Recombinant Mouse Chloride intracellular channel protein 3 (Clic3)

What is Recombinant Mouse Chloride intracellular channel protein 3 (Clic3)?

Recombinant Mouse Chloride intracellular channel protein 3 (Clic3) is a genetically engineered version of the native mouse Clic3 protein, which belongs to the chloride intracellular channel (CLIC) family. This protein is produced through biotechnological methods to enable controlled expression and purification for research applications. In its recombinant form, Clic3 maintains the functional characteristics of the native protein while providing researchers with a standardized reagent for experimental investigations. The protein is involved in various cellular processes, including cell growth, differentiation, and ion transport across cell membranes.

What are the primary biological functions of Clic3?

Clic3 participates in several crucial biological processes:

• Osteoblast Differentiation: Clic3 significantly enhances osteoblast differentiation and bone formation. Studies demonstrate that overexpression of Clic3 in human mesenchymal stem cells substantially increases mineralization, suggesting potential applications in treating bone disorders.

• Cancer Progression: Clic3 has been implicated in cancer cell invasion and metastasis mechanisms. For instance, in breast cancer, Clic3 regulates the trafficking of late endosomal MT1-MMP, which influences tumor invasion capabilities.

• Cellular Signaling and Ion Transport: As part of the CLIC family, Clic3 plays essential roles in maintaining membrane structure and facilitating ion transport, which underpins numerous cellular functions .

• Endosomal Trafficking: Clic3 is involved in endosomal trafficking processes, particularly those related to promoting invasive cell behavior .

How does Clic3 differ from other CLIC family proteins?

Clic3 exhibits distinct characteristics compared to other members of the CLIC family:

While all CLIC proteins share structural similarities, Clic3 specifically localizes in muscles, heart, lung, and kidney tissues, functioning both as a component of anion channels and as a channel regulator. Uniquely, Clic3 promotes invasive behavior in cells, distinguishing it from CLIC2, which inhibits MMP14 activity and metastasis .

What are the optimal storage and handling conditions for Recombinant Mouse Clic3?

For optimal storage and handling of Recombinant Mouse Clic3:

• Temperature Management: Store the main stock at -80°C for long-term preservation of protein stability and activity.

• Aliquoting Strategy: Upon receipt, divide the protein into single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise protein integrity and function.

• Working Storage: Maintain working aliquots at 4°C for up to one week to preserve activity while allowing convenient access for ongoing experiments.

• Freeze-Thaw Avoidance: Minimize freeze-thaw cycles as they can cause protein denaturation, aggregation, and activity loss. Each cycle potentially reduces the functional capacity of the protein.

• Buffer Compatibility: When designing experiments, consider buffer compositions that maintain Clic3's native conformation and functional properties.

How should researchers design reproducible experiments using Recombinant Mouse Clic3?

To ensure reproducible experiments with Recombinant Mouse Clic3:

• Strain Selection: Choose appropriate mouse strains for in vivo experiments carefully, recognizing that different strains exhibit variable phenotypic responses even to genetically identical treatments .

• Environmental Controls: Standardize housing conditions, including temperature, humidity, light cycles, and cage enrichment to minimize variability in mouse models .

• Technical Replication: Implement technical replicates to account for procedural variations, with a minimum of three replicates per experimental condition.

• Biological Replication: Design experiments with sufficient biological replicates to account for inter-individual variability, adhering to power analysis recommendations.

• Controls: Include both positive and negative controls in all experiments to validate assay functionality and establish baseline measurements.

• Experimental Blinding: Employ blinded analysis techniques to prevent unconscious bias in data collection and interpretation.

• Standardized Protocols: Develop and strictly adhere to standardized protocols for protein handling, assay conditions, and data analysis workflows.

• Consistency in Reagents: Maintain consistency in reagent sources, including recombinant protein lots, antibodies, and detection systems across experimental series .

How can Recombinant Mouse Clic3 be used to investigate osteoblast differentiation mechanisms?

Investigating osteoblast differentiation with Recombinant Mouse Clic3 requires sophisticated experimental approaches:

What methodological approaches should be used to study Clic3's role in cancer progression?

To investigate Clic3's role in cancer progression, researchers should implement these methodological approaches:

• Invasion Assays: Utilize transwell invasion chambers with extracellular matrix components to quantify the invasive capacity of cancer cells with manipulated Clic3 expression.

• Endosomal Trafficking Analysis: Employ live-cell imaging with fluorescently tagged endosomal markers to visualize and quantify Clic3's effects on endosomal dynamics, particularly focusing on late endosomal MT1-MMP trafficking.

• 3D Organoid Models: Develop three-dimensional tumor organoids that better recapitulate the in vivo tumor microenvironment to assess Clic3's influence on invasive morphology and behavior.

• Co-immunoprecipitation Studies: Identify Clic3's protein interaction partners in cancer cells, particularly focusing on matrix metalloproteinases and endosomal trafficking machinery.

• In Vivo Metastasis Models: Establish orthotopic tumor models with Clic3-manipulated cancer cell lines to evaluate effects on primary tumor growth, local invasion, and distant metastasis formation.

• Comparative Expression Analysis: Compare Clic3 expression levels across benign and malignant tumors of the same tissue origin to establish correlations with disease aggressiveness and patient outcomes.

How does Clic3 interact with matrix metalloproteinases in cancer progression?

The interaction between Clic3 and matrix metalloproteinases represents a critical mechanism in cancer progression:

• Molecular Interactions: While CLIC2 binds directly to MMP14 (MT1-MMP) and inhibits its activity, CLIC3 appears to modulate MMP activity through different mechanisms, potentially by controlling the trafficking of late endosomal MT1-MMP, which significantly influences tumor invasion capabilities .

• Endosomal Regulation: Clic3 regulates endosomal trafficking pathways that control the surface presentation and activity of MMPs, particularly MT1-MMP, which is crucial for extracellular matrix degradation during cancer invasion.

• Contrasting Effects: This represents an interesting functional divergence within the CLIC family, as CLIC3 appears to promote invasive behavior while CLIC2 inhibits MMP14 activity and potentially suppresses invasion. CLIC4, on the other hand, stimulates MMP14 activity .

• Methodological Assessment: To study these interactions, researchers should employ:

  • Co-localization studies using confocal microscopy

  • FRET (Fluorescence Resonance Energy Transfer) analysis to detect close molecular associations

  • Gelatin zymography to quantify MMP activity in the presence of varying Clic3 levels

  • Surface biotinylation assays to measure MMP surface presentation

What are the challenges in distinguishing membrane-bound versus soluble functions of Clic3?

Distinguishing between membrane-bound and soluble functions of Clic3 presents significant experimental challenges:

• Dual Localization: Like other CLIC family members, Clic3 exists in both soluble cytosolic forms and membrane-integrated configurations, with the transition between these states influenced by pH and redox conditions .

• Methodological Separation: Researchers must develop techniques to specifically isolate and study these distinct pools:

  • Ultracentrifugation to separate membrane fractions from cytosolic components

  • Selective permeabilization protocols to access specific cellular compartments

  • Construction of mutant Clic3 variants with altered membrane-integration capabilities

• Functional Assessment: Different experimental approaches are needed to evaluate:

  • Ion channel activity in membrane-integrated forms using electrophysiological methods

  • Protein-protein interactions in soluble forms through co-immunoprecipitation

  • Localization patterns via subcellular fractionation and immunofluorescence microscopy

• Integration-Transition Factors: Studies should address the environmental conditions that promote transition between soluble and membrane-bound states, including:

  • Redox status (oxidative conditions promote membrane integration)

  • pH changes (acidic conditions may favor channel formation)

  • Lipid composition of target membranes

  • Post-translational modifications affecting conformation

What are the significant differences between human CLIC3 and mouse Clic3 that researchers should consider?

Researchers working with mouse models must consider these important differences between human CLIC3 and mouse Clic3:

• Sequence Homology: While human and mouse CLIC3 share significant sequence homology, subtle structural differences may influence protein-protein interactions and functional capabilities.

• Expression Patterns: Tissue distribution patterns may vary between species, potentially affecting the translational relevance of mouse model findings to human conditions.

• Functional Conservation: Core functions appear conserved across species, including roles in endosomal trafficking and promotion of invasive behavior, but species-specific nuances may exist in regulatory mechanisms .

• Experimental Implications: When designing experiments, researchers should:

  • Validate key findings in both mouse and human cell systems

  • Consider using humanized mouse models for specific applications

  • Account for potential differences in binding partners and signaling networks

• Antibody Selection: Ensure antibodies used for detection have been validated for the appropriate species, as epitope differences can affect recognition efficiency.

How does our understanding of other CLIC family members inform research approaches for Clic3?

Insights from other CLIC family members provide valuable context for Clic3 research:

• Structural Insights: The crystal structures determined for other CLIC proteins, particularly CLIC1 and CLIC2, provide templates for modeling Clic3 structure and predicting functional domains .

• Functional Parallels: CLIC4's established roles in TGF-β signaling and angiogenesis suggest potential parallel functions for Clic3 that warrant investigation .

• Methodological Adaptation: Experimental approaches successful in characterizing other CLIC proteins can be adapted for Clic3, including:

  • Artificial membrane integration assays used for CLIC1 and CLIC2

  • Protein-protein interaction screens employed for CLIC4

  • Cellular localization studies developed for multiple CLIC family members

• Divergent Functions: The contrasting roles of different CLICs in cancer progression (CLIC2 as inhibitory versus CLIC3 and CLIC4 as promoting) highlight the importance of precise member-specific investigations .

• Absence of Mouse CLIC2: The notable absence of the CLIC2 gene in mice limits comparative studies between these family members in mouse models and underscores the unique evolutionary history of this protein family .

What are common pitfalls in experimental design when working with Recombinant Mouse Clic3?

Researchers should be aware of these common pitfalls when designing experiments with Recombinant Mouse Clic3:

• Protein Stability Issues: Recombinant Clic3 may exhibit structural changes or activity loss over time or through improper handling. Implement regular activity assays to verify protein functionality throughout experimental timelines.

• Expression System Artifacts: Different expression systems (bacterial, insect, mammalian) may produce Clic3 with varying post-translational modifications, potentially affecting activity. Document and maintain consistency in the expression system used.

• Tag Interference: His-tags or other fusion tags may interfere with protein function or interactions. When possible, compare tagged and cleaved versions of the protein to identify any tag-induced artifacts.

• Buffer Incompatibility: Some buffer components may affect Clic3's conformation or activity. Optimize buffer conditions through systematic testing of pH ranges and salt concentrations.

• Inconsistent Results Between Models: Findings from cell lines may not translate to primary cells or in vivo models. Design experiments with graduated complexity, validating key findings across multiple experimental systems .

• Inadequate Controls: Failure to include appropriate controls can lead to misinterpretation of results. Always include both positive controls (known Clic3 effects) and negative controls (unrelated proteins with similar structure) .

How can researchers distinguish between direct and indirect effects of Clic3 manipulation in experimental systems?

To differentiate between direct and indirect effects of Clic3 manipulation:

• Temporal Analysis: Implement time-course experiments to identify immediate versus delayed responses following Clic3 manipulation, as direct effects typically manifest more rapidly.

• Dose-Response Relationships: Establish dose-response curves for Clic3 effects, as direct interactions often show proportional relationships to concentration.

• Mutational Analysis: Generate function-specific mutants of Clic3 that selectively disrupt particular domains or activities to isolate specific functions.

• Reconstitution Experiments: In knockdown systems, reintroduce wild-type or mutant Clic3 to determine which phenotypes can be rescued, indicating direct functional relationships.

• Proximity Labeling: Employ BioID or APEX2 proximity labeling techniques to identify proteins that directly interact with Clic3 in living cells.

• Pharmacological Validation: Use specific inhibitors of suspected downstream pathways to determine if blocking these pathways prevents Clic3-induced effects, suggesting an indirect mechanism.

• In Vitro Reconstitution: Develop purified component systems to test direct biochemical activities of Clic3 in the absence of cellular complexity.