Recombinant Xenopus laevis CDGSH iron-sulfur domain-containing protein 2B (cisd2-b), partial

What is CISD2-B and what is its significance in Xenopus laevis research?

CISD2-B is one of the homeologs of the CDGSH iron-sulfur domain-containing protein 2 found in the allotetraploid Xenopus laevis genome. This protein belongs to the CDGSH iron-sulfur domain family and contains iron-sulfur clusters that participate in various cellular processes. In Xenopus research, CISD2-B serves as a valuable model for understanding the gene's function across vertebrates. The presence of both L (long) and S (short) chromosomal versions in X. laevis makes it particularly useful for studying gene evolution following genome duplication events. Xenbase, the dedicated Xenopus model organism database, provides comprehensive genomic information about this gene, facilitating comparative analyses across species .

How is CISD2-B expression regulated during Xenopus development?

CISD2-B expression varies significantly throughout Xenopus development, with specific temporal patterns that can be visualized using the developmental stage profiles available in resources like Xenbase. RNA-Seq data displays transcripts per million (TPM) values from early oocyte stages through later developmental stages (up to NF stage 40). The expression profiles of X. laevis L and S homeologs can be compared through interactive graphing tools, which plot expression levels against developmental stages. These profiles reveal stage-specific regulation that likely corresponds to the protein's role in developmental processes . The expression of CISD2 genes is often tissue-specific, with dynamic changes during embryonic development that reflect its functional importance in various developmental pathways.

How do the L and S homeologs of CISD2-B differ in Xenopus laevis?

In Xenopus laevis, the genome duplication has resulted in two homeologs of many genes, referred to as L (long chromosome) and S (short chromosome) versions. For CISD2-B, these homeologs can exhibit different expression patterns across tissues and developmental stages. Xenbase provides heatmap visualizations comparing L versus S homeolog expression in various tissues. These differences often reflect subfunctionalization or neofunctionalization processes that occurred after genome duplication. Researchers can examine these comparative expression patterns through RNA-Seq data available on Xenbase, which allows plotting of both homeologs simultaneously to visualize their relative expression levels throughout development .

What role does CISD2-B play in cellular signaling pathways?

Research on CISD2 in various models suggests significant involvement in multiple signaling pathways, particularly the Wnt/β-catenin pathway. In pancreatic cancer studies, CISD2 has been shown to regulate β-catenin activation and subsequent signaling cascades. CISD2 deficit leads to inactivation of the Wnt/β-catenin pathway, which contributes to decreased cell survival . In Xenopus, this protein likely plays similar roles in developmental contexts, potentially influencing cell fate determination, proliferation, and morphogenetic movements. The protein may interact with GSK3β, influencing its phosphorylation state and thus regulating β-catenin stability. Western blot analyses using anti-GSK3β, anti-p-GSK3β, anti-β-catenin, and anti-p-β-catenin antibodies can help elucidate these interactions in Xenopus models .

How can CISD2-B be used to model human disease processes?

CISD2 has been implicated in several human pathologies, making CISD2-B in Xenopus a valuable model for disease-related research. Studies have shown that CISD2 is involved in cancer progression, particularly in pancreatic cancer where high levels correlate with advanced clinical stage, positive vascular invasion, distant metastasis, and larger tumor size . Researchers can use Xenopus CISD2-B to model these disease processes by:

• Creating transgenic Xenopus lines with altered CISD2-B expression

• Using morpholinos or CRISPR-Cas9 to knock down or knock out the gene

• Studying resulting phenotypes for relevance to human disease conditions

• Performing rescue experiments with human CISD2 to assess functional conservation

These approaches allow researchers to investigate the conserved mechanisms between amphibian and human systems, potentially revealing novel therapeutic targets.

What is known about the relationship between CISD2-B and epithelial-to-mesenchymal transition (EMT)?

Evidence from human cancer studies suggests that CISD2 significantly influences epithelial-to-mesenchymal transition (EMT), a process critical in both development and cancer metastasis. CISD2 silencing has been shown to inhibit EMT via the Wnt/β-catenin pathway . In Xenopus, EMT processes are essential during gastrulation and neural crest formation, suggesting potential developmental roles for CISD2-B. Researchers investigating this relationship should examine markers such as E-cadherin, N-cadherin, vimentin, and γ-catenin, which can be detected through immunoblotting techniques. The regulatory mechanisms may involve direct or indirect effects on these EMT markers through modulation of the Wnt/β-catenin pathway activity. Understanding these mechanisms in Xenopus can provide valuable insights into evolutionary conserved processes relevant to human development and disease .

What are the most effective techniques for isolating and purifying recombinant Xenopus laevis CISD2-B?

For effective isolation and purification of recombinant Xenopus laevis CISD2-B, researchers should consider the following methodology:

• Cloning strategy: The partial CISD2-B coding sequence should be amplified from Xenopus laevis cDNA using PCR with gene-specific primers. This can be facilitated by sequence information available through Xenbase .

• Expression system selection: Bacterial expression systems (typically E. coli BL21(DE3)) using vectors such as pET or pGEX are commonly employed for recombinant protein production. For CISD2-B specifically, a vector containing a 6xHis or GST tag facilitates purification.

• Protein expression optimization:

  • Induce expression at OD600 of 0.6-0.8

  • Test various IPTG concentrations (0.1-1 mM)

  • Optimize temperature (typically 18-25°C for iron-sulfur proteins)

  • Include additives such as iron ammonium sulfate and L-cysteine in the growth medium

• Purification protocol:

  • Lyse cells using sonication in buffer containing protease inhibitors

  • Perform immobilized metal affinity chromatography (for His-tagged proteins)

  • Consider additional purification steps such as ion exchange or gel filtration chromatography

  • For iron-sulfur proteins, include reducing agents such as DTT or β-mercaptoethanol

The purified protein should be validated using SDS-PAGE, Western blotting with anti-CISD2 antibodies, and mass spectrometry to confirm identity and integrity .

How can gene expression analysis of CISD2-B be optimized in Xenopus studies?

Optimizing gene expression analysis of CISD2-B requires multiple complementary approaches:

• Quantitative RT-PCR (qRT-PCR):

  • Extract total RNA using standardized kits (e.g., Allprep DNA/RNA Kit)

  • Generate cDNA using random hexamer priming and M-MLV reverse transcriptase

  • Use CISD2-B specific TaqMan Gene Expression Assay or design SYBR Green primers

  • Select appropriate endogenous controls (e.g., ABL or HPRT) for normalization

  • Run reactions in duplicate or triplicate on real-time PCR systems

• RNA-Seq analysis:

  • Generate stage-specific transcriptome data across developmental stages

  • Plot transcripts per million (TPM) values against developmental stages

  • Compare expression between L and S homeologs using tools available on Xenbase

  • Apply log2 transformation for better visualization of data points

• In situ hybridization (ISH):

  • Design RNA probes specific to CISD2-B

  • Perform whole-mount ISH to visualize spatial expression patterns

  • Document results with high-quality images across developmental stages

  • Submit images to community databases like Xenbase

• Protein detection:

  • Use Western blotting with anti-CISD2 antibodies

  • Normalize to housekeeping proteins like GAPDH

  • Quantify band intensity using software like ImageJ

These complementary approaches provide comprehensive insight into both the temporal and spatial expression patterns of CISD2-B throughout Xenopus development.

What are effective strategies for CISD2-B gene manipulation in Xenopus models?

Several strategies exist for manipulating CISD2-B expression in Xenopus models:

• Lentiviral-mediated gene manipulation:

  • Utilize lentiviral vectors carrying scramble controls, overexpression constructs (Lv-oeCISD2), or shRNA constructs (Lv-shCISD2)

  • Optimize multiplicity of infection (MOI); previous studies with similar genes used MOI of 15

  • Confirm successful transduction through fluorescent markers and expression validation

• Morpholino antisense oligonucleotides:

  • Design splice-blocking or translation-blocking morpholinos specific to CISD2-B

  • Microinject morpholinos at early developmental stages (1-2 cell)

  • Use 5-25 ng as standard dosage, optimizing based on phenotypic readouts

  • Include standard control morpholinos and rescue experiments to validate specificity

• CRISPR-Cas9 genome editing:

  • Design guide RNAs targeting specific regions of CISD2-B

  • Synthesize Cas9 mRNA and sgRNA through in vitro transcription

  • Inject into fertilized Xenopus eggs at one-cell stage

  • Screen F0 embryos for mutations using T7 endonuclease assay or direct sequencing

• mRNA overexpression:

  • Clone CISD2-B into appropriate vectors for in vitro transcription

  • Synthesize capped mRNA using appropriate kits

  • Inject synthesized mRNA into embryos at early cleavage stages

  • Monitor phenotypic changes and validate expression through qRT-PCR or Western blotting

For all manipulation approaches, careful experimental design including appropriate controls and rescue experiments is essential to ensure specificity and minimize off-target effects.

How should RNA-Seq data for CISD2-B be analyzed across developmental stages?

RNA-Seq analysis of CISD2-B expression requires a structured analytical approach:

• Data preprocessing and normalization:

  • Quality control using FastQC to assess sequence quality

  • Trimming of low-quality reads and adapter sequences

  • Alignment to the Xenopus laevis genome (available through Xenbase)

  • Normalization to transcripts per million (TPM) for cross-sample comparability

• Developmental trajectory analysis:

  • Plot expression values against developmental stages (oocyte stage 1-2 to NF stage 40)

  • Generate interactive graphs comparing L and S homeologs

  • Apply appropriate transformations (raw or log2) for optimal visualization

  • Identify key developmental transitions with significant expression changes

• Comparative analysis with related genes:

  • Select related genes or pathway components for co-expression analysis

  • Plot multiple genes simultaneously to identify correlated expression patterns

  • Focus on genes identified through co-citation in literature

  • Examine potential co-regulation during developmental processes

• Statistical validation:

  • Apply appropriate statistical tests to identify significant changes

  • Use replicates to establish confidence intervals

  • Consider time-series analysis methods for developmental data

  • Validate key findings with alternative methods (qRT-PCR, Western blot)

Xenbase provides tools for visualizing RNA-Seq data, allowing researchers to examine specific developmental stages and compare homeologs within an interactive interface. This facilitates the identification of stage-specific regulation patterns that may correspond to critical developmental events .

What bioinformatic approaches can identify potential interaction partners of CISD2-B?

Multiple bioinformatic approaches can help identify potential CISD2-B interaction partners:

• Co-expression analysis:

  • Analyze RNA-Seq datasets across developmental stages to identify genes with similar expression patterns

  • Calculate Pearson or Spearman correlation coefficients to quantify co-expression relationships

  • Construct gene co-expression networks to visualize potential functional relationships

  • Focus on genes involved in pathways known to interact with CISD2, such as Wnt/β-catenin signaling

• Protein-protein interaction prediction:

  • Utilize structural information to predict potential binding partners

  • Apply homology-based approaches using known interactions of CISD2 in other species

  • Employ algorithms that consider domain compatibility and conservation

  • Integrate information from interaction databases like STRING or BioGRID

• Pathway enrichment analysis:

  • Conduct Gene Ontology (GO) enrichment analysis for potential interactors

  • Identify overrepresented pathways using KEGG or Reactome databases

  • Focus on pathways related to iron metabolism, Wnt signaling, and EMT processes

  • Visualize potential pathway connections using tools like Cytoscape

• Cross-species comparative analysis:

  • Compare CISD2 interactions across species (human, mouse, zebrafish)

  • Identify evolutionarily conserved interaction partners

  • Leverage Xenbase cross-species resources to identify orthologs

  • Prioritize conserved interactions for experimental validation

These computational approaches provide testable hypotheses about CISD2-B interactors that can be subsequently validated through experimental techniques such as co-immunoprecipitation, proximity ligation assays, or yeast two-hybrid screening.

How can tissue-specific expression data of CISD2-B be effectively interpreted?

Interpreting tissue-specific expression data for CISD2-B requires systematic analysis:

• Heatmap visualization and analysis:

  • Generate heatmaps comparing CISD2-B expression across tissues

  • Include both L and S homeologs to identify potential subfunctionalization

  • Cluster tissues based on expression patterns to identify functional groupings

  • Compare with known developmental or physiological processes in those tissues

• Integration with histological data:

  • Correlate expression levels with in situ hybridization or immunohistochemistry images

  • Identify cell-type specific expression within heterogeneous tissues

  • Document expression changes during tissue development or remodeling

  • Analyze community-submitted and literature-derived images available through Xenbase

• Comparative analysis with disease-relevant tissues:

  • Compare expression patterns with tissues affected in CISD2-related human diseases

  • Focus on tissues where CISD2 has known roles in pathogenesis, such as pancreatic tissue

  • Identify conservation of expression patterns across species

  • Correlate expression with tissue-specific phenotypes in genetic models

• Functional interpretation:

  • Associate tissue-specific expression with known tissue functions

  • Consider metabolic requirements and iron utilization of different tissues

  • Evaluate expression in the context of Wnt/β-catenin signaling activity

  • Connect expression patterns to EMT processes during development or regeneration

Effective interpretation requires integrating these analyses with existing knowledge about tissue-specific requirements for CISD2 function, potentially revealing novel insights into its role in development and disease.

What assays can determine the effect of CISD2-B on cellular proliferation?

Based on CISD2's established role in proliferation, several assays can assess its function:

• Cell proliferation assays:

  • MTT/MTS/WST-1 colorimetric assays to measure metabolic activity

  • BrdU incorporation to detect DNA synthesis

  • Ki-67 immunostaining to identify proliferating cells

  • Colony formation assays to assess long-term proliferative capacity

• Cell cycle analysis:

  • Flow cytometry with propidium iodide staining to determine cell cycle distribution

  • EdU pulse-chase experiments to track S-phase progression

  • Cyclin expression analysis via Western blotting

  • Assessment of cell cycle checkpoint proteins

• In vivo proliferation studies:

  • CISD2-B manipulation in Xenopus embryos followed by:

  • Phospho-histone H3 immunostaining to mark mitotic cells

  • Analysis of tissue-specific growth and development

  • Quantification of organ size and cell number

  • Assessment of developmental timing and progression

• Molecular pathway analysis:

  • Evaluation of Wnt/β-catenin pathway activity using TOPFlash reporter assays

  • Western blot analysis of c-Myc and other proliferation-associated targets

  • Assessment of GSK3β phosphorylation status

  • Examination of nuclear β-catenin localization

These complementary approaches can provide comprehensive insight into how CISD2-B influences cellular proliferation in different contexts and developmental stages.

How can the role of CISD2-B in epithelial-to-mesenchymal transition be experimentally validated?

To validate CISD2-B's role in EMT, researchers should employ multi-faceted approaches:

• Molecular marker analysis:

  • Western blot analysis of epithelial markers (E-cadherin, γ-catenin)

  • Assessment of mesenchymal markers (N-cadherin, vimentin)

  • Quantification of transcription factors driving EMT (Snail, Slug, Twist)

  • Normalization to housekeeping proteins such as GAPDH

• Morphological and behavioral assays:

  • Cell morphology assessment using phase-contrast microscopy

  • Migration assays (wound healing, transwell)

  • Invasion assays using Matrigel-coated chambers

  • Time-lapse microscopy to track cellular behavior changes

• In vivo developmental analysis:

  • CISD2-B manipulation in Xenopus embryos followed by:

  • Assessment of neural crest migration (a classic EMT process)

  • Analysis of gastrulation movements

  • Examination of tissue boundary formation

  • Histological analysis of tissue architecture

• Molecular pathway interrogation:

  • Nuclear/cytoplasmic fractionation to assess β-catenin localization

  • Analysis of Wnt/β-catenin pathway components

  • Rescue experiments with pathway activators or inhibitors

  • Co-immunoprecipitation to detect physical interactions between CISD2-B and EMT regulators

Integration of these approaches provides robust evidence for CISD2-B's role in EMT processes, connecting molecular mechanisms to cellular and developmental phenotypes.

What techniques can assess CISD2-B's interaction with the Wnt/β-catenin signaling pathway?

Several complementary techniques can evaluate CISD2-B's interaction with Wnt/β-catenin signaling:

• Protein interaction studies:

  • Co-immunoprecipitation of CISD2-B with pathway components

  • Proximity ligation assays to detect in situ protein interactions

  • FRET or BRET analysis for real-time interaction monitoring

  • Pull-down assays with purified recombinant proteins

• Pathway activity measurements:

  • TOPFlash/FOPFlash reporter assays to quantify β-catenin-mediated transcription

  • Western blot analysis of pathway components (β-catenin, GSK3β, p-GSK3β)

  • Nuclear/cytoplasmic fractionation to assess β-catenin translocation

  • qRT-PCR analysis of Wnt target genes

• Genetic interaction approaches:

  • Epistasis experiments combining CISD2-B manipulation with Wnt pathway modulators

  • Rescue experiments using constitutively active β-catenin

  • Double knockdown/overexpression studies

  • CRISPR-Cas9 editing of potential interaction domains

• Structural biology approaches:

  • Molecular modeling of potential interaction interfaces

  • Mutagenesis of key residues to disrupt specific interactions

  • X-ray crystallography or cryo-EM of protein complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

These approaches can establish whether CISD2-B directly or indirectly influences Wnt/β-catenin signaling, potentially revealing novel regulatory mechanisms relevant to both development and disease .

How does Xenopus laevis CISD2-B compare with orthologs in other model organisms?

Comparative analysis of CISD2 across species reveals important evolutionary insights:

• Sequence conservation analysis:

  • Amino acid sequence comparison shows high conservation of the CDGSH domain

  • Phylogenetic analysis reveals evolutionary relationships between vertebrate CISD2 proteins

  • Conservation of critical functional residues, particularly those coordinating iron-sulfur clusters

  • Divergence in regulatory regions suggesting species-specific expression patterns

• Functional conservation assessment:

  • Similar roles in Wnt/β-catenin signaling across vertebrates

  • Conservation of EMT-related functions from amphibians to mammals

  • Consistent involvement in proliferation regulation

  • Species-specific adaptations in certain functional domains

• Expression pattern comparison:

  • Xenbase tools allow comparison with other vertebrate expression databases

  • Assessment of temporal expression differences during development

  • Evaluation of tissue-specific expression conservation

  • Identification of species-specific expression features

• Disease relevance comparison:

  • Correlation between Xenopus phenotypes and mammalian disease models

  • Comparative analysis of CISD2 mutations and their phenotypic consequences

  • Evaluation of protein interactions conserved between species

  • Assessment of therapeutic target potential based on evolutionary conservation

This comparative approach provides context for understanding fundamental versus species-specific aspects of CISD2-B function, facilitating translation between model systems and human applications.

What are the key differences between L and S homeologs of CISD2-B in Xenopus laevis?

The allotetraploid nature of the Xenopus laevis genome results in two distinct CISD2-B homeologs with several notable differences:

• Expression pattern divergence:

  • RNA-Seq data reveals differential expression patterns between L and S homeologs

  • Developmental stage-specific expression differences can be visualized through interactive graphs

  • Tissue-specific expression variations shown in heatmaps available through Xenbase

  • Potential subfunctionalization reflected in complementary expression domains

• Sequence and structural variations:

  • Nucleotide and amino acid substitutions between homeologs

  • Potential differences in protein domain organization

  • Variations in regulatory regions affecting expression control

  • Possible alterations in post-translational modification sites

• Functional specialization:

  • Differential interactions with signaling pathways

  • Varying contributions to developmental processes

  • Potential redundancy or complementation between homeologs

  • Different responses to experimental manipulation

• Evolutionary trajectory:

  • Different rates of sequence evolution between L and S homeologs

  • Varying selection pressures suggesting functional divergence

  • Comparison with single CISD2 orthologs in diploid species

  • Assessment of retention versus loss of function in duplicate genes

Understanding these differences provides insight into the evolutionary consequences of genome duplication and the functional diversification of duplicate genes in polyploid organisms.

How might single-cell sequencing advance our understanding of CISD2-B function?

Single-cell sequencing technologies offer unprecedented resolution for studying CISD2-B function:

• Cell-type specific expression profiling:

  • Identification of specific cell populations expressing CISD2-B

  • Correlation with cell states during developmental transitions

  • Detection of rare cell populations with unique CISD2-B expression patterns

  • Integration with spatial information through spatial transcriptomics

• Developmental trajectory mapping:

  • Reconstruction of developmental lineages expressing CISD2-B

  • Identification of branch points where CISD2-B influences cell fate decisions

  • Correlation with EMT transitions at single-cell resolution

  • Integration with pseudotime analysis to map temporal dynamics

• Perturbation-response analysis:

  • CRISPR-Cas9 screening combined with single-cell readouts

  • Assessment of compensatory responses to CISD2-B manipulation

  • Identification of cell-type specific dependencies on CISD2-B

  • Determination of primary versus secondary effects of CISD2-B perturbation

• Multi-omics integration:

  • Combined analysis of transcriptome, epigenome, and proteome at single-cell level

  • Correlation of CISD2-B expression with chromatin accessibility

  • Integration with single-cell protein measurements

  • Construction of gene regulatory networks at single-cell resolution

These approaches would reveal heterogeneity in CISD2-B function across different cell populations and developmental contexts, potentially identifying novel roles that are obscured in bulk analyses.