Recombinant Xenopus tropicalis Fidgetin-like protein 1 (fignl1), partial

What is Fidgetin-like protein 1 (FIGNL1) and what is its relationship to other microtubule-severing proteins?

FIGNL1 belongs to the AAA ATPase family of microtubule-severing proteins that includes katanin, spastin, and fidgetin. These proteins regulate microtubule dynamics during cell division and development. In the context of mitotic spindles, while katanin functions at kinetochores to destabilize kinetochore-MT plus-ends during anaphase A, related severing proteins like fidgetin and spastin destabilize microtubules at the spindle pole during metaphase . FIGNL1 is believed to share functional similarities with these proteins but possesses distinctive regulatory mechanisms and expression patterns that differentiate it from other family members.

How conserved is FIGNL1 across Xenopus species and other vertebrates?

FIGNL1 exhibits high conservation across vertebrate species, similar to other developmental proteins in Xenopus. Comparable to the 95% identity observed between Xenopus laevis and Xenopus tropicalis katanin p60 sequences , FIGNL1 sequences show significant conservation especially within functional domains. This conservation makes Xenopus tropicalis an excellent model for studying FIGNL1 function relevant to human development and disease. When considering conservation for experimental design, researchers should focus on the MT-binding and ATPase domains which typically display the highest degree of evolutionary conservation within this protein family.

What are the typical expression patterns of FIGNL1 during Xenopus tropicalis development?

FIGNL1 expression in X. tropicalis can be visualized using whole-mount in situ hybridization (WMISH) techniques, similar to those used for other developmental genes like sf3b4 . FIGNL1 is typically expressed in tissues undergoing active cell division and morphogenesis. Expression analysis throughout development shows patterns in neural tissues, developing somites, and potentially in neural crest cells. Understanding these expression patterns provides crucial context for functional studies and phenotypic analysis of FIGNL1 mutants.

What are the optimal methods for expressing recombinant Xenopus tropicalis FIGNL1?

For efficient expression of recombinant X. tropicalis FIGNL1, a Maltose Binding Protein (MBP) tagging strategy similar to that used for katanin p60 is recommended . The protocol includes:

• Cloning the X. tropicalis FIGNL1 coding sequence into an expression vector containing an MBP tag

• Transforming the construct into a suitable E. coli strain (BL21 or Rosetta)

• Inducing protein expression with IPTG at lower temperatures (16-18°C) to enhance solubility

• Optimizing expression conditions including IPTG concentration (0.1-0.5 mM) and induction time (4-16 hours)

Expression levels should be verified by SDS-PAGE and Western blotting using antibodies against the MBP tag or FIGNL1 itself.

What purification strategies yield highest activity for recombinant FIGNL1?

Based on successful approaches with related proteins in Xenopus, the following purification protocol is recommended :

• Affinity chromatography using amylose resin for MBP-tagged FIGNL1

• Buffer optimization to maintain protein stability (typically containing 20-50 mM HEPES pH 7.5, 100-300 mM NaCl, 1 mM DTT, 1 mM MgCl₂)

• Optional: Second purification step using ion exchange or size exclusion chromatography

• Activity preservation by including 10% glycerol and flash-freezing purified protein

Importantly, purification should be performed at 4°C with protease inhibitors to prevent degradation. ATP analogues (ATPγS) may be included during certain steps to stabilize the protein in its ATP-bound conformation.

How can researchers assess the purity and activity of recombinant FIGNL1?

A multi-faceted approach to quality assessment includes:

• SDS-PAGE analysis for purity assessment (>90% purity recommended)

• Western blotting with anti-FIGNL1 antibodies for identity confirmation

• Microtubule co-sedimentation assays to determine MT binding affinity (similar to those used for katanin p60)

• ATPase activity assays using colorimetric phosphate detection methods

• In vitro microtubule severing assays using fluorescently labeled microtubules and real-time imaging

The microtubule severing activity assay is particularly important, as it directly measures the protein's functional activity. Activity should be ATP-dependent, similar to what has been observed for katanin .

What experimental approaches can determine FIGNL1's specific role in microtubule dynamics?

To characterize FIGNL1's role in microtubule dynamics, researchers should employ a combination of in vitro and in vivo approaches:

• In vitro microtubule severing assays using purified recombinant protein and fluorescently labeled microtubules

• Real-time imaging of microtubule networks in Xenopus egg extracts supplemented with recombinant FIGNL1

• Spindle assembly assays in egg extracts with and without immunodepletion of endogenous FIGNL1

• Comparison of MT severing kinetics between FIGNL1 and other severing proteins like katanin and spastin

This multi-method approach will help distinguish FIGNL1's specific effects from those of other microtubule-severing proteins. Quantitative parameters should include MT half-life, severing frequency, and location preferences along microtubules .

How does post-translational modification regulate FIGNL1 activity in Xenopus tropicalis?

Based on findings with related proteins like katanin p60, FIGNL1 activity is likely regulated by post-translational modifications. To investigate this:

• Analyze potential phosphorylation sites using bioinformatics tools and comparison to known regulatory sites in related proteins (such as Ser131 in X. laevis katanin)

• Perform phosphorylation assays using X. tropicalis egg extracts and recombinant FIGNL1

• Generate phosphomimetic and phospho-null mutations of candidate regulatory sites

• Compare activities of wild-type and mutant FIGNL1 proteins in in vitro assays

• Identify kinases responsible for modifications using specific inhibitors or immunodepletion from egg extracts

Comparing regulation between X. tropicalis and X. laevis can provide additional insights, as species-specific differences in post-translational modification (like the phosphorylation at Ser131 in X. laevis katanin) may contribute to differential protein activity .

What phenotypes are associated with FIGNL1 depletion or overexpression in Xenopus tropicalis embryos?

To characterize FIGNL1-associated phenotypes:

• Deplete FIGNL1 using morpholinos or CRISPR/Cas9 gene editing

• Overexpress wild-type or mutant forms using mRNA injection

• Analyze developmental phenotypes focusing on:

  • Spindle morphology and cell division defects

  • Neural tube and neural crest cell migration (similar to analysis for sf3b4)

  • Embryonic development progression

• Perform rescue experiments by co-injecting morpholinos with wild-type or mutant FIGNL1 mRNA

The unilateral injection method at the 2-cell stage provides an excellent internal control, with one half of the embryo serving as a within-animal control while the other half carries the mutation of interest .

What is the optimal CRISPR/Cas9 strategy for generating Xenopus tropicalis FIGNL1 mutants?

Based on successful CRISPR/Cas9 applications in X. tropicalis:

• Design 2-3 guide RNAs targeting conserved functional domains (ATPase domain recommended)

• Use established X. tropicalis CRISPR protocols with ribonucleoprotein complexes

• For F0 analysis, inject one cell at the 2-cell stage to create mosaic embryos with an internal control

• For stable lines, inject both cells and raise founder animals

• Confirm mutations by PCR amplification and sequencing of the targeted region

The unilateral injection technique is particularly valuable for high-throughput phenotypic screening, allowing researchers to generate and analyze thousands of mutant embryos in a short timeframe .

How can researchers efficiently screen and characterize FIGNL1 CRISPR mutants?

An efficient screening strategy includes:

• T7 endonuclease I assay or high-resolution melt analysis for initial identification of mutations

• Targeted amplicon sequencing for precise mutation characterization

• Western blotting to confirm protein reduction/absence

• Functional assays such as immunofluorescence of mitotic spindles

• Phenotypic analysis, comparing homozygous and heterozygous mutants

For phenotypic analysis, examine both early (neural tube closure, gastrulation) and later (organogenesis) developmental stages. Similar to observations with sf3b4 mutants, heterozygous FIGNL1 mutants may show minimal phenotypes while homozygous mutants could display more severe developmental defects .

What strategies can address potential embryonic lethality of FIGNL1 mutations?

If complete FIGNL1 knockout causes embryonic lethality, consider:

• Creating conditional knockouts using inducible Cas9 systems

• Generating domain-specific mutations that affect certain functions while preserving others

• Using the unilateral injection approach to study cell-autonomous effects in F0 embryos

• Creating tissue-specific knockdowns using targeted morpholinos

• Utilizing partial protein expression with hypomorphic alleles

Partial depletion approaches may reveal dose-dependent functions without causing complete developmental failure, similar to the different phenotypes observed between heterozygous and homozygous sf3b4 mutants .

How does FIGNL1 function compare to katanin and spastin in Xenopus tropicalis?

To compare FIGNL1 with related proteins:

• Conduct parallel biochemical assays of purified recombinant proteins including:

  • Microtubule binding affinities

  • ATP hydrolysis rates

  • Microtubule severing activities and kinetics

• Perform co-depletion experiments to identify redundant or synergistic functions

• Compare localization patterns using immunofluorescence or tagged proteins

• Analyze structural similarities and differences using bioinformatics tools

Based on studies of katanin and related proteins, expect some functional overlap but with distinct regulatory mechanisms and tissue-specific roles . Quantitative comparisons should include MT severing rates, ATP hydrolysis efficiency, and binding affinities to different MT populations.

What are the key differences in regulatory mechanisms between FIGNL1 and other AAA ATPases in X. tropicalis versus X. laevis?

To explore species-specific differences in regulation:

• Align protein sequences to identify potential regulatory sites that differ between species

• Compare post-translational modifications between X. tropicalis and X. laevis FIGNL1

• Examine interspecies differences in binding partners using co-immunoprecipitation

• Analyze expression patterns and subcellular localization in both species

• Perform reciprocal protein replacement experiments in egg extracts

Similar to the regulatory differences observed with katanin p60, where X. laevis contains an inhibitory phosphorylation site (Ser131) absent in X. tropicalis , FIGNL1 may exhibit species-specific regulatory mechanisms that contribute to differences in activity or function.

How do FIGNL1 interaction networks differ from those of other AAA ATPases in Xenopus tropicalis?

To characterize FIGNL1-specific interaction networks:

• Perform immunoprecipitation followed by mass spectrometry to identify binding partners

• Use yeast two-hybrid screens to detect direct protein interactions

• Validate key interactions with co-immunoprecipitation and co-localization studies

• Compare interactomes between FIGNL1, katanin, and spastin

• Conduct functional studies to determine the significance of identified interactions

Analysis should focus on identifying unique binding partners that may confer FIGNL1-specific functions distinct from other severing proteins, potentially explaining their non-redundant roles despite similar enzymatic activities.

How can FIGNL1 studies in Xenopus tropicalis inform human disease mechanisms?

To translate findings to human disease relevance:

• Analyze conservation of functional domains between X. tropicalis and human FIGNL1

• Identify human disease variants in FIGNL1 and model them in X. tropicalis

• Use CRISPR/Cas9 to introduce precise human patient mutations

• Compare phenotypes with known human developmental disorders

• Analyze potential connections to cell division defects in human pathologies

The ability to rapidly generate and analyze mutants in X. tropicalis makes it an excellent system for modelling human genetic disorders . Findings may be particularly relevant to microcephaly, growth disorders, or neurodevelopmental conditions associated with cell division defects.

What are the methodological challenges in studying FIGNL1 interactions with the mitotic spindle in Xenopus tropicalis?

Advanced technical considerations include:

• Developing methods to visualize dynamic FIGNL1-microtubule interactions in real-time

• Creating fluorescently tagged versions that retain full activity

• Optimizing extract systems for studying specific stages of cell division

• Implementing super-resolution microscopy techniques to detect localized severing events

• Developing quantitative models of microtubule dynamics incorporating FIGNL1 activity

Researchers should carefully validate that tagged constructs maintain normal activity levels, as tags may interfere with oligomerization or substrate binding, similar to considerations with other AAA ATPases .

How can high-throughput approaches be applied to study FIGNL1 function in developmental contexts?

For larger-scale investigations:

• Develop CRISPR libraries targeting different FIGNL1 domains for parallel phenotypic screening

• Implement automated imaging and analysis pipelines for quantifying developmental phenotypes

• Perform temporal transcriptomics to identify downstream effects of FIGNL1 depletion

• Use proteomics to characterize changes in the microtubule-associated proteome

• Apply the unilateral injection approach to efficiently test multiple conditions in parallel

The ability to generate thousands of mutant embryos in a single day makes X. tropicalis particularly suitable for parallelized analysis, enabling the investigation of subtle phenotypic effects across multiple experimental conditions .