Recombinant Xenopus tropicalis Apoptosis-inducing factor 2 (aifm2)

What is AIFM2 and what are its primary functions?

AIFM2 (also known as AMID or PRG3) is a flavoprotein oxidoreductase with NADH/NAD oxidoreductase activity. Initially characterized as a p53 target involved in caspase-independent cell death, recent research has revealed its broader physiological roles. AIFM2 functions primarily as:

• A NADH oxidase that supports the maintenance of the NAD+/NADH ratio, which is crucial for sustaining glycolysis

• A regulator of mitochondrial metabolism, particularly in oxidative phosphorylation

• A protein that dynamically relocates between lipid droplets and mitochondria in response to cellular stimulation

Contrary to its name, recent studies indicate that AIFM2 may not induce apoptosis in certain cell types such as brown adipose tissue (BAT) cells, suggesting tissue-specific functions . In Xenopus, AIFM2 is likely involved in developmental processes and potentially in regeneration, though species-specific functions require further characterization.

How is AIFM2 distributed across tissues and organisms?

AIFM2 demonstrates distinct expression patterns across tissues and organisms:

In mammals:

• Highly and specifically expressed in brown adipose tissue (BAT)

• Induced upon cold exposure/β-adrenergic stimulation in BAT and inguinal white adipose tissue (iWAT)

• Low expression in various other organs

• Upregulated in certain cancers, including hepatocellular carcinoma (HCC)

In Xenopus tropicalis:

• While specific expression data for X. tropicalis AIFM2 is limited in the search results, the protein likely plays roles in developmental processes and potentially in tissue regeneration contexts

• Expression may be detected in various embryonic and tadpole tissues, with possible enrichment in metabolically active tissues

This tissue-specific expression pattern suggests that AIFM2 functions may be contextually regulated and potentially specialized across different cell types and developmental stages.

What are the key structural domains and localization features of AIFM2?

AIFM2 contains several important structural domains that dictate its function and localization:

• N-terminal hydrophobic region (aa1-27): Essential for both lipid droplet and mitochondrial localization

• NADH/NAD oxidoreductase domain: Critical for its enzymatic function in oxidizing NADH to NAD+

• C-terminal region (aa308-373): Not essential for lipid droplet localization

• N-myristoylation site at the glycine-2 residue: Required specifically for lipid droplet association

Deletion studies have revealed:

• Deletion of the N-terminal domain (Aifm2 ΔN) results in cytosolic localization

• G2A mutation prevents lipid droplet association but maintains mitochondrial localization

• Deletion of the C-terminal domain (Aifm2 ΔC) does not affect lipid droplet localization

AIFM2 lacks a canonical mitochondrial targeting signal sequence but can still associate with mitochondria upon stimulation, likely interacting with the outer side of the inner mitochondrial membrane . This unique localization behavior suggests a specialized mechanism for conditionally targeting mitochondria in response to cellular cues.

How do researchers isolate specific cell populations from Xenopus tropicalis for AIFM2 studies?

Isolating specific cell populations from Xenopus tropicalis for AIFM2 studies requires addressing unique challenges, particularly the presence of maternal yolk platelets that can introduce light scatter and false positives in FACS analysis. A recommended protocol includes:

• Tissue disaggregation procedure:

  • Dissect target tissues (e.g., tail, limb buds) from transgenic Xenopus tropicalis

  • Implement enzymatic digestion with appropriate proteases

  • Create single-cell suspensions through mechanical disruption and filtration

• FACS optimization for Xenopus cells:

  • Gate against both nontransgenic and ubiquitously transgenic animals to reduce false positives and negatives

  • Account for yolk platelet interference in light scatter patterns

  • Use the Xtr.Tg(pax6:GFP;cryga:RFP;actc1:RFP) Papal transgenic line as a control to validate sorting parameters

• Nucleic acid quality preservation:

  • Process sorted cells promptly to maintain RNA integrity

  • Use appropriate preservation buffers during the sorting process

  • Validate RNA quality with quantitative methods before downstream applications

This optimized approach ensures high-quality, specific cell isolation from complex Xenopus tissues, enabling precise analysis of AIFM2 expression and function in target cell populations.

How does AIFM2 regulate NAD+/NADH balance and its impact on cellular metabolism?

AIFM2 functions as a critical NADH oxidase that significantly influences the NAD+/NADH ratio, with profound implications for cellular metabolism:

Mechanism of action:

• Oxidizes NADH to NAD+ in the cytoplasm

• Transfers electrons to the mitochondrial electron transport chain

• Supports maintenance of cytosolic NAD+ pool necessary for glycolysis

Metabolic consequences:

• In brown adipose tissue, β-adrenergic stimulation increases NAD+/NADH ratio approximately 3-fold, which is prevented by AIFM2 knockdown

• AIFM2 knockdown reduces NAD+/NADH ratio by approximately 20% in unstimulated conditions

• This NAD+ regulation supports robust glycolysis, particularly important in metabolically active tissues

Experimental evidence for metabolic impact:

• Oxygen consumption rate (OCR) is significantly decreased with AIFM2 knockdown and increased with AIFM2 overexpression

• Activities of oxidative phosphorylation (OXPHOS) complexes follow the same pattern

• ATP production correlates with AIFM2 expression levels

Interestingly, despite these effects on oxidative metabolism, AIFM2 does not significantly alter glucose uptake or lactate production , suggesting it specifically regulates the efficiency of mitochondrial metabolism rather than substrate utilization. In Xenopus tropicalis, this metabolic regulation may be particularly important during energy-intensive developmental processes or regeneration events.

What is the relationship between AIFM2 and mitochondrial dynamics?

AIFM2 exhibits a complex relationship with mitochondrial dynamics, influencing multiple aspects of mitochondrial structure and function:

Effects on mitochondrial morphology and biogenesis:

• AIFM2 knockdown significantly decreases mitochondrial mass, while overexpression increases it

• AIFM2 regulates mitochondrial DNA (mtDNA) content, with knockdown decreasing and overexpression increasing mtDNA levels

• These effects suggest AIFM2 is a positive regulator of mitochondrial biogenesis

Mitochondrial membrane potential regulation:

• AIFM2 expression levels correlate with mitochondrial membrane potential

• Knockdown reduces membrane potential while overexpression enhances it

Mitochondrial localization dynamics:

• Without stimulation, AIFM2 primarily associates with lipid droplets

• Upon β-adrenergic stimulation, AIFM2 redistributes to mitochondria

• In mitochondria, AIFM2 specifically localizes to mitoplasts (inner membrane and matrix) rather than the outer membrane or intermembrane space

• This localization pattern intensifies with cold exposure in vivo

This dynamic relationship with mitochondria positions AIFM2 as a conditional regulator of mitochondrial function, responding to cellular energetic demands and environmental stimuli. For Xenopus tropicalis research, understanding these dynamics may provide insights into developmental energy regulation and tissue-specific metabolic adaptations.

How does AIFM2 interact with the SIRT1/PGC-1α signaling pathway?

AIFM2 exerts significant influence on cellular metabolism through its interaction with the SIRT1/PGC-1α signaling pathway:

Mechanism of interaction:

• AIFM2 increases NAD+ levels, which serves as a crucial substrate for SIRT1 activity

• AIFM2 post-transcriptionally upregulates PGC-1α protein expression without affecting its mRNA levels

• This suggests AIFM2 may enhance PGC-1α protein stability through SIRT1-mediated deacetylation

Experimental evidence:

• AIFM2 knockdown markedly decreases PGC-1α protein levels while overexpression increases them

• A significant positive correlation exists between AIFM2 and PGC-1α protein expressions in hepatocellular carcinoma tissues

• Overexpression of PGC-1α rescues the inhibitory effects of AIFM2 knockdown on mitochondrial function

• Conversely, PGC-1α knockdown attenuates the promotive effects of AIFM2 overexpression on mitochondrial function

Functional consequences:

• This signaling axis regulates mitochondrial biogenesis

• Controls oxidative phosphorylation capacity

• Influences ATP production

• May promote cellular migration and invasion in certain contexts

This AIFM2-SIRT1-PGC-1α axis represents a metabolic regulatory pathway that could be conserved across species, including Xenopus tropicalis. Understanding these interactions provides potential targets for manipulating cellular metabolism in experimental systems and could illuminate evolutionary conservation of metabolic regulation.

What methodological approaches can detect changes in AIFM2 subcellular localization?

Detecting AIFM2's dynamic subcellular localization requires specialized techniques that can distinguish between its lipid droplet and mitochondrial associations:

Biochemical fractionation approaches:

• Sucrose step gradient centrifugation (60%, 20%, 5%) to isolate lipid droplet fractions

• Differential centrifugation to separate pure cytosolic fractions from mitochondria

• Preparation of mitoplasts by ultracentrifugation of digitonin-treated mitochondria to distinguish between outer membrane/intermembrane space and inner membrane/matrix localization

Live cell imaging methods:

• GFP-tagged AIFM2 lentivirus transduction for dynamic visualization

• Co-staining with LipidTox for lipid droplet visualization

• MitoTracker Red for mitochondrial co-localization

• Quantification of co-localization percentages before and after stimulation (e.g., with isoproterenol)

Stimulation protocols to induce relocalization:

• β-adrenergic agonists (isoproterenol, CL-316,248)

• Cold exposure in vivo (for animal models)

• These stimuli trigger AIFM2 translocation from lipid droplets to mitochondria

Domain mapping strategies:

• Expression of deletion constructs (ΔN, ΔC) and point mutants (G2A) tagged with GFP

• Analysis of their subcellular distribution using the techniques above

• This approach has revealed the N-terminal domain and N-myristoylation as critical for proper localization

These methods could be adapted for Xenopus tropicalis systems with appropriate consideration of species-specific cell biology and available reagents. Visualization of AIFM2 dynamics in developing embryos or regenerating tissues could provide unique insights into its role in these contexts.

What are optimal approaches for expressing and purifying recombinant Xenopus tropicalis AIFM2?

Recombinant expression and purification of Xenopus tropicalis AIFM2 requires attention to its structural features and biochemical properties:

Expression system selection:

• Prokaryotic (E. coli): Suitable for basic structural studies but may lack post-translational modifications

• Eukaryotic (insect cells): Preferable for functional studies due to appropriate post-translational modifications, especially N-myristoylation

• Mammalian cells: Optimal for studying cellular localization and interactions but with lower yield

Construct design considerations:

• Include the complete open reading frame (ORF) with intact N-terminus to preserve myristoylation sites

• Consider expressing domain-specific constructs (N-terminal, NADH oxidoreductase domain, C-terminal) for structure-function studies

• For visualization and purification, C-terminal tags are preferable to avoid interfering with N-terminal modifications

Purification strategy:

• Affinity chromatography using appropriate tags (His, GST, or FLAG)

• Ion exchange chromatography as an intermediate purification step

• Size exclusion chromatography for final polishing and to ensure monomeric state

• Include reducing agents throughout purification to maintain the redox-active flavin cofactor

Quality control considerations:

• Verify proper flavin incorporation through spectroscopic analysis (absorption at 450nm)

• Confirm enzymatic activity through NADH oxidation assays

• For N-myristoylated protein, mass spectrometry can confirm the presence of the lipid modification

Commercial cDNA clones are available as starting materials for recombinant expression, with prices starting from $99.00 . These approaches enable production of functionally active AIFM2 suitable for biochemical, structural, and cellular studies.

How can researchers assess AIFM2's impact on mitochondrial function?

Comprehensive assessment of AIFM2's impact on mitochondrial function requires multiple complementary approaches:

Oxygen consumption measurements:

• Seahorse XF analysis to measure oxygen consumption rate (OCR)

• Parameters to measure: basal respiration, ATP-linked respiration, maximal respiratory capacity, and spare respiratory capacity

• Compare these parameters between AIFM2 knockdown, overexpression, and control conditions

OXPHOS complex activity assays:

• Spectrophotometric assays for individual respiratory chain complexes (I-V)

• Blue native PAGE combined with in-gel activity assays

• Results typically show corresponding changes in OXPHOS complex activities with AIFM2 expression levels

ATP production quantification:

• Luminescence-based ATP assays

• Real-time ATP production measurements using genetically encoded sensors

• AIFM2 expression positively correlates with cellular ATP levels

Mitochondrial membrane potential assessment:

• Fluorescent dyes such as TMRM, JC-1, or Rhodamine 123

• Flow cytometry or microscopy-based quantification

• AIFM2 knockdown typically reduces membrane potential while overexpression enhances it

Mitochondrial morphology and mass analysis:

• Confocal microscopy of mitochondrial networks using MitoTracker or mitochondrially-targeted fluorescent proteins

• Quantification of mitochondrial DNA content by qPCR

• Both parameters are positively regulated by AIFM2 expression

NAD+/NADH ratio measurement:

• Enzymatic cycling assays based on lactate dehydrogenase

• NAD+ biosensors for real-time measurements

• AIFM2 is essential for maintaining NAD+/NADH ratio, especially upon β-adrenergic stimulation

These methods provide a comprehensive assessment of how AIFM2 influences multiple aspects of mitochondrial biology and cellular energetics in experimental systems.

What approaches enable AIFM2 functional studies in Xenopus tropicalis?

Studying AIFM2 function in Xenopus tropicalis requires specialized approaches that leverage the unique advantages of this model system:

Transgenic animal generation:

• Create fluorescent reporter lines that express fluorescent proteins under the AIFM2 promoter

• Develop tissue-specific AIFM2 overexpression or knockdown lines

• Consider using the established Xtr.Tg(pax6:GFP;cryga:RFP;actc1:RFP) Papal transgenic line methodology as a template

Tissue-specific isolation techniques:

• Adapt the established protocol for disaggregation of complex tissues like tail and limb buds

• Account for yolk platelets that can introduce light scatter in FACS analysis

• Gate against both nontransgenic and ubiquitously transgenic animals to reduce false positives and negatives

Microinjection of morpholinos or CRISPR components:

• Target AIFM2 at early developmental stages

• Analyze phenotypic consequences on development, metabolism, and tissue patterning

• Rescue experiments with wild-type or mutant AIFM2 mRNA to confirm specificity

Ex vivo tissue culture:

• Establish explant cultures from transgenic animals

• Manipulate AIFM2 expression or activity using pharmacological agents

• Monitor metabolic parameters in real-time

Regeneration models:

• Leverage Xenopus tropicalis tail regeneration model

• Assess AIFM2 expression changes during regeneration

• Manipulate AIFM2 levels to determine impact on regenerative capacity

Genomic approaches:

• ATAC-Seq to determine chromatin accessibility changes in AIFM2-manipulated tissues

• RNA-Seq to identify transcriptional changes

• These approaches require high-quality nucleic acid preparations from sorted cells

These Xenopus-specific approaches enable investigation of AIFM2 function in developmental contexts, offering insights that complement studies in mammalian systems.

How can researchers investigate AIFM2 domain function through mutagenesis?

Strategic mutagenesis approaches can dissect the functional importance of specific AIFM2 domains:

Key domains for targeted mutagenesis:

• N-terminal domain (aa1-27): Critical for both lipid droplet and mitochondrial localization

• NADH/NAD oxidoreductase domain: Essential for enzymatic activity

• C-terminal domain (aa308-373): May influence protein stability or interactions

• N-myristoylation site (G2): Specifically required for lipid droplet association

Specific mutations and their predicted effects:

Functional readouts to assess mutant effects:

• Subcellular localization by fluorescence microscopy

• Biochemical fractionation to quantify distribution

• NADH oxidase activity measurements

• Impact on NAD+/NADH ratio

• Effects on mitochondrial biogenesis markers

• Ability to enhance PGC-1α protein levels

Expression systems for mutant analysis:

• Lentiviral transduction for stable expression in cell cultures

• Transient transfection for rapid screening

• mRNA injection into Xenopus embryos for developmental studies

• CRISPR/Cas9 knock-in strategies for physiological expression levels

This systematic mutagenesis approach enables precise mapping of structure-function relationships in AIFM2 and can reveal species-specific differences when applied across experimental models.