Aurora-A kinase and what is the functional significance?

The interaction between CEP192 and Aurora-A represents a critical regulatory nexus in centrosome function. Recent structural studies have elucidated both the structure of the human Aurora-A:CEP192 complex and its critical role at the apex of Aurora-A activation in mitosis .

CEP192 localizes mitotic Aurora-A activity by priming its interaction with downstream effectors. When CEP192 is depleted, the intensity of phosphorylated Aurora-A (pT288) signal is diminished, resulting in reduced phosphorylation of downstream substrates like LATS2 (at S83) . This indicates that CEP192 is required for proper Aurora-A activation at the centrosome.

Experimental evidence reveals distinct spatial patterns of Aurora-A activation depending on its activators:

• In CEP192-depleted cells: Weak and diffuse pT288 signal detectable at spindle poles

• In TPX2-depleted cells: Tight dot-like pT288 signal at the centrosome

• In control cells: Clear centrosomal signals for both pT288 and phospho-LATS2-S83

This indicates that CEP192 and TPX2 activate Aurora-A in spatially distinct compartments (centrosome and spindle microtubules, respectively), pointing to compartmentalized regulation of Aurora-A activity during mitosis .

What methods are effective for studying CEP192 protein-protein interactions?

Several methodological approaches have proven valuable for investigating CEP192 interactions with other centrosomal proteins:

• BioID proximity labeling: This technique has emerged as the most effective method for determining the centrosomal interactome . BioID involves fusing a protein of interest (like CEP192) to a promiscuous biotinylase, which biotinylates lysine residues within a proximity of up to 10 nm. This approach is particularly advantageous for centrosomal proteins because:

  • It does not require solubility of the interaction partners

  • It detects close proximity under in vivo conditions

  • It overcomes the challenge that centrosomal proteins are often virtually absent from soluble cell extracts

• Expansion microscopy (ExM): This technique allows for high-resolution visualization of protein co-localization. The classical pre-expansion staining ExM method, when combined with small tags and small fluorescent probes, provides accurate dimensions that match those observed in electron microscopy .

• Knock-in approaches: Using knock-in strategies for tagging CEP192 (e.g., with GFP or BioID2) ensures expression under endogenous promoter control, avoiding artifacts associated with overexpression .

Using BioID with CEP192, researchers have identified CDK5RAP2 and potentially CP91 as direct interaction partners. Reciprocally, CEP192 was biotinylated by both CDK5RAP2-BioID2 and CP91-BioID2, confirming mutual interactions between these proteins .

How does CEP192 depletion affect centrosome structure and function?

CEP192 depletion produces distinct phenotypic effects on centrosome structure and function:

• Supernumerary MTOCs: Approximately 39% of CEP192-depleted cells display supernumerary microtubule organizing centers (MTOCs) . Importantly, these supernumerary MTOCs differ from those caused by CEP192 overexpression:

  • They lack detectable core layer components

  • They contain corona components like CDK5RAP2, CP148, and CP224

  • They are essentially MTOCs rather than bona fide centrosomes

• Ultrastructural changes: Electron microscopy of CEP192-depleted cells reveals:

  • Supernumerary cytosolic MTOCs devoid of discernible layered core structure

  • Nucleus-attached MTOCs often lacking clearly discernible layered core structure

  • Nucleus-attached centrosomes with outer layers showing lower electron density

  • Reduced diameter of outer layers compared to the central layer

• Functional consequences: CEP192 depletion impacts centrosome function by:

  • Disrupting centrosome maturation

  • Affecting microtubule organization

  • Potentially leading to abnormal cell division and genome instability

These findings collectively suggest that CEP192 is essential for maintaining proper centrosome structure and function, with its depletion causing both structural abnormalities and functional defects.

What are the best approaches for visualizing CEP192 localization during different cell cycle stages?

Optimizing visualization of CEP192 throughout the cell cycle requires careful consideration of methodology:

• Widefield deconvolution microscopy: Effective for visualizing endogenous CEP192 association with centrosomes during both interphase and throughout all mitotic stages . This approach, when combined with specific anti-CEP192 antibodies, provides reliable detection of the protein's localization.

• Live-cell imaging: Using knock-in cell lines expressing Cep192-GFP under the control of the endogenous promoter enables dynamic tracking of CEP192 localization. Time-lapse imaging confirms that CEP192 remains located at the centrosome from G2/M transition until the next interphase .

• Expansion microscopy (ExM): This super-resolution approach provides enhanced spatial resolution for analyzing CEP192 distribution relative to other centrosomal components. The dimensions of labeled Cep192 structures match the dimensions of outer core layers observed in electron microscopic images .

• Immunofluorescence with phospho-specific markers: Combining CEP192 detection with markers of mitotic progression (such as phosphorylated Aurora-A) allows correlation of CEP192 dynamics with specific cell cycle events .

For optimal results, researchers should consider:

• Using knock-in cell lines to avoid overexpression artifacts

• Employing high-resolution microscopy techniques

• Including multiple centrosomal markers for contextual analysis

• Implementing cell cycle synchronization methods for stage-specific examination

What are the optimal fixation and staining protocols for CEP192 immunofluorescence?

Effective immunofluorescence detection of CEP192 requires careful attention to sample preparation:

• Fixation method:

  • For optimal preservation of centrosomal structures, use 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature.

  • Alternative approach: methanol fixation (-20°C for 10 minutes) may better preserve some epitopes while removing cytoplasmic background.

  • For combined preservation of cytoskeletal and centrosomal structures, a pre-extraction protocol using 0.5% Triton X-100 in PHEM buffer prior to PFA fixation is recommended .

• Antibody selection and dilution:

  • For immunofluorescence applications, CEP192 antibodies are typically used at dilutions ranging from 1:50 to 1:200 .

  • When selecting an antibody, prioritize those validated specifically for immunofluorescence applications.

  • Consider the epitope location - antibodies targeting different regions of CEP192 may yield varying results.

• Signal amplification:

  • For enhanced detection of low-abundance epitopes, consider using fluorescent secondary antibodies with higher quantum yields.

  • Tyramide signal amplification (TSA) can be employed when working with challenging samples.

• Co-staining considerations:

  • When examining CEP192 in relation to other centrosomal proteins, carefully select compatible antibody combinations (host species, isotypes).

  • Include α-tubulin staining to visualize microtubule organization in relation to CEP192-labeled centrosomes.

These protocols should be optimized for specific experimental contexts and cell types to achieve optimal signal-to-noise ratios.

How can I quantitatively assess CEP192 levels and distribution in experimental samples?

Quantitative analysis of CEP192 provides valuable insights into centrosome biology and cell cycle regulation:

• Western blot quantification:

  • For comparing CEP192 protein levels across different conditions or cell types.

  • Loading controls should include both housekeeping proteins and another centrosomal protein with stable expression.

  • Densitometric analysis should be performed using software that allows background subtraction and normalization .

• Immunofluorescence intensity measurements:

  • When analyzing the effect of genetic manipulations (e.g., RNAi), mix experimental cells with control cells (e.g., GFP-α-tubulin expressing cells) to provide internal reference standards .

  • For CEP192 depletion studies, expect approximately 28.5% reduction in staining intensity (SD = 9.9%) based on previous RNAi experiments .

  • Use 3D confocal z-stacks to capture the entire centrosome volume for accurate intensity measurements.

• High-content screening approaches:

  • Automated image acquisition and analysis can be employed for large-scale studies.

  • Machine learning algorithms can be trained to identify centrosomes and quantify associated CEP192 signals.

  • This approach is particularly valuable for genetic or chemical screens targeting centrosome biology.

• Cell cycle-specific analysis:

  • Combine CEP192 staining with cell cycle markers (e.g., DAPI for DNA content, cyclin B1 for G2/M).

  • Sort cells by cell cycle stage for population-level analysis of CEP192 dynamics.

  • Remember that CEP192 expression peaks during G2/M phase , making this a critical period for analysis.

For all quantitative approaches, statistical analysis should account for both biological and technical variability.

What are common issues when working with CEP192 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with CEP192 antibodies:

• High background in immunofluorescence:

  • Optimize blocking conditions (try 5% BSA or 10% normal serum from secondary antibody host species)

  • Increase washing steps in duration and number

  • Consider using detergent (0.1-0.3% Triton X-100) in wash buffers

  • Try alternative fixation methods as epitope accessibility may be fixation-dependent

• Weak or absent centrosomal signal:

  • Pre-extraction with detergent before fixation can improve centrosome visibility

  • Ensure cells are properly permeabilized

  • Consider antigen retrieval methods for certain fixation protocols

  • Check if antibody recognizes post-translationally modified forms of CEP192

• Multiple bands in Western blot:

  • CEP192 can exist in multiple isoforms or undergo post-translational modifications

  • Verify expected molecular weight (~192 kDa)

  • Include positive control samples with known CEP192 expression

  • Consider using gradient gels for better resolution of high molecular weight proteins

• Inconsistent results across experiments:

  • Standardize cell culture conditions, as CEP192 expression varies with cell cycle

  • Consider cell synchronization protocols for more consistent results

  • Establish rigorous quality control for antibody lots

  • Include internal controls in each experiment

How can CEP192 antibodies be utilized to study centrosome abnormalities in disease models?

CEP192 antibodies offer valuable insights into centrosome biology in disease contexts:

• Cancer research applications:

  • Centrosome amplification is a hallmark of many cancers

  • CEP192 antibodies can assess centrosome number, size, and maturation status in tumor samples

  • Quantitative analysis of CEP192 levels may correlate with tumor aggressiveness or treatment response

  • The dysregulation of CEP192 has been linked to abnormal cell division and genomic instability , both relevant to cancer biology

• Neurodevelopmental disorder studies:

  • Centrosome dysfunction is implicated in microcephaly and other neurodevelopmental conditions

  • CEP192 antibodies can examine centrosome structure in patient-derived cells or model organisms

  • Co-staining with other centrosomal markers can reveal specific defects in centrosome organization

• Ciliopathy research:

  • As centrosomes serve as basal bodies for cilia formation, CEP192 detection can provide insights into ciliopathy mechanisms

  • Combined staining for CEP192 and ciliary markers allows assessment of centriole-to-basal body transition

• Drug discovery applications:

  • CEP192 antibodies can be employed in high-content screens for compounds affecting centrosome structure or function

  • Evaluation of centrosome integrity following drug treatment provides mechanistic insights

  • Quantitative analysis of CEP192 localization serves as a readout for centrosome-targeting therapies

These applications benefit from combining CEP192 detection with other centrosomal markers to provide comprehensive insights into centrosome structure and function in disease contexts.

What emerging technologies might enhance our understanding of CEP192 biology?

Several cutting-edge technologies hold promise for advancing CEP192 research:

• Super-resolution microscopy beyond expansion microscopy:

  • STORM, PALM, and STED microscopy provide nanoscale resolution without physical sample expansion

  • These approaches could further resolve the spatial organization of CEP192 relative to other centrosomal components

  • Live-cell super-resolution imaging could capture dynamic aspects of CEP192 function

• Proximity proteomics beyond BioID:

  • Newer proximity labeling approaches like TurboID offer faster labeling kinetics

  • APEX2-based proximity labeling provides temporal resolution for studying dynamic interactions

  • These methods could uncover transient or cell cycle-specific CEP192 interaction partners

• Cryo-electron tomography:

  • This technique could provide structural insights into CEP192 organization within intact centrosomes

  • Combined with immunogold labeling, it could precisely localize CEP192 within the 3D ultrastructure of centrosomes

• CRISPR-based genomic tagging:

  • Endogenous tagging of CEP192 with split fluorescent proteins could enable visualization of specific protein-protein interactions in live cells

  • Degron tagging systems would allow rapid, conditional depletion of CEP192 for acute functional studies

  • Base editing approaches could introduce specific point mutations to study structure-function relationships

• Single-cell analysis technologies:

  • Single-cell transcriptomics combined with spatial information could reveal cell-specific regulation of CEP192

  • Mass cytometry with CEP192 antibodies could profile centrosome status across heterogeneous cell populations

These emerging technologies promise to deepen our understanding of CEP192's role in centrosome biology and cellular function.

What knowledge gaps remain in our understanding of CEP192 function?

Despite significant advances, several important questions about CEP192 remain unanswered:

• Structural organization:

  • How does CEP192 oligomerize to create a scaffold for other centrosomal proteins?

  • What is the detailed structure of CEP192 and how does it change during centrosome maturation?

  • How does CEP192 simultaneously interact with multiple binding partners?

• Regulatory mechanisms:

  • What controls CEP192 expression peaking during G2/M phase?

  • Which post-translational modifications regulate CEP192 function?

  • How is CEP192 turnover regulated at the centrosome?

• Functional redundancy:

  • To what extent can other centrosomal scaffolds compensate for CEP192 deficiency?

  • Are there tissue-specific or context-dependent functions of CEP192?

  • How does CEP192 function differ between cycling and quiescent cells?

• Evolutionary conservation:

  • How do CEP192 functions compare across different model organisms?

  • What aspects of CEP192 regulation are evolutionarily conserved?

  • Did CEP192 acquire new functions during the evolution of complex organisms?

• Disease relevance:

  • Are CEP192 mutations or dysregulation directly linked to human diseases?

  • Could CEP192 serve as a therapeutic target in cancers with centrosome amplification?

  • Does CEP192 play a role in age-related centrosome dysfunction?