Recombinant Xenopus laevis Apoptosis-inducing factor 2 (aifm2)

What is the molecular structure and basic function of Xenopus laevis Aifm2?

Xenopus laevis Aifm2 is a 374-amino acid protein with DNA-binding oxoreductase activity that promotes apoptosis. The full-length recombinant protein can be expressed with an N-terminal His tag in E. coli expression systems . Its amino acid sequence begins with MGSKVSVEESVRVVIVGGGFAGIAAASQLKS and contains functional domains typical of the AIF family. As an oxoreductase, it likely participates in redox reactions within cells while also having the capacity to translocate to the nucleus during apoptosis where it can bind DNA and participate in chromatin condensation and DNA fragmentation .

How does recombinant Xenopus laevis Aifm2 differ from its mammalian counterparts?

While the core functions of Aifm2 are conserved across species, Xenopus laevis Aifm2 exists in a phylogenetically intermediate position between aquatic vertebrates and land tetrapods . This makes it valuable for evolutionary studies of apoptotic mechanisms. The Xenopus variant maintains the characteristic oxoreductase domain and DNA-binding capabilities but may exhibit species-specific regulatory mechanisms. Unlike mammalian systems where multiple AIF family members have been extensively characterized, Xenopus Aifm2 research is still emerging, offering opportunities to discover novel functional aspects not observed in mammals.

What expression systems are recommended for producing functional recombinant Xenopus laevis Aifm2?

The most common and effective expression system for recombinant Xenopus laevis Aifm2 is E. coli . When expressing the full-length protein (1-374 amino acids), an N-terminal His tag is typically added to facilitate purification through affinity chromatography. The protein is often recovered as a lyophilized powder after purification. For functional studies, researchers should consider that post-translational modifications present in native Aifm2 may be absent in bacterial expression systems, potentially affecting certain activities. For applications requiring post-translational modifications, insect cell (baculovirus) or mammalian expression systems might be preferable, though these approaches require optimization for Xenopus proteins.

What are the recommended protocols for effective knockdown of Aifm2 in Xenopus laevis models?

For Aifm2 knockdown in Xenopus models, RNA interference (RNAi) using short interfering RNAs (siRNAs) has proven effective. Based on protocols adapted from erythroid cell studies, transfection of siRNAs targeting Aifm2 for 24 hours can achieve significant knockdown . For Xenopus oocytes or early embryos, microinjection of siRNAs (typically 10-50 ng) directly into the cytoplasm is recommended. Alternatively, morpholino oligonucleotides can be designed to block Aifm2 translation and injected at the 1-2 cell stage. Validation of knockdown should be performed via Western blotting and qRT-PCR, with careful attention to designing primers specific to Xenopus laevis Aifm2 versus related family members.

How should researchers assess Aifm2 activity in Xenopus oocytes and embryos?

To assess Aifm2 activity in Xenopus systems, researchers can employ a combination of approaches:

• For apoptotic function assessment, near-infrared fluorescent caspase substrates can be microinjected into oocytes to monitor downstream caspase activation in real-time .

• Oxoreductase activity can be measured using NAD(P)H consumption assays adapted for whole cells or tissue extracts.

• DNA binding capability can be assessed through chromatin immunoprecipitation followed by sequencing (ChIP-seq).

• Subcellular localization should be tracked using immunofluorescence or by expressing fluorescently-tagged Aifm2 to observe translocation between mitochondria and nucleus during apoptosis.

• For effects on gene expression, researchers should examine changes in erythropoietic factors like Klf1 and globin genes, which have been shown to be regulated in relation to Aifm2 function in other systems .

What purification methods yield the highest activity for recombinant Xenopus laevis Aifm2?

A multi-step purification approach is recommended for obtaining high-activity recombinant Aifm2:

Maintaining reducing conditions (1-5 mM DTT or 2-10 mM β-mercaptoethanol) throughout purification is critical for preserving enzyme activity. The purified protein should be stored with 10% glycerol at -80°C, avoiding repeated freeze-thaw cycles which significantly reduce activity .

How can researchers resolve the apparently contradictory findings regarding Aifm2's role in apoptosis versus differentiation?

The dual role of Aifm2 in apoptosis and differentiation represents an important research paradox. To address this contradiction, researchers should:

• Conduct temporal studies to determine if Aifm2 functions differently at various developmental stages in Xenopus.

• Perform domain-specific mutagenesis to separate the oxoreductase function from the DNA-binding capability, determining which domain correlates with each cellular outcome.

• Investigate context-dependent protein interactions using proximity labeling approaches (BioID or APEX) to identify different Aifm2 binding partners under apoptotic versus differentiation conditions.

• Examine post-translational modifications of Aifm2 that might direct its function toward either pathway.

Evidence from erythroid studies suggests that Aifm2 knockdown increases expression of the erythropoietic transcription factor Klf1 while decreasing globin expression, indicating a role in differentiation that may be independent of its apoptotic function . In Xenopus, researchers should exploit the developmental transitions (particularly metamorphosis) to examine how Aifm2 function changes during periods of programmed cell death versus cellular differentiation.

What techniques can distinguish between caspase-dependent and Aifm2-mediated (caspase-independent) cell death in Xenopus models?

To distinguish between these pathways, researchers should implement a multi-faceted approach:

• Utilize the near-infrared caspase substrate imaging technique in Xenopus oocytes to monitor caspase activity in real-time while simultaneously tracking Aifm2 localization with fluorescently-tagged protein .

• Apply pharmacological inhibitors selectively: z-VAD-fmk for pan-caspase inhibition versus specific Aifm2 inhibitors.

• Perform genetic manipulations where both pathways are independently modulated:

  • Cytochrome c microinjection (50-100 nM threshold) to trigger caspase-dependent death

  • Forced Aifm2 nuclear localization to induce caspase-independent death

• Analyze nuclear morphology changes specific to each pathway:

  • Large-scale DNA fragmentation (50 kb fragments) is characteristic of Aifm2-mediated death

  • Oligonucleosomal fragmentation (180 bp ladder) is typical of caspase-dependent apoptosis

How does cell cycle stage influence Aifm2 function in Xenopus oocytes and early embryos?

Cell cycle stage significantly impacts Aifm2 function in Xenopus models. Research approaches should include:

• Synchronize oocytes at different meiotic stages using progesterone (PG) treatment and compare Aifm2 localization and activity .

• Examine the influence of M-phase versus interphase on Aifm2-induced apoptosis using cell cycle inhibitors.

• Test the hypothesis that M-phase arrest confers resistance to Aifm2-mediated death, similar to the observed resistance to cytochrome c-induced apoptosis in meiotic oocytes .

• Investigate if the eventual death of meiosis-arrested oocytes proceeds through Aifm2-dependent mechanisms since this death appears to be caspase-independent .

The unique cell cycle transitions in Xenopus development provide an exceptional system for studying how Aifm2 function is modulated by cell cycle regulators, potentially revealing novel regulatory mechanisms not easily observed in other models.

How can Xenopus laevis Aifm2 studies inform our understanding of apoptotic mechanisms across vertebrate evolution?

Xenopus laevis occupies a phylogenetically intermediate position between aquatic vertebrates and land tetrapods, making it invaluable for evolutionary studies of apoptotic mechanisms . Research approaches should:

• Perform comparative sequence and structural analyses of Aifm2 across species ranging from fish to mammals.

• Conduct cross-species functional complementation experiments to determine if Xenopus Aifm2 can rescue mammalian Aifm2-deficient systems.

• Examine conservation of protein-protein interactions, particularly with IAPs (Inhibitor of Apoptosis Proteins), which play critical roles in determining sensitivity to apoptotic stimuli .

• Investigate whether the relationship between Aifm2 and erythropoietic transcription factors like Klf1 is conserved across vertebrate evolution .

These approaches can reveal which aspects of Aifm2 function represent ancestral characteristics and which are species-specific adaptations, providing insight into the evolution of programmed cell death pathways.

How does Aifm2 function differ between Xenopus oocytes, embryos, and adult tissues?

Understanding the tissue and developmental specificity of Aifm2 function requires:

• Comprehensive expression profiling of Aifm2 across developmental stages and tissues using RNA-seq and protein quantification.

• Functional assays in isolated tissues using recombinant Aifm2 to determine tissue-specific sensitivities.

• Comparison of Aifm2-interacting partners between developmental stages using co-immunoprecipitation followed by mass spectrometry.

• Investigation of whether the non-apoptotic functions of Aifm2 (like regulation of erythropoietic factors) are restricted to specific developmental contexts .

Of particular interest is whether Aifm2 plays specialized roles during the dramatic tissue remodeling that occurs during Xenopus metamorphosis, a period characterized by extensive programmed cell death and differentiation .

What are the critical quality control parameters for recombinant Xenopus laevis Aifm2 preparations?

Researchers should verify the following quality parameters for recombinant Aifm2:

For functional studies, the oxidoreductase activity should be specifically measured using appropriate substrates, and DNA binding capability confirmed before experimental use .

How can researchers address experimental variability when studying Aifm2-mediated effects in Xenopus oocytes?

To minimize variability in Xenopus oocyte experiments:

• Use sibling oocytes from the same female whenever possible to reduce genetic and developmental variability.

• Implement standardized staging criteria for oocyte selection, particularly when studying meiotic transitions.

• When microinjecting cytochrome c or Aifm2 to induce apoptosis, maintain strict control over injection volume and protein concentration; calibrate microinjection equipment regularly.

• For caspase activity monitoring, standardize the concentration of near-infrared substrate and image acquisition parameters .

• Include appropriate controls for each experiment:

  • Buffer-injected negative controls

  • Positive controls with known apoptosis inducers

  • Dose-response curves to establish thresholds (e.g., 50-100 nM cytochrome c threshold)

Careful documentation of the source and health status of Xenopus females is also critical, as seasonal variations and housing conditions can affect oocyte quality and response to experimental manipulations.