SSX2IP Antibody

What is SSX2IP and what are its main cellular functions?

SSX2IP is a multifunctional protein that acts as a centrosome maturation factor by maintaining the integrity of the pericentriolar material and enabling proper microtubule nucleation at mitotic spindle poles . It plays essential roles in:

• Promoting centrosome maturation and maintenance during early vertebrate development

• Preserving centrosome integrity during rapid cleavage divisions

• Supporting proper mitotic spindle assembly in somatic cells

• Facilitating microtubule nucleation at spindle poles

• Contributing to ciliogenesis by recruiting components like the BBSome, CEP290, RAB8, and SSTR3 to cilia

• Participating in cell movement by localizing to the leading edge of moving cells

The protein accumulates at spindle poles in a Dynein-dependent manner and interacts with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1 .

What are the recommended applications for SSX2IP antibodies?

SSX2IP antibodies can be utilized in multiple experimental approaches:

When working with SSX2IP antibodies, researchers should note that optimal dilution should be experimentally determined for each application . For immunohistochemistry and immunocytochemistry experiments, Mouse-On-Mouse blocking reagents may be necessary to reduce high background signal .

How does SSX2IP localize during the cell cycle?

SSX2IP demonstrates dynamic localization patterns throughout the cell cycle:

• During interphase: Localizes to centriolar satellites and centrosomes

• At M-phase onset: Expression increases and accumulates at microtubule minus ends

• During mitosis: Concentrates at spindle poles in both oocytes and chromatin-induced, centrosome-free spindles

• In dividing blastomeres: Strongly associates with centrosomes

This localization is Dynein-dependent, as inhibition of this minus-end directed microtubule motor abolishes SSX2IP accumulation at spindle poles . For accurate visualization, researchers should use fixation methods that preserve centrosome structure and consider co-staining with markers like γ-tubulin to confirm centrosomal localization.

What protein interactions does SSX2IP participate in?

SSX2IP engages in multiple protein interactions that contribute to its cellular functions:

• γ-Tubulin Ring Complex (γ-TuRC): SSX2IP directly interacts with components of the γ-TuRC, including γ-tubulin, XGrip109/GCP3, and XGrip210/GCP6, facilitating their recruitment to centrosomes

• PCM-1: Associates with this centriolar satellite protein

• WRAP73: Forms a complex that regulates spindle anchoring at mitotic centrosomes

• Afadin and α-actinin: Connects the nectin-afadin and E-cadherin-catenin systems at adherens junctions

• Wtip: Physical association that may be essential for cell junction remodeling and morphogenetic processes during neurulation

Notably, the N-terminal domain of Wtip (WtipN) co-precipitates with SSX2IP more efficiently than full-length Wtip, suggesting domain-specific interactions .

What are the consequences of SSX2IP depletion on centrosome function?

Immunodepletion of SSX2IP from Xenopus laevis egg extracts reveals significant functional impairments in centrosome activity:

• Reduced γ-TuRC recruitment: Depletion results in decreased loading of γ-tubulin ring complex components (γ-tubulin, XGrip109/GCP3, XGrip210/GCP6) onto centrosomes

• Impaired microtubule nucleation: Centrosomes show diminished capacity to nucleate microtubules both in the absence and presence of RanGTP

• Spindle assembly defects: Most structures display half-spindle or aster-like morphology rather than proper bipolar spindles

• Chromosome segregation abnormalities: Visualized in live embryos using Histone2B-GFP, SSX2IP inhibition leads to defects in chromosome segregation

These defects can be significantly rescued by re-expression of GFP-SSX2IP, confirming the specificity of the depletion phenotype . Researchers investigating centrosome function should consider SSX2IP as a key factor in experimental designs focused on centrosome maturation and spindle assembly.

How can researchers distinguish between centrosomal and centriolar satellite pools of SSX2IP?

Differentiating between centrosomal and centriolar satellite pools of SSX2IP requires careful experimental design:

Methodological approach:

• Co-localization analysis: Perform triple immunofluorescence with antibodies against SSX2IP, a centrosome marker (γ-tubulin), and a centriolar satellite marker (PCM-1)

• Cold treatment: Brief exposure to cold temperatures (4°C) depolymerizes dynamic microtubules while preserving stable centrosomal microtubules, allowing better visualization of centriolar satellites

• Super-resolution microscopy: Techniques such as structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) provide sufficient resolution to distinguish between these proximal structures

• Biochemical fractionation: Differential centrifugation can separate centrosome-enriched fractions from centriolar satellite components for western blot analysis

• Domain mutant analysis: Expression of SSX2IP mutants lacking specific domains may show differential localization to centrosomes versus centriolar satellites

In medaka embryos, researchers have successfully used two different SSX2IP antibodies to confirm centrosomal association and co-localization with both γ-tubulin and PCM-1 at blastula stage 10-11 .

What experimental approaches can resolve contradictory findings about SSX2IP function?

When faced with contradictory findings about SSX2IP function, researchers should consider several experimental approaches:

• Model system differences: Compare SSX2IP function across different model systems, as observed differences between Xenopus laevis egg extracts and intact embryos may reveal context-dependent functions

• Temporal analysis: Investigate SSX2IP function at different cell cycle stages, as its role may vary throughout the cell cycle or developmental stages

• Functional domain mapping: Generate and test truncation or point mutants to identify which domains are responsible for specific functions:

  • Centrosome localization

  • γ-TuRC interaction

  • PCM-1 binding

  • Dynein-dependent transport

• Rescue experiments: Perform complementation with:

  • Species-specific orthologs to identify evolutionarily conserved functions

  • Domain mutants to pinpoint functional regions

  • GFP-tagged versus untagged proteins to rule out tag interference

• Combined approaches: Integrate in vitro biochemical assays with in vivo functional studies to build a comprehensive understanding

For example, in Xenopus laevis egg extracts, spindles assemble normally after SSX2IP immunodepletion in chromatin-driven spindle formation, but fail in bipolar spindle formation when using sperm nuclei . This contradiction was resolved by determining that SSX2IP primarily affects centrosome-dependent rather than chromatin-dependent spindle assembly pathways.

What is the relationship between SSX2IP and the γ-tubulin ring complex?

SSX2IP plays a crucial role in recruiting the γ-tubulin ring complex (γ-TuRC) to centrosomes, which is essential for microtubule nucleation and proper spindle assembly:

• Physical interaction: SSX2IP directly interacts with γ-TuRC components, including γ-tubulin, XGrip109/GCP3, and XGrip210/GCP6

• Recruitment function: Immunodepletion of SSX2IP from Xenopus laevis egg extracts impedes γ-TuRC loading onto centrosomes

• Functional consequence: Reduced γ-TuRC recruitment correlates with diminished microtubule nucleation capacity at centrosomes

• Rescue capability: Re-expression of GFP-SSX2IP restores both γ-TuRC recruitment and microtubule nucleation

• Phenocopy: Direct depletion of γ-TuRC (by 90%) from Xenopus egg extracts abolishes bipolar spindle formation and produces astral arrays or half spindles similar to those seen after SSX2IP depletion

To study this relationship, researchers can employ co-immunoprecipitation assays, proximity ligation assays (PLA), or fluorescence resonance energy transfer (FRET) approaches to characterize the molecular details of this interaction.

What are the technical considerations for using SSX2IP antibodies in developmental biology models?

When using SSX2IP antibodies in developmental biology models, researchers should consider:

• Species cross-reactivity: Verify antibody recognition of your specific model organism's SSX2IP ortholog. While human and Xenopus SSX2IP have been well-characterized, cross-reactivity with other species should be experimentally confirmed

• Developmental timing: SSX2IP expression may be regulated developmentally. In Xenopus, SSX2IP is expressed at M-phase onset , so timing sample collection appropriately is crucial

• Fixation methods: For embryonic tissues:

  • Paraformaldehyde (4%) for 20-30 minutes preserves structure while maintaining epitope accessibility

  • Methanol fixation may better preserve centrosomal epitopes but can disrupt membrane structures

• Alternative approaches: Consider using antisense morpholino oligonucleotides (MOs) for targeted inhibition of SSX2IP function, as successfully employed in medaka embryos

• Live imaging strategies: For monitoring SSX2IP dynamics during development, mRNA encoding fluorescently-tagged SSX2IP can be injected into early embryos, as demonstrated with GFP-SSX2IP mRNA in Xenopus egg extracts

• Validation controls: Include both positive controls (known SSX2IP-expressing tissues) and negative controls (morpholino-treated or CRISPR-edited embryos lacking SSX2IP)

Successful examples include visualization of centrosomal SSX2IP in medaka blastomeres using two different antibodies and monitoring chromosome segregation with Histone2B-GFP in SSX2IP-depleted embryos via digital scanned laser light-sheet fluorescence microscopy .

How does SSX2IP's association with Wtip influence cell junction remodeling?

The physical association between SSX2IP and Wtip appears to be important for cell junction remodeling and morphogenetic processes during neurulation :

• Binding specificity: The N-terminal domain of Wtip (WtipN) co-precipitates with SSX2IP more efficiently than full-length Wtip, suggesting domain-specific interactions

• Co-localization: When co-expressed in ectoderm cells, GFP-SSX2IP and RFP-WtipN form mixed cytoplasmic aggregates, indicating their physical association in vivo

• Developmental regulation: The interaction appears to be stage-specific, with different localization patterns observed at stages 10.5 and 12.5

• Functional implications: The association is suggested to be essential for cell junction remodeling and morphogenetic processes accompanying neurulation

To study this interaction, researchers can employ:

• Co-immunoprecipitation assays with tagged constructs (as demonstrated with GFP-hSSX2IP and Flag-WtipN or Flag-Wtip)

• Fluorescence microscopy to visualize co-localization in fixed or live tissues

• Functional assays to assess junction integrity after disrupting the interaction

• Domain mapping to identify the precise regions mediating the interaction

This emerging research area represents an important new direction for understanding SSX2IP's roles beyond centrosome maturation.

How can researchers troubleshoot non-specific binding when using SSX2IP antibodies?

When encountering non-specific binding with SSX2IP antibodies, researchers should implement these troubleshooting strategies:

• Optimizing antibody dilution: Experimentally determine the optimal antibody concentration for each application and tissue type

• Blocking improvements:

  • For mouse monoclonal antibodies on mouse tissues, use Mouse-On-Mouse blocking reagents

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to 1-2 hours at room temperature

• Validation strategies:

  • Use immunogen-affinity purified antibodies for higher specificity

  • Compare staining patterns with different antibodies targeting distinct epitopes of SSX2IP

  • Include SSX2IP-depleted or knockout samples as negative controls

• Sample preparation optimization:

  • Adjust fixation conditions to preserve epitope accessibility

  • For paraffin-embedded tissues, optimize antigen retrieval methods

  • Use fresh samples when possible, as storage can affect epitope integrity

• Detection system considerations:

  • For immunofluorescence, try directly conjugated antibodies (e.g., FITC-labeled)

  • Consider signal amplification systems for low-abundance targets

  • Optimize incubation times and temperatures

Implementing these strategies systematically can significantly improve specificity when working with SSX2IP antibodies.

What are the recommended controls for studying SSX2IP function in spindle assembly?

When investigating SSX2IP's role in spindle assembly, include these essential controls:

• Depletion controls:

  • Mock-depleted extracts using non-specific IgG

  • Complementation/rescue with GFP-SSX2IP mRNA to confirm specificity

  • Partial depletion to establish dose-dependency

• Comparison controls:

  • Parallel depletion of known spindle assembly factors (e.g., TPX2) to distinguish pathway-specific effects

  • γ-TuRC depletion to compare phenotypes with SSX2IP depletion

• Pathway isolation controls:

  • RanT24N addition to block chromatin-dependent spindle assembly, isolating centrosome-dependent effects

  • Dynein inhibition to verify dependency of SSX2IP localization on minus-end directed transport

• Visualization controls:

  • Co-staining for multiple markers (tubulin, chromatin, centrosome markers)

  • Time-lapse imaging to distinguish assembly defects from stability issues

• Quantification metrics:

  • Spindle morphology classification (bipolar, monopolar, aster-like)

  • Microtubule density measurements at centrosomes

  • Chromosome segregation error rates

In published studies, these controls have been effectively used to demonstrate that SSX2IP specifically affects centrosome-dependent rather than chromatin-dependent spindle assembly pathways .

What methodologies are recommended for studying SSX2IP's role in ciliogenesis?

To investigate SSX2IP's function in ciliogenesis, researchers should consider these methodological approaches:

• Cell culture models:

  • Serum starvation protocols to induce primary cilia formation

  • Cell types with prominent cilia (e.g., RPE-1, IMCD3 cells)

  • 3D culture systems to study cilia in a more physiological context

• Knockdown/knockout strategies:

  • siRNA or shRNA for transient/stable reduction

  • CRISPR-Cas9 genome editing for complete knockout

  • Inducible depletion systems to study temporal requirements

• Imaging approaches:

  • Immunofluorescence for cilia markers (acetylated tubulin, Arl13b)

  • Super-resolution microscopy to resolve cilia substructures

  • Live imaging with fluorescently-tagged SSX2IP to track dynamics

• Functional assays:

  • Ciliary trafficking assays (e.g., monitoring Smoothened translocation)

  • Measure cilia length, frequency, and morphology

  • Assess ciliary signaling pathways (Hedgehog, PDGF, etc.)

• Biochemical techniques:

  • Proximity labeling to identify ciliary interaction partners

  • Immunoprecipitation to confirm interactions with known ciliogenesis factors

  • Fractionation to isolate ciliary versus non-ciliary SSX2IP pools

SSX2IP is required for targeted recruitment of the BBSome, CEP290, RAB8, and SSTR3 to cilia , making these components important markers in functional studies.

What are emerging areas of research involving SSX2IP beyond centrosome function?

Several promising research areas are emerging around SSX2IP beyond its established centrosomal functions:

• Cell junction biology: The association between SSX2IP and Wtip suggests important roles in cell junction remodeling during morphogenetic processes

• Cell migration mechanisms: SSX2IP localizes to the leading edge of moving cells in response to PDGF and may promote cell movement through Rac signaling activation

• Developmental morphogenesis: SSX2IP's involvement in neurulation processes suggests broader developmental roles worthy of investigation

• Ciliopathy connections: Given its role in ciliogenesis and interactions with established ciliopathy proteins (BBSome, CEP290), SSX2IP may have unexplored connections to ciliopathies

• Cancer biology implications: The original identification of SSX2IP as an interactor of the synovial sarcoma-associated protein SSX2 suggests potential roles in cancer pathogenesis that remain underexplored

• Evolutionary conservation analysis: Comparative studies across species could reveal evolutionarily conserved versus specialized functions of SSX2IP

Each of these areas represents valuable opportunities for researchers to explore new facets of SSX2IP biology beyond its established centrosomal roles.

What technical advances might enhance SSX2IP antibody applications in research?

Future technical advances that could enhance SSX2IP antibody applications include:

• Domain-specific antibodies: Development of antibodies recognizing specific functional domains of SSX2IP would allow more precise analysis of its diverse cellular roles

• Phospho-specific antibodies: Antibodies detecting post-translational modifications could reveal regulatory mechanisms controlling SSX2IP function

• Super-resolution compatible probes: Optimized fluorescent antibodies for STORM, PALM, or expansion microscopy would improve visualization of SSX2IP in centrosomes and centriolar satellites

• Live-cell nanobodies: Small, genetically encoded antibody fragments that work in living cells could enable real-time tracking of endogenous SSX2IP

• Degradation-targeting antibody conjugates: Antibody-based targeted protein degradation tools would allow acute, reversible inactivation of SSX2IP

• Single-molecule analysis tools: Techniques for monitoring individual SSX2IP molecules could reveal dynamic behaviors currently masked in population studies

• Cryo-EM compatible probes: Antibody fragments suitable for cryo-electron microscopy would enable structural studies of SSX2IP complexes