Recombinant Mouse CAP-Gly domain-containing linker protein 3 (Clip3)

What is CAP-Gly domain-containing linker protein 3 (CLIP3) and what cellular functions does it serve?

CLIP3, also known as CLIPR-59, is a member of the cytoplasmic linker protein 170 family. This protein contains specialized cytoskeleton-associated protein glycine-rich (CAP-Gly) domains that mediate interactions between microtubules and cellular organelles . CLIP3 plays multiple critical roles in cellular function:

• Functions as a cytoplasmic linker protein involved in TGN-endosome dynamics

• Plays a role in T cell apoptosis by facilitating the association between tubulin and lipid raft ganglioside GD3

• Acts as a scaffold protein that mediates membrane localization of phosphorylated protein kinase B (AKT)

• Modulates the cellular compartmentalization of AKT kinase family

• Promotes cell membrane localization of glucose transporters, contributing to glucose transport regulation in adipocytes and potentially in neuronal cells

CLIP3 is primarily expressed in the human brain but has functional roles across multiple tissues . Its gene is conserved across numerous species, including human, mouse, rat, cat, horse, rabbit, cow, sheep, and naked mole-rat .

How is CLIP3 gene expression regulated at the transcriptional level?

Transcriptional regulation of CLIP3 involves several key mechanisms:

• The promoter region of CLIP3, particularly the 300 bp upstream of the transcription start site, contains important regulatory elements

• Evidence suggests Nuclear Respiratory Factor 1 (NRF1) may be involved in CLIP3 transcriptional regulation, as demonstrated through luciferase reporter gene assays

• The Speedy/RINGO cell cycle regulator family member A (Spy1) appears to negatively regulate CLIP3 expression, with an inverse correlation between their expression levels observed in glioblastoma tissues

• NFAT (Nuclear Factor of Activated T-cells) transcription factors may also participate in CLIP3 regulation, as indicated by the use of pGL3-NFAT luciferase vectors in reporter assays

For researchers investigating CLIP3 transcriptional regulation, it is recommended to analyze both the core promoter region and potential enhancer elements using techniques such as chromatin immunoprecipitation (ChIP), reporter gene assays, and CRISPR-based transcriptional modulation approaches.

What is known about CLIP3 protein structure and its functional domains?

While the search results don't provide complete structural information, we can infer CLIP3's key structural elements:

• CAP-Gly domains: These specialized regions enable microtubule binding and are characteristic of the cytoplasmic linker protein family

• Protein-protein interaction domains: Enable CLIP3 to function as a scaffold protein, particularly for AKT kinase family members

• Membrane-association regions: Allow CLIP3 to interact with cellular membranes and participate in vesicle trafficking

The multi-domain structure of CLIP3 facilitates its diverse cellular functions, from cytoskeletal organization to vesicular transport and metabolic regulation. Researchers should consider these domains when designing truncation or mutation experiments to dissect domain-specific functions.

What are the optimal methods for expressing and purifying recombinant mouse CLIP3 protein?

Multiple expression systems have been successfully employed for producing recombinant mouse CLIP3, each with distinct advantages:

Optimal Storage and Handling Recommendations:

• Store at -20°C for routine use, or -80°C for extended storage

• Avoid repeated freeze-thaw cycles; working aliquots can be stored at 4°C for up to one week

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

• Add 5-50% glycerol (final concentration) for long-term storage

Stability Considerations:

• Liquid form shelf life: approximately 6 months at -20°C/-80°C

• Lyophilized form shelf life: approximately 12 months at -20°C/-80°C

When selecting an expression system, consider your experimental requirements. The yeast system may be sufficient for basic binding studies, while the baculovirus system may be preferable for functional assays where post-translational modifications are critical.

What considerations should be made when designing CRISPR-Cas9 experiments targeting CLIP3?

When designing CRISPR-Cas9 experiments for CLIP3 gene editing, researchers should consider:

Guide RNA Design Principles:

• Use established design tools, such as those developed by Feng Zhang's laboratory at the Broad Institute

• Select guide RNAs that efficiently target CLIP3 while minimizing off-target effects

• Order at least two gRNA constructs per target to increase success probability

Vector Selection Criteria:

• Ensure vectors contain all necessary elements: U6 promoter, spacer (target) sequence, gRNA scaffold, and terminator

• Consider vectors with appropriate selection markers for your experimental system

Experimental Design Recommendations:

• Verify gRNA sequences against your specific target gene sequence before ordering

• Consider which exons or splice variants to target based on your research questions

• Validate editing efficiency using sequencing, T7 endonuclease assays, or similar methods

• Confirm protein knockdown via Western blot with validated antibodies

For CLIP3 specifically, targeting conserved regions of functional importance (such as the CAP-Gly domains) may yield more dramatic phenotypes than targeting other regions of the protein.

What antibodies and detection methods are most reliable for studying CLIP3 in mouse models?

Based on the available information, the following approaches are recommended:

Validated Antibodies:

• Rabbit Recombinant Monoclonal CLIPR-59 antibody [EPR28002-60] has been validated for Western blot and immunoprecipitation applications with mouse, rat, and human samples

Recommended Applications and Dilutions:

• Western blotting: 1/1000 dilution

• Immunoprecipitation: 1/30 dilution (2μg in 0.35mg lysates)

Control Recommendations:

• Include isotype controls (e.g., Rabbit IgG monoclonal [EPR25A])

• Use appropriate positive control tissues (brain tissue lysate is particularly suitable)

• Include CLIP3 knockdown/knockout samples as negative controls

Complementary Detection Methods:

• qRT-PCR for mRNA quantification, normalizing to GAPDH using the 2^-ΔΔCt method

• Immunofluorescence microscopy for localization studies

• Proximity ligation assays for detecting protein-protein interactions in situ

When working with novel antibodies, validation is critical. Cross-validate findings using multiple detection methods and include appropriate controls to ensure specificity.

How does CLIP3 influence glucose transport and metabolism?

CLIP3 plays a significant role in glucose transport across multiple cell types, with important implications for metabolism:

Mechanistically, CLIP3 appears to function as a scaffold that facilitates the association of glucose transporters with trafficking machinery . This involves:

• AKT kinase family compartmentalization

• Dynamic regulation of glucose transporter translocation to the plasma membrane

• Coordination with other trafficking proteins like Rab11a, which is involved in recycling endosome formation

The relationship between CLIP3 and glucose metabolism is particularly significant in cancer, where downregulation of CLIP3 contributes to the Warburg effect (preferential use of glycolysis even in the presence of oxygen) .

What is the significance of the Spy1-CLIP3 axis in cellular function and disease?

The Spy1-CLIP3 axis represents an important regulatory mechanism with significant implications for cell cycle control, apoptosis, and metabolism:

Spy1 Functions:

• Regulates cell division by directly binding to and activating cyclin-dependent kinases (CDKs)

• Activates CDKs independently of phosphorylation, bypassing normal cell cycle checkpoints

• Is upregulated in glioblastoma compared to lower-grade gliomas

Spy1-CLIP3 Interaction:

• Negative correlation between Spy1 and CLIP3 expression observed in glioblastoma tissues

• This interaction confers resistance to TNF-α-induced apoptosis

• May enable glioblastoma stem cells to escape radiation-induced cell death

Downstream Effects:

• Metabolic reprogramming: Favors glycolysis over oxidative phosphorylation

• Enhanced stemness: Contributes to cancer stem cell maintenance

• Therapeutic resistance: Particularly to radiation therapy in glioblastoma

Understanding this axis provides potential therapeutic opportunities. For instance, disrupting the Spy1-mediated suppression of CLIP3 or directly enhancing CLIP3 function (as with glimepiride) could sensitize resistant cancer cells to treatment .

How does CLIP3 participate in microtubule dynamics and vesicle trafficking?

CLIP3's role in microtubule dynamics and vesicle trafficking stems from its structural and functional properties:

Microtubule Interactions:

• The CAP-Gly domains enable direct binding to microtubules

• This binding allows CLIP3 to mediate interactions between microtubules and cellular organelles

• May influence microtubule stability and organization

Vesicle Trafficking Functions:

• Involved in TGN-endosome dynamics

• Participates in the trafficking of glucose transporters (GLUT3, GLUT4) to the plasma membrane

• May function in recycling endosome formation, potentially through interaction with Rab11a

Methodological Approaches to Study These Functions:

• Live-cell imaging using fluorescently tagged CLIP3 and relevant markers (e.g., Rab11a-OFP for recycling endosomes)

• Co-immunoprecipitation to identify trafficking machinery components that interact with CLIP3

• Subcellular fractionation to determine CLIP3 distribution across cellular compartments

These functions position CLIP3 as an important coordinator of cellular architecture and vesicular transport, with implications for numerous cellular processes including metabolism and signal transduction.

What is the evidence for CLIP3's role in glioblastoma progression and radioresistance?

Substantial evidence supports CLIP3's involvement in glioblastoma (GBM) biology:

The mechanistic model suggests that radioresistant GBM cells, particularly glioblastoma stem-like cells (GSCs), downregulate CLIP3, which leads to:

• Enhanced GLUT3 trafficking to the plasma membrane

• Increased glucose uptake and glycolytic metabolism

• Metabolic adaptation that supports survival following radiation therapy

These findings position CLIP3 as a tumor suppressor in GBM, with its downregulation contributing to therapeutic resistance and disease progression.

How might pharmacological modulation of CLIP3 function impact cancer treatment outcomes?

Pharmacological targeting of CLIP3 represents a promising therapeutic strategy:

Glimepiride as a CLIP3-Targeting Agent:

• Glimepiride, an FDA-approved medication for type 2 diabetes, restores CLIP3 expression in GBM cells

• This restoration disrupts GSCs maintenance and suppresses glycolytic activity

• When combined with radiotherapy, glimepiride significantly reduced GBM growth and improved survival in preclinical models

Mechanistic Basis for Therapeutic Efficacy:

• Restored CLIP3 function normalizes glucose transporter trafficking

• Reverses the metabolic shift toward glycolysis seen in radioresistant cancer cells

• Reduces stemness characteristics, making cells more susceptible to radiation therapy

Advantages of Drug Repositioning Approach:

• Uses an existing FDA-approved medication with established safety profile

• May accelerate clinical translation compared to novel compound development

• Targets a mechanism distinct from current GBM treatments, which primarily focus on cell proliferation

This approach demonstrates how understanding the fundamental biology of CLIP3 can lead to novel therapeutic strategies through targeting previously unrecognized cancer vulnerabilities.

What animal models and experimental systems are most appropriate for CLIP3 research?

Several model systems have been successfully employed in CLIP3 research:

In Vivo Models:

• GBM orthotopic xenograft mouse model: Used to demonstrate the efficacy of combined radiotherapy and glimepiride treatment

• Genetic mouse models: While not explicitly mentioned in the search results, could be developed for tissue-specific CLIP3 modulation

In Vitro Systems:

• Primary GBM cell cultures: Used to study CLIP3's role in radioresistance

• Cell lines expressing fluorescently tagged CLIP3 or interacting proteins (e.g., GLUT3-GFP, Rab11a-OFP): Enable dynamic trafficking studies

Model Selection Considerations:

• Research question specificity: Match the model to the aspect of CLIP3 function being investigated

• Translational potential: Consider models that best recapitulate human disease when studying pathological roles

• Technical requirements: For trafficking studies, models amenable to live-cell imaging are preferred

When designing studies, researchers should consider whether genetically modified animals (e.g., CLIP3 knockout or overexpression models) or wild-type animals with pharmacological modulation of CLIP3 function would best address their specific research questions.

What emerging technologies could enhance our understanding of CLIP3 dynamics in living cells?

Several cutting-edge technologies offer promising approaches for studying CLIP3:

Advanced Imaging Techniques:

• Live-cell confocal microscopy in environmentally controlled chambers (as used for GLUT3-GFP tracking)

• Super-resolution microscopy to visualize CLIP3 distribution at nanometer resolution

• FRAP (Fluorescence Recovery After Photobleaching) to assess CLIP3 mobility and binding dynamics

• Photoactivatable or photoconvertible tags to track specific CLIP3 subpopulations

Proximity Labeling Methods:

• BioID, TurboID or APEX2 systems to map the CLIP3 proximal proteome in living cells

• Allows identification of transient or weak interactions often missed by traditional co-immunoprecipitation

CRISPR-Based Technologies:

• CRISPR activation/inhibition systems for precise temporal control of CLIP3 expression

• CRISPR-based endogenous tagging to avoid overexpression artifacts

• Base editing or prime editing for precise introduction of specific CLIP3 mutations

Single-Cell Approaches:

• Single-cell RNA-seq to understand CLIP3 expression heterogeneity

• Single-cell metabolomics to link CLIP3 status to cellular metabolic profiles

These technologies would enable more dynamic, spatially resolved information about CLIP3 function in physiological and pathological contexts.

What are the critical controls needed when investigating CLIP3-protein interactions?

Robust controls are essential when studying CLIP3 interactions:

For Co-Immunoprecipitation Experiments:

• Negative controls:

  • Isotype control antibody (e.g., Rabbit IgG monoclonal [EPR25A])

  • Lysates from cells with CLIP3 knockdown

  • Non-specific binding controls (e.g., protein A/G beads alone)

• Positive controls:

  • Known interaction partners (e.g., Spy1)

  • Total lysate input samples (typically 5-10% of IP amount)

For Binding Assays:

• Domain deletion/mutation variants to map interaction interfaces

• Competitive binding assays with purified components

• Reciprocal pulldowns to confirm directionality of interactions

For In Vivo Interaction Studies:

• Proximity ligation assays with appropriate antibody controls

• FRET/BRET approaches with non-interacting protein pairs as negative controls

• Cross-linking prior to lysis to capture transient interactions

When reporting interaction studies, researchers should clearly describe all controls used and their results to enable proper interpretation of the findings.

How can researchers distinguish between direct and indirect effects when studying CLIP3 function?

Distinguishing direct from indirect effects is a common challenge in CLIP3 research:

Experimental Approaches:

• Acute vs. chronic manipulation: Acute depletion or activation (e.g., using degron systems) can help identify direct effects before compensatory mechanisms engage

• Rescue experiments: Re-expression of wild-type CLIP3 in knockout cells should reverse direct effects

• Domain-specific mutations: Targeted mutations affecting specific functions can help dissect direct roles

• In vitro reconstitution: Purified components in cell-free systems can demonstrate direct biochemical activities

For Trafficking Studies:

• Direct microscopic observation of CLIP3 with cargo proteins (e.g., GLUT3-GFP)

• Careful temporal analysis to establish order of events

• Use of cargo mutants that cannot bind CLIP3 to distinguish scaffold vs. active roles

For Signaling Pathway Analysis:

• Phosphoproteomic analysis with short time-courses after CLIP3 modulation

• Correlation of CLIP3 localization with signaling events

• Use of specific pathway inhibitors to delineate where CLIP3 acts within a cascade

These approaches, used in combination, can help establish causality and distinguish CLIP3's direct functions from secondary consequences of its manipulation.

What are common technical challenges when working with recombinant CLIP3 protein and how can they be addressed?

Several technical issues may arise when working with recombinant CLIP3:

Solubility and Stability Issues:

• Challenge: Recombinant CLIP3 may aggregate or show reduced stability

• Solutions:

  • Include glycerol (5-50%) in storage buffer

  • Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

  • Consider testing different buffer compositions to optimize stability

Expression Challenges:

• Challenge: Obtaining full-length, properly folded protein

• Solutions:

  • Compare expression systems (yeast vs. baculovirus)

  • Consider expressing functional domains separately if full-length protein is problematic

  • Optimize expression conditions (temperature, induction time, etc.)

Functional Activity Verification:

• Challenge: Confirming that recombinant CLIP3 retains native function

• Solutions:

  • Develop binding assays for known interaction partners

  • Verify microtubule binding activity using in vitro assays

  • Compare activity of proteins produced in different expression systems

Storage and Reconstitution:

• Challenge: Maintaining activity during storage

• Solutions:

  • Store at -20°C for routine use, or -80°C for extended storage

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Consider flash-freezing small aliquots to minimize freeze-thaw cycles

Addressing these challenges requires systematic optimization and characterization of your specific CLIP3 preparation.

What strategies can minimize off-target effects when modulating CLIP3 expression in experimental models?

Minimizing off-target effects is crucial for reliable CLIP3 research:

For RNAi-Based Approaches:

• Use multiple siRNA sequences targeting different regions of CLIP3 mRNA

• Include non-targeting control siRNAs

• Validate knockdown efficiency at both mRNA (qRT-PCR) and protein (Western blot) levels

• Perform rescue experiments with RNAi-resistant CLIP3 constructs

For CRISPR-Based Approaches:

• Use guide RNAs designed by established algorithms to minimize off-target effects

• Employ multiple guide RNAs targeting different regions

• Consider using nickase variants of Cas9 for increased specificity

• Perform whole-genome or targeted sequencing to identify potential off-target modifications

For Pharmacological Manipulation:

• Use multiple chemically distinct compounds when available

• Include appropriate vehicle controls

• Establish clear dose-response relationships

• Verify target engagement using cellular or biochemical assays

• Consider genetic approaches to validate pharmacological findings

For Overexpression Studies:

• Use inducible expression systems to control expression levels

• Include appropriate empty vector controls

• Consider endogenous tagging approaches when possible

• Verify that overexpression doesn't cause protein mislocalization or aggregation

These approaches help ensure that observed phenotypes can be confidently attributed to specific modulation of CLIP3 rather than experimental artifacts.

How can researchers effectively study CLIP3 in brain tissues, considering its predominant expression in this organ?

Studying CLIP3 in brain tissues presents unique challenges:

Tissue Processing and Preservation:

• Fresh tissue is optimal for protein and RNA analyses

• For fixed tissues, consider post-fixation variables that might affect epitope recognition

• Use brain region-specific approaches, as CLIP3 expression may vary across regions

Immunohistochemical Detection:

• Validate antibodies specifically for brain tissue applications

• Include positive controls (wild-type brain) and negative controls (CLIP3 knockout tissue if available)

• Consider antigen retrieval methods to enhance detection in fixed tissues

Cell Type-Specific Analysis:

• Use co-labeling with cell type-specific markers to identify CLIP3 expression patterns

• Consider single-cell approaches to address cellular heterogeneity

• For in vitro studies, use appropriate neural cell models (primary cultures, differentiated stem cells)

Functional Studies in Brain Context:

• Consider stereotactic injection of viral vectors for region-specific CLIP3 modulation

• Use brain-penetrant compounds (like glimepiride) for pharmacological studies

• Employ slice cultures for ex vivo manipulation and imaging

These specialized approaches can help overcome the challenges inherent in studying brain-expressed proteins like CLIP3, enabling more accurate and physiologically relevant characterization of its functions in the central nervous system.