Recombinant Xenopus laevis Phosphatidylinositol 3-kinase catalytic subunit type 3 (pik3c3), partial

What is PIK3C3 and what are its primary functions in Xenopus laevis?

PIK3C3, also known as VPS34, is the sole class III member of the phosphatidylinositol 3-kinase family. In Xenopus laevis, as in other vertebrates, PIK3C3 phosphorylates phosphatidylinositol (PI) to produce phosphatidylinositol 3-phosphate [PI(3)P], which recruits downstream effectors containing FYVE and Phox domains to membranes .

PIK3C3 plays critical roles in several cellular processes:

• Regulation of autophagy and autophagosome formation

• Endocytosis and vesicular trafficking

• Nutrient sensing and signaling to mTOR

• Transport of lysosomal enzyme precursors to lysosomes

• Regulation of degradative endocytic trafficking

• The abscission step in cytokinesis in the context of PI3KC3-C2

In Xenopus laevis specifically, PIK3C3 is involved in glucose metabolism regulation as demonstrated by experiments with PI 3-kinase inhibitor wortmannin, which inhibits insulin-like growth factor-1-stimulated deoxyglucose uptake in Xenopus oocytes .

How has the PIK3C3 gene evolved in Xenopus laevis compared to other vertebrates?

Xenopus laevis presents a unique evolutionary case for studying PIK3C3 due to its allotetraploid nature. The species arose through hybridization of two diploid species approximately 17-18 million years ago (MYA), resulting in duplicated genes across its genome .

The two subgenomes in Xenopus laevis have evolved asymmetrically:

• The shorter (S) chromosomes have undergone more interchromosome rearrangements, gene losses, and silenced gene expression than the longer (L) chromosomes

• Many genes involved in immune function have been lost in S homoeologous chromosomes

• Multiple genes encoding proteins involved in signaling (including kinases like PIK3C3) have been retained, likely due to stoichiometrically controlled expression or subfunctionalization

While PIK3C3 (flt3) is well conserved across all jawed vertebrates, studies of homoeologous genes in Xenopus laevis suggest potential subfunctionalization of duplicated copies, making it an excellent model for studying gene evolution after whole genome duplication .

What are the recommended methods for producing recombinant Xenopus laevis PIK3C3?

Based on established protocols for recombinant protein production in Xenopus systems, the following methodology is recommended:

Expression System Selection:

• Insect cell expression systems (particularly Sf9 cells) have been successfully used for producing recombinant proteins from Xenopus laevis

• Bacterial expression systems may be suitable for partial domains but often struggle with full-length kinases due to post-translational modification requirements

Cloning and Vector Construction:

• Amplify the PIK3C3 cDNA by PCR using appropriate primers (design primers based on the Xenopus laevis PIK3C3 sequence)

• Insert restriction enzyme sites (such as HindIII and XhoI) for subsequent cloning

• Perform restriction digestion of the PCR product and vector

• Ligate the digested PCR product into an appropriate expression vector

Transfection and Protein Production:

• Transfect Sf9 insect cells using cellfectin II (or similar transfection reagent) and 1 μg of plasmid containing the PIK3C3 sequence

• For large-scale protein production, culture cells in Sf-900™ II SFM supplemented with 10 μg/ml of Gentamicin

• Purify the recombinant protein using NiNTA-Agarose Chromatography if a His-tag was incorporated into the design

Protein Stabilization:

• Supplement purified protein fractions with 0.02% NaN₃ and cOmplete™ EDTA-free Protease Inhibitor Cocktail

• Store at 4°C for short-term or -20°C for long-term storage

What functional assays can verify the activity of recombinant Xenopus laevis PIK3C3?

Several functional assays can be used to verify the kinase activity and cellular functions of recombinant PIK3C3:

Kinase Activity Assay:

• Measure phosphorylation of phosphatidylinositol (PI) to produce phosphatidylinositol 3-phosphate [PI(3)P] using radioactive ATP (³²P-ATP) or non-radioactive methods with fluorescent substrates

• Quantify PI(3)P production using thin-layer chromatography or ELISA-based detection systems

Cellular Functional Assays:

• Autophagy Induction Assay: Add recombinant PIK3C3 to autophagy-deficient cells (such as PIK3C3-knockout cells) and measure LC3-II conversion by western blot

• Glucose Transport Assay: Measure deoxyglucose uptake in Xenopus oocytes after introduction of recombinant PIK3C3, with and without wortmannin inhibition

• Binding Partner Interaction: Use co-immunoprecipitation to verify interaction with known binding partners (such as BECN1 or ATG14L)

Verification Using Inhibitors:

• Compare activity in the presence and absence of selective PIK3C3 inhibitors like SAR405, which has been demonstrated to delay disease progression in experimental autoimmune encephalomyelitis models

• Measure half-maximal inhibition of PIK3C3 activity, which occurs at approximately 20 nM for wortmannin in Xenopus oocytes

How does PIK3C3 expression vary across tissues and developmental stages in Xenopus laevis?

PIK3C3 expression in Xenopus laevis shows distinct patterns across tissues and developmental stages:

Developmental Expression:

• In early embryos and oocytes of Xenopus laevis, certain histone variants like H2A.X are highly abundant (50% or more of the H2A) , suggesting active chromatin remodeling that may involve PIK3C3-mediated signaling

• During tadpole development, homoeologous PIK3C3 genes show evidence of subfunctionalization

Tissue-Specific Expression:
Based on comparative studies with mammalian PIK3C3, expression likely varies significantly across tissues. In mouse models, distinct tissue-specific expression patterns have been observed:

• Highest expression in glomerular podocytes in the kidney

• High expression in renal proximal tubule cells

• Nearly undetectable expression in glomerular mesangial cells, endothelial cells, and renal interstitial cells

• Differential expression in white and brown adipose tissues

Expression in Immune Cells:

• Studies in mouse models show PIK3C3 is crucial for myeloid cell function, suggesting similar importance in Xenopus immune system

• Macrophages deficient in PIK3C3 show increased surface levels of MHC class I and II molecules

While these patterns are derived partly from mammalian models, the evolutionary conservation of PIK3C3 suggests similar tissue-specific expression patterns likely exist in Xenopus laevis, particularly given the critical role of PIK3C3 in fundamental cellular processes.

How do the L and S homoeologs of PIK3C3 differ in Xenopus laevis, and what methodologies can detect these differences?

The allotetraploid nature of Xenopus laevis provides a unique opportunity to study homoeologous PIK3C3 genes (from L and S chromosomes). These differences can be detected and characterized using the following methods:

Sequence Analysis:

• Perform whole genome sequencing or targeted sequencing of PIK3C3 loci from both L and S chromosomes

• Use bioinformatic tools to identify single nucleotide polymorphisms (SNPs) and other sequence variations between homoeologs

Expression Analysis:

• Homoeolog-Specific RT-PCR:

  • Design primers that specifically amplify either the L or S homoeolog

  • Perform quantitative RT-PCR to measure relative expression levels across tissues and developmental stages

• RNA-Seq with Homoeolog Resolution:

  • Perform RNA-seq and use computational methods to assign reads to specific homoeologs

  • Analyze differential expression between homoeologs across conditions

Functional Characterization:

• Generate recombinant proteins from both L and S homoeologs

• Compare enzymatic activities, binding affinities, and cellular effects

• Use CRISPR-Cas9 to selectively knock out individual homoeologs and assess phenotypic consequences

A study examining FLT3 and FLT3LG homoeologs in Xenopus laevis found evidence of subfunctionalization, with both homoeologs (S and L) retaining functional activity but showing differential expression patterns . Similar methodologies could be applied to PIK3C3 homoeologs:

• Produce tagged recombinant proteins from both homoeologs

• Assess cellular binding and activity profiles

• Compare expression patterns across tissues and developmental stages

How does PIK3C3 regulate autophagy in Xenopus laevis cells and how can this process be experimentally manipulated?

PIK3C3 plays a central role in autophagy initiation and progression in Xenopus laevis cells through the following mechanisms:

Autophagy Initiation and Regulation:

• PIK3C3 forms a complex with p150 (PIK3R4), BECN1 (Beclin 1), and ATG14L to generate phosphatidylinositol 3-phosphate [PI(3)P] at the phagophore assembly site

• PI(3)P recruits effector proteins containing FYVE and Phox domains to initiate autophagosome formation

• This complex (Complex I) is crucial for initial autophagosome formation

• A separate complex (Complex II) involving PIK3C3, p150, BECN1, and UVRAG regulates endolysosomal and autophagolysosomal maturation

Experimental Manipulation Methods:

• Pharmacological Inhibition:

  • Use wortmannin (IC₅₀ ~20 nM in Xenopus oocytes)

  • Apply SAR405, a selective PIK3C3 inhibitor

  • Employ rapamycin to induce autophagy upstream of PIK3C3 activity

• Genetic Manipulation:

  • Use CRISPR-Cas9 gene editing to generate conditional knockout models

  • Apply siRNA or morpholinos for transient knockdown

  • Express dominant-negative PIK3C3 mutants

• Autophagy Monitoring:

  • Track LC3-II conversion via western blotting

  • Use fluorescent reporters like RFP-EGFP-LC3 to monitor autophagosome-lysosome fusion

  • Measure autophagic flux using chloroquine treatment to block autophagosome-lysosome fusion

Comparative Studies:
Studies in PIK3C3-deficient podocytes show:

• Substantial perinuclear vacuolization

• Accumulation of LC3 and SQSTM1 (p62)

• Disrupted autophagic flux, as evidenced by minimal increase in LC3-II levels upon chloroquine treatment

These methodologies can be adapted to Xenopus laevis cell systems to characterize the specific role of PIK3C3 in amphibian autophagy processes.

What is the relationship between PIK3C3 and metabolism in Xenopus laevis, particularly during stress conditions?

PIK3C3 plays important roles in metabolism in Xenopus laevis, particularly during stress conditions:

Glucose Metabolism:

• PIK3C3 regulates glucose transport in Xenopus laevis oocytes

• Inhibition with wortmannin blocks insulin-like growth factor-1-stimulated deoxyglucose uptake

• Conversely, peptides that activate PI 3-kinase stimulate glucose transport

Stress Adaptation:
Xenopus laevis can tolerate dehydration when their environments evaporate. Phosphoproteomic analysis reveals:

• Enrichment of phosphoproteins related to glycolysis/gluconeogenesis, TCA cycle, and protein metabolism during dehydration

• Hypoxia-inducible PFKFB3 enzyme becomes highly phosphorylated and activated during dehydration

• All four transcript variants of the pfkfb3 gene are upregulated during dehydration

While specific roles of PIK3C3 during dehydration have not been directly established in Xenopus, the involvement of PIK3C3 in nutrient sensing and autophagy suggests it likely plays a role in metabolic adaptation to stress conditions.

Table 1: Key Metabolic Pathways Associated with PIK3C3 Function in Xenopus laevis

How can recombinant Xenopus laevis PIK3C3 be used to study evolutionary divergence of signaling pathways?

Recombinant Xenopus laevis PIK3C3 provides a unique tool for studying evolutionary divergence of signaling pathways due to several factors:

Allotetraploidy as an Evolutionary Window:

• Xenopus laevis is an allotetraploid species that arose through hybridization approximately 17-18 MYA

• The asymmetric evolution of its L and S subgenomes allows examination of gene retention and subfunctionalization

• Comparing homoeologous PIK3C3 genes (L and S versions) can reveal evolutionary processes following genome duplication

Methodological Approaches:

• Comparative Biochemistry:

  • Produce recombinant PIK3C3 from Xenopus laevis (both L and S homoeologs), Xenopus tropicalis (diploid relative), and other vertebrate species

  • Compare enzymatic activities, substrate specificities, and inhibitor sensitivities

  • Identify functional divergence through protein domain swapping experiments

• Interspecies Complementation:

  • Express Xenopus laevis PIK3C3 in PIK3C3-deficient mammalian, fish, or invertebrate cells

  • Assess functional conservation by measuring rescue of autophagy and other cellular processes

  • Identify species-specific regulatory partners and mechanisms

• Evolutionary Rate Analysis:

  • Measure evolutionary rates of PIK3C3 sequences across vertebrate lineages

  • Compare rates between duplicated genes in Xenopus laevis to test models of subfunctionalization versus neofunctionalization

  • Identify positively selected amino acid residues that may confer adaptive advantages

Research Applications:

• Study the evolution of autophagy regulation across vertebrate lineages

• Investigate how signaling pathway components adapt following genome duplication

• Examine convergent evolution of stress responses in amphibians compared to other vertebrates

Similar approaches examining FLT3 and FLT3LG homoeologs in Xenopus laevis revealed subfunctionalization of these duplicated genes, suggesting a common pattern that may extend to PIK3C3 .

What methodologies are most effective for studying PIK3C3 interaction networks in Xenopus laevis systems?

Studying the PIK3C3 interactome in Xenopus laevis requires specialized approaches that account for its unique genome and cellular characteristics:

Protein-Protein Interaction Methods:

• Proximity-dependent Biotin Identification (BioID):

  • Express PIK3C3-BirA* fusion protein in Xenopus cells or embryos

  • Biotinylated proteins represent proximal interactors

  • Identify interactors using streptavidin pulldown followed by mass spectrometry

  • Advantage: Captures transient and weak interactions in living cells

• Co-immunoprecipitation Combined with Mass Spectrometry:

  • Generate antibodies specific to Xenopus laevis PIK3C3 or use tagged recombinant versions

  • Perform co-IP from different tissues or developmental stages

  • Identify binding partners through mass spectrometry analysis

  • Compare interactomes between L and S homoeologs

• Yeast Two-Hybrid Screening:

  • Use PIK3C3 or its domains as bait against Xenopus laevis cDNA libraries

  • Validate interactions in Xenopus cellular systems

  • Compare interaction profiles with mammalian PIK3C3 to identify conserved and divergent partners

Functional Genomics Approaches:

• CRISPR-Cas9 Screens:

  • Design sgRNA libraries targeting Xenopus genes

  • Screen for modifiers of PIK3C3-dependent processes (autophagy, endocytosis)

  • Identify genetic interactions using combinatorial CRISPR approaches

• Phosphoproteomics:

  • Compare phosphorylation profiles in wild-type and PIK3C3-deficient Xenopus cells

  • Identify downstream signaling targets and feedback regulation

  • Example: Phosphoproteomic analysis of Xenopus laevis revealed enrichment in cellular functions related to glycolysis/gluconeogenesis and TCA cycle during dehydration

Imaging-Based Methods:

• Fluorescence Resonance Energy Transfer (FRET):

  • Generate fluorescent protein fusions with PIK3C3 and candidate interactors

  • Measure FRET signals in live Xenopus cells or embryos

  • Track dynamic interactions during different cellular processes

• Split-GFP Complementation:

  • Fuse complementary GFP fragments to PIK3C3 and candidate interactors

  • Fluorescence occurs only when proteins interact

  • Useful for validating interactions in Xenopus cells and monitoring subcellular localization

These methodologies can be employed to build comprehensive interaction maps for PIK3C3 in Xenopus laevis, allowing comparison with other vertebrate systems and providing insights into the evolution of autophagy and vesicular trafficking networks.

What are common challenges in working with recombinant Xenopus laevis PIK3C3 and how can they be addressed?

Working with recombinant PIK3C3 presents several technical challenges that can be addressed through specific methodological approaches:

Challenge 1: Low Expression Levels

• Solution: Optimize codon usage for expression system (insect cells, mammalian cells, etc.)

• Method: Design synthetic genes with optimized codons while maintaining the amino acid sequence

• Alternative: Use stronger promoters or inducible expression systems to enhance yield

Challenge 2: Protein Insolubility/Aggregation

• Solution: Express individual domains rather than full-length protein

• Method: Based on structural predictions, express catalytic domain separately from regulatory domains

• Alternative: Use solubility-enhancing fusion tags (MBP, SUMO, TRX) and optimize buffer conditions

Challenge 3: Loss of Enzymatic Activity

• Solution: Ensure proper post-translational modifications

• Method: Express in eukaryotic systems (insect cells preferred) rather than bacterial systems

• Verification: Include positive controls in kinase assays (known active kinases) and verify ATP binding

Challenge 4: Instability During Storage

• Solution: Optimize storage conditions with stabilizing agents

• Method: Add 0.02% NaN₃ and protease inhibitor cocktails as used for other Xenopus recombinant proteins

• Alternative: Flash-freeze small aliquots in storage buffer containing 10-20% glycerol

Challenge 5: Distinguishing Between L and S Homoeologs

• Solution: Design homoeolog-specific strategies

• Method: Use homoeolog-specific primers for cloning and verification

• Verification: Sequence verification and homoeolog-specific antibodies if available

Table 2: Troubleshooting Guide for Recombinant Xenopus laevis PIK3C3 Production

How can researchers analyze PIK3C3 function in the context of Xenopus laevis tissue-specific expression?

Analyzing PIK3C3 function in a tissue-specific context requires specialized approaches that account for the unique aspects of Xenopus laevis biology:

Tissue-Specific Expression Analysis:

• Single-Cell RNA Sequencing:

  • Dissociate tissues to single cells and perform scRNA-seq

  • Identify cell types expressing PIK3C3 and its regulatory partners

  • Compare expression patterns between tissues and developmental stages

  • Bioinformatically distinguish between L and S homoeologs

• Spatial Transcriptomics:

  • Apply in situ hybridization techniques with probes specific to PIK3C3

  • Design probes that can distinguish between L and S homoeologs

  • Combine with immunohistochemistry for protein-level validation

• Reporter Constructs:

  • Generate transgenic Xenopus with PIK3C3 promoter-driven fluorescent reporters

  • Create separate reporters for L and S homoeologs to track differential expression

  • Observe expression patterns in live embryos during development

Functional Analysis in Specific Tissues:

• Tissue-Specific CRISPR Knockout:

  • Use tissue-specific promoters to drive Cas9 expression

  • Design gRNAs targeting PIK3C3 (separate strategies for L and S homoeologs)

  • Example approach: The triple-knockout strategy used to establish colorless and immunodeficient amphibian models of Xenopus tropicalis could be adapted for PIK3C3

• Cell Type-Specific Inhibition:

  • Apply cell type-specific expression of dominant-negative PIK3C3

  • Use inducible systems (like CreERT2) for temporal control

  • Validate specificity using tissue-specific markers

• Ex Vivo Tissue Culture:

  • Establish primary cultures from specific Xenopus tissues

  • Apply PIK3C3 inhibitors or siRNA knockdown

  • Measure tissue-specific responses (e.g., insulin response in muscle, immune function in hematopoietic cells)

Analytical Methods for Tissue-Specific Function:

• Phosphoproteomic Analysis:

  • Compare phosphorylation profiles in tissues with high vs. low PIK3C3 expression

  • Identify tissue-specific signaling targets

  • Examples from mouse studies show PIK3C3 deficiency in myeloid cells affects surface expression of MHC molecules

• Metabolomic Analysis:

  • Profile metabolic changes in tissues with manipulated PIK3C3 levels

  • Focus on pathways known to be affected by PIK3C3 (glycerophospholipids, cardiolipins, etc.)

  • Connect to amphibian-specific adaptations like dehydration tolerance

• Autophagy Flux Measurement:

  • Compare autophagic activity across tissues with varying PIK3C3 expression

  • Use biochemical (LC3-II/LC3-I ratio) and imaging methods (GFP-LC3 puncta formation)

  • Connect to tissue-specific stress responses

These approaches can reveal how PIK3C3 function is adapted to the specific needs of different tissues in Xenopus laevis, potentially uncovering novel roles in amphibian-specific physiological adaptations.