Recombinant Xenopus tropicalis CLIP-associating protein 1 (clasp1), partial

What is CLASP1 and what are its primary functions in Xenopus tropicalis?

CLASP1 (Cytoplasmic Linker Associated Protein 1) belongs to a class of microtubule plus end-binding proteins (+TIPs) that contribute to microtubule dynamics and organization during various cellular processes, particularly mitosis. In Xenopus tropicalis, as in other vertebrates, CLASP proteins regulate microtubule polymerization, stabilization, and spindle assembly. CLASP1 contains multiple microtubule-binding domains, including TOG (Tumor Over-expressed Gene) domains and a Ser-x-Ile-Pro (SxIP) motif that interacts with end-binding protein 1 (EB1) . The protein plays essential roles in mitotic spindle assembly and function, with its depletion leading to dramatic spindle phenotypes .

Why is Xenopus tropicalis used as a model system for CLASP1 studies?

Xenopus tropicalis offers several advantages as a model organism for studying CLASP1 and other proteins:

• It possesses a diploid genome that is highly conserved between frogs and humans, making the identification of orthologous genes more straightforward than in models with less conservation or duplicated genomes .

• Its genome has high synteny with the human genome, facilitating comparative genomic analyses .

• Xenopus egg extracts provide a powerful biochemical system for studying mitotic spindle assembly and microtubule dynamics in vitro .

• The model organism database Xenbase provides user-friendly access to the annotated reference genome with excellent tools for genetic analysis .

• X. tropicalis can be maintained at significantly lower costs than rodent models, with a single pair capable of producing over 4,000 embryos in a day .

• CRISPR/Cas9 mutagenesis is well-established and cost-effective in X. tropicalis, enabling high-throughput functional studies .

What is the domain structure of X. tropicalis CLASP1 and how does it compare to human CLASP1?

X. tropicalis CLASP1 shares a similar domain architecture with human CLASP1, featuring:

• Multiple TOG domains in the N-terminal region that make the strongest contribution to microtubule polymerization and bundling .

• A SxIP motif that mediates interaction with EB1 and is required for plus-end tracking .

• A C-terminal coiled-coil domain that mediates dimerization and association with other factors, including kinetochore motor centromere protein E (CENP-E) and the chromokinesin Xkid .

The high conservation between X. tropicalis and human proteins means that findings in the frog model are often applicable to understanding human protein function. This conservation also facilitates the use of antibodies and other reagents developed for human or X. laevis proteins in X. tropicalis experiments .

How can I express and purify recombinant X. tropicalis CLASP1?

For the expression and purification of recombinant X. tropicalis CLASP1:

• Vector selection: Choose an expression vector appropriate for your experimental needs, incorporating suitable affinity tags (His, GST, or MBP) to facilitate purification.

• Expression system: Based on published approaches with human CLASP1, bacterial expression systems can be used for individual domains, while full-length protein or larger fragments may require insect cell or mammalian expression systems due to size and potential post-translational modifications.

• Purification strategy:

  • Perform affinity chromatography using the incorporated tag

  • Follow with size-exclusion chromatography to improve purity

  • Consider ion-exchange chromatography as an additional purification step

  • Verify protein integrity using SDS-PAGE and Western blotting

• Quality control: Assess microtubule-binding activity using in vitro microtubule co-sedimentation assays to confirm functionality of the purified protein.

How do the multiple domains of CLASP1 contribute to microtubule dynamics in Xenopus egg extracts?

Research using human CLASP1 truncation mutants in Xenopus egg extracts has revealed distinct contributions of different domains to microtubule dynamics:

• N-terminal TOG domains: The two N-terminal TOG domains make the strongest contribution to microtubule polymerization and bundling . A third TOG domain further contributes to CLASP activity .

• SxIP motif: This motif is required for plus-end tracking through interaction with EB1 . When this interaction is disrupted, CLASP1 fails to properly localize to microtubule plus ends.

• C-terminal coiled-coil domain: This domain mediates dimerization and association with multiple binding partners, including CENP-E and Xkid . Deletion of this domain causes aberrant microtubule polymerization and dramatic spindle phenotypes, even when small amounts of protein are added to egg extracts . This indicates that proper localization of CLASP activity is essential for controlling microtubule polymerization during mitosis.

Full-length CLASP1 is required to rescue spindle assembly in Xenopus egg extracts depleted of endogenous CLASP, demonstrating that all domains must work together for proper function .

What are the differences in microtubule dynamics between X. tropicalis and X. laevis, and how might this impact CLASP1 functional studies?

Comparative studies between X. tropicalis and X. laevis egg extracts have revealed significant differences in microtubule dynamics that could impact CLASP1 functional studies:

Key differences include:

• Microtubules polymerize significantly slower in X. tropicalis extracts compared to X. laevis (14.7 μm/min vs. 18.5 μm/min, p=0.013) .

• X. tropicalis spindles are approximately 30% shorter than X. laevis spindles .

• Mixing experiments have revealed a dynamic, dose-dependent regulation of spindle size by cytoplasmic factors .

These differences suggest that CLASP1 may function in a slightly different microtubule dynamic environment in X. tropicalis compared to X. laevis, potentially affecting experimental outcomes when studying its function.

How can CRISPR/Cas9 genome editing be optimized for studying CLASP1 function in X. tropicalis?

CRISPR/Cas9 genome editing in X. tropicalis provides a powerful approach for studying CLASP1 function in vivo. Optimization strategies include:

• Guide RNA design:

  • Target conserved functional domains of CLASP1 for maximum disruption

  • Design sgRNAs with minimal off-target effects using Xenbase tools

  • Consider targeting early exons to ensure frameshift mutations affect all protein isoforms

• Delivery method:

  • Inject ribonucleoprotein complexes (pre-assembled Cas9 protein and sgRNA) for high efficiency and reduced toxicity

  • For tissue-specific studies, utilize the developmental fate map to target injections to specific blastomeres

  • For unilateral mutants, inject one of the two cells at 2-cell stage to create an internal control within the same animal

• Validation strategies:

  • Perform T7 endonuclease I assay or direct sequencing to confirm mutations

  • Verify reduced protein expression through Western blot or immunofluorescence

  • Assess functional consequences through phenotypic analysis

• Phenotypic analysis:

  • Examine spindle morphology and microtubule dynamics in affected tissues

  • Assess cell division defects and developmental abnormalities

  • Compare phenotypes to those observed after CLASP1 depletion in egg extracts

The high tolerance of X. tropicalis embryos to injected ribonucleoprotein complexes makes this approach particularly suitable for studying CLASP1 function .

What are the differences between recombinant full-length and partial CLASP1 proteins in functional assays?

Research comparing full-length and partial CLASP1 proteins has revealed significant functional differences:

• Spindle assembly rescue: Only full-length recombinant CLASP1 can rescue spindle assembly in Xenopus egg extracts depleted of endogenous CLASP . This indicates that all domains are required for complete functionality in spindle assembly.

• Microtubule binding: N-terminal fragments containing TOG domains can bind microtubules but lack the ability to properly localize within the spindle due to the absence of the C-terminal domain .

• Dominant-negative effects: Truncated forms lacking the C-terminal domain can cause aberrant microtubule polymerization and dramatic spindle phenotypes, even when added at small amounts . This suggests these fragments may compete with endogenous CLASP for microtubule binding without proper localization or regulation.

• Protein interactions: Partial proteins lacking the C-terminal coiled-coil domain fail to dimerize and cannot interact with binding partners such as CENP-E and Xkid . This affects their localization and function within the cell.

When using recombinant partial CLASP1 proteins, researchers should carefully consider which domains are included and how their absence might affect experimental outcomes.

How can proteomics approaches be used to identify CLASP1 interacting partners in X. tropicalis?

Proteomics approaches for identifying CLASP1 interacting partners in X. tropicalis can be implemented through the following strategies:

• Immunoprecipitation coupled with mass spectrometry:

  • Express tagged recombinant CLASP1 in X. tropicalis egg extracts

  • Perform immunoprecipitation using antibodies against the tag or against CLASP1 directly

  • Analyze co-precipitated proteins by mass spectrometry

  • Cross-reference results with both X. tropicalis and X. laevis protein databases for improved identification

• Proximity labeling techniques:

  • Express CLASP1 fused to proximity labeling enzymes (BioID or APEX2)

  • Allow biotinylation of proximal proteins in egg extracts or embryos

  • Purify biotinylated proteins and identify them by mass spectrometry

• Domain-specific interaction studies:

  • Generate recombinant proteins representing specific CLASP1 domains

  • Use these as baits in pull-down assays followed by mass spectrometry

  • Compare interactomes of different domains to understand domain-specific functions

• Cross-species validation:

  • Compare interacting partners identified in X. tropicalis with those known from human or X. laevis studies

  • Focus on conserved interactions that are likely to be functionally significant

Research has shown that while X. tropicalis and X. laevis proteins are highly conserved, only about 45% of tryptic peptides have identical masses between orthologous proteins . Therefore, using species-specific databases for mass spectrometry analysis is important for optimal protein identification.

What are common challenges when working with recombinant X. tropicalis CLASP1 and how can they be addressed?

Common challenges when working with recombinant X. tropicalis CLASP1 include:

• Protein solubility and stability issues:

  • Challenge: Full-length CLASP1 is a large protein (~165 kDa) that may have solubility problems.

  • Solution: Express the protein at lower temperatures (16-18°C), use solubility-enhancing tags like MBP, and include stabilizing agents such as glycerol and reducing agents in buffers.

• Preserving microtubule-binding activity:

  • Challenge: Purified CLASP1 may lose microtubule-binding activity during purification.

  • Solution: Minimize freeze-thaw cycles, purify in the presence of ATP, and verify activity with microtubule co-sedimentation assays after purification.

• Species-specific antibody recognition:

  • Challenge: Antibodies raised against human or X. laevis CLASP1 may not recognize X. tropicalis protein with equal efficiency.

  • Solution: Test antibody cross-reactivity before experiments and consider generating X. tropicalis-specific antibodies if needed. Fortunately, many antibodies against X. laevis proteins give identical staining patterns in X. tropicalis extracts .

• Protein degradation during purification:

  • Challenge: CLASP1 may be susceptible to proteolytic degradation.

  • Solution: Include protease inhibitors throughout purification, work at 4°C, and minimize handling time.

How do spindle assembly assays differ when using X. tropicalis versus X. laevis egg extracts?

When performing spindle assembly assays, researchers should be aware of several key differences between X. tropicalis and X. laevis egg extracts:

• Spindle size differences:

  • X. tropicalis spindles are approximately 30% shorter than X. laevis spindles .

  • This difference is primarily due to cytoplasmic factors, as demonstrated by mixing experiments .

  • When designing experiments, account for these baseline differences in spindle morphology.

• Microtubule dynamics:

  • Microtubules polymerize slower in X. tropicalis extracts (14.7 μm/min) compared to X. laevis (18.5 μm/min) .

  • These differences may affect the kinetics of spindle assembly and the response to perturbations.

• Dynamic regulation:

  • Spindle size regulation is highly dynamic, as demonstrated by experiments where addition of X. laevis extract to preformed X. tropicalis spindles caused them to rapidly grow in length .

  • This dynamic regulation should be considered when interpreting results from mixed or complemented extracts.

• Chromatin contribution:

  • Chromatin mass does influence spindle length, with spindles assembled around X. tropicalis chromosomes being approximately 10% shorter in all extract conditions tested .

  • Consider the source of chromatin/DNA when comparing results across different experimental systems.

• Protein complexity:

  • X. laevis has a pseudotetraploid genome, which often results in multiple protein isoforms, whereas X. tropicalis typically has a single protein isoform .

  • This can simplify protein analysis in X. tropicalis and improve interpretation of depletion or overexpression experiments.

How can X. tropicalis CLASP1 studies contribute to understanding human disease mechanisms?

X. tropicalis CLASP1 studies can significantly contribute to understanding human disease mechanisms through several approaches:

• Modeling disease-associated variants:

  • CRISPR/Cas9 technology can be used to introduce specific mutations in X. tropicalis clasp1 that correspond to human disease variants .

  • The unilateral mutation approach in X. tropicalis (mutating one side of the embryo while leaving the other as an internal control) provides a powerful system for assessing the effects of these variants .

• High-throughput phenotypic screening:

  • The ability to generate thousands of mutant embryos per day makes X. tropicalis ideal for screening multiple disease-associated variants .

  • This approach can help identify which variants cause functional defects and prioritize them for more detailed mechanistic studies.

• Understanding genetic interactions:

  • X. tropicalis can be used to study how CLASP1 interacts with other disease-associated genes in the same pathway or network.

  • The speed and cost-effectiveness of X. tropicalis experiments allow for combinatorial approaches that would be prohibitively expensive in mammalian models .

• Developmental context:

  • X. tropicalis provides a vertebrate developmental context that allows researchers to study how CLASP1 dysfunction affects organ and tissue development.

  • This can provide insights into developmental disorders associated with microtubule dysfunction.

• Drug screening and therapeutic development:

  • The X. tropicalis system can be used to screen for compounds that rescue CLASP1 mutant phenotypes, potentially identifying therapeutic approaches for related human diseases.

The high conservation of CLASP1 function between X. tropicalis and humans makes this model system particularly valuable for translational research into diseases involving microtubule dysregulation.

What are emerging techniques for studying CLASP1 dynamics in live X. tropicalis embryos?

Several emerging techniques are enhancing our ability to study CLASP1 dynamics in live X. tropicalis embryos:

• Fluorescent protein fusions with genome editing:

  • CRISPR/Cas9-mediated homology-directed repair can be used to tag endogenous CLASP1 with fluorescent proteins.

  • This allows visualization of CLASP1 dynamics at physiological expression levels without overexpression artifacts.

• Optogenetic approaches:

  • Light-inducible protein interaction systems can be applied to control CLASP1 activity or localization with high spatiotemporal precision.

  • This allows researchers to perturb CLASP1 function in specific cells or tissues at defined developmental stages.

• Advanced microscopy techniques:

  • Light sheet microscopy is particularly suitable for X. tropicalis embryos, allowing long-term imaging with minimal phototoxicity.

  • Super-resolution microscopy techniques can provide nanoscale details of CLASP1 localization and dynamics at microtubule plus ends.

• Biosensors for microtubule dynamics:

  • Fluorescent biosensors can be used alongside labeled CLASP1 to simultaneously monitor microtubule dynamics and CLASP1 activity.

  • This correlative approach helps establish cause-effect relationships between CLASP1 localization and changes in microtubule behavior.

• Single-cell transcriptomics integration:

  • Combining live imaging of CLASP1 dynamics with subsequent single-cell RNA sequencing can link protein behavior to transcriptional responses.

  • Existing X. tropicalis single-cell transcriptomic atlases provide valuable reference data for these analyses .

These techniques leverage the experimental advantages of X. tropicalis, including its external development, optical transparency at early stages, and amenability to genetic manipulation.