Recombinant Danio rerio FUN14 domain-containing protein 1 (fundc1)

How is DrFundc1 expression distributed across zebrafish tissues and developmental stages?

DrFundc1 shows tissue-specific and temporally regulated expression patterns. The highest expression is found in the brain, followed by moderate expression in the liver, ovary, testis, and kidney, while the lowest expression levels are detected in heart and muscle tissues .

During embryonic development, DrFundc1 expression follows a dynamic pattern:

• Present in zygotes throughout embryogenesis

• Expression increases from the 1-cell stage

• Peaks at the gastrula stage (6 hours post-fertilization)

• Decreases at 12 hours post-fertilization (hpf)

• Maintained at relatively low levels from 24 hpf until hatching

This temporal expression pattern suggests critical roles during specific developmental windows, particularly during gastrulation when body axis formation occurs .

What is the evolutionary conservation of Fundc1 across species?

Phylogenetic analysis reveals that FUN14 domain-containing proteins are remarkably ancient and widespread, present across all three domains of life: archaea, bacteria, and eukaryotes . This exceptional evolutionary conservation suggests fundamental biological importance. In contrast, other mitophagy receptors like the BNIP3 domain-containing protein family evolved more recently and are present only in animals .

Multiple sequence alignments show that FUNDC1 is highly conserved among vertebrates, with critical functional residues maintaining conservation patterns across species. This conservation extends to the key regulatory mechanisms, such as the phosphorylation sites and the LC3-interacting region (LIR) motif that mediates interaction with autophagy machinery .

What are the optimal conditions for expressing and purifying recombinant DrFundc1?

For recombinant DrFundc1 expression, E. coli systems have been successfully employed. The full-length protein (amino acids 1-152) with an N-terminal His-tag produces good yields with high purity (>90% as determined by SDS-PAGE) . The recommended protocol includes:

• Expression in E. coli using a suitable expression vector containing the full DrFundc1 coding sequence

• Purification using nickel affinity chromatography leveraging the N-terminal His-tag

• Storage of the purified protein as a lyophilized powder

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol (5-50% final concentration) for long-term storage

• Storage at -20°C/-80°C with aliquoting to avoid freeze-thaw cycles

To maintain protein integrity, working aliquots should be stored at 4°C for no more than one week, as repeated freezing and thawing can compromise protein function .

How can researchers effectively knockdown and rescue DrFundc1 expression in zebrafish embryos?

For functional studies of DrFundc1 in zebrafish embryos, researchers have successfully employed the following methodological approach:

• Knockdown strategy: Short hairpin RNA (shRNA) targeting DrFundc1 has been effectively used to reduce expression levels. This approach led to observable phenotypes including midline bifurcation with two notochords and two spinal cords in zebrafish embryos .

• Rescue experiments: Co-injection of DrFundc1 mRNA alongside the shRNA successfully repaired the defects resulting from the knockdown, confirming the specificity of the observed phenotypes to DrFundc1 depletion .

• Validation: Verification of knockdown and rescue efficiency should be performed using both quantitative RT-PCR to measure transcript levels and Western blotting to assess protein levels, when antibodies are available.

• Phenotypic assessment: Whole-mount in situ hybridization (WISH) can be used to detect spatial expression patterns, while developmental abnormalities should be documented through high-resolution microscopy .

This experimental design allows for robust investigation of DrFundc1 function during zebrafish embryogenesis and can be adapted to study specific developmental processes.

How does DrFundc1 regulate mitophagy and how can this process be monitored experimentally?

DrFundc1 plays a crucial role in mitophagy regulation through its interaction with both mitochondria and autophagy machinery. Experimental evidence shows co-localization of DrFundc1 with MitoTracker (a mitochondrial marker) and CellLight Lysosomes-GFP (a lysosomal marker) in transfected cells . To study DrFundc1-mediated mitophagy, researchers can employ the following methodological approaches:

• Monitoring LC3B conversion: DrFundc1 expression induces the conversion of LC3B-I to LC3B-II, which is a key step in autophagy induction. This conversion can be detected by Western blotting .

• Co-localization studies: Fluorescently tagged DrFundc1 can be co-expressed with mitochondrial and lysosomal markers to visualize mitophagy events using confocal microscopy.

• Mitophagy flux assessment: Treatment with lysosomal inhibitors (e.g., bafilomycin A1) can help distinguish between increased autophagosome formation and impaired degradation.

• Expression analysis of mitophagy-related genes: DrFundc1 expression influences several autophagy- and apoptosis-related genes, including ATG5, ATG7, LC3B, BECLIN1, and BAX. qRT-PCR can be used to monitor these changes .

The experimental data shows that deliberate expression of DrFundc1 causes conversion of LC3B-I to LC3B-II, inducing autophagy, while co-localization with lysosomal markers indicates mitophagy occurrence .

What molecular mechanisms underlie DrFundc1's role in body axis formation during zebrafish development?

The molecular mechanisms through which DrFundc1 influences body axis formation involve complex interplay between mitochondrial function, autophagy, apoptosis, and developmental signaling. Research has revealed several key pathways:

• Regulation of cell death and proliferation: DrFundc1 expression affects cell viability, as demonstrated by Trypan blue staining, TUNEL assays, and BrdU incorporation studies in cell culture models. These processes are critical during embryonic patterning .

• Modulation of developmental gene expression: Knockdown of DrFundc1 results in altered expression of neural genes, including cyclinD1, suggesting a role in neural development regulation .

• Impact on midline structures: Depletion of DrFundc1 using shRNA leads to midline bifurcation with the formation of two notochords and two spinal cords, indicating its importance in establishing proper body symmetry and axial development .

• Cellular basis of developmental defects: When DrFundc1 is knocked down, both autophagy- and apoptosis-related genes show dysregulated expression, linking mitochondrial quality control to proper developmental patterning .

Experimentally, these mechanisms can be investigated through:

• Targeted gene expression analysis of developmental markers

• Live imaging of developing embryos following DrFundc1 manipulation

• Cellular assays measuring apoptosis, proliferation, and differentiation

• Rescue experiments with pathway-specific modulators

What are common challenges in working with recombinant DrFundc1 and how can they be overcome?

Researchers working with recombinant DrFundc1 may encounter several challenges that can be addressed through specific methodological approaches:

• Protein solubility issues: As a transmembrane protein, DrFundc1 may show limited solubility. This can be improved by:

  • Expressing only the soluble domains

  • Using detergents appropriate for membrane proteins

  • Adding solubility-enhancing tags

  • Optimizing buffer conditions

• Protein stability concerns: The recombinant protein requires careful handling to maintain activity:

  • Store as recommended at -20°C/-80°C with proper aliquoting

  • Avoid repeated freeze-thaw cycles

  • Use working aliquots at 4°C for no more than one week

  • Consider adding stabilizing agents like trehalose (6% is used in commercial preparations)

• Functional validation: Confirming that recombinant DrFundc1 maintains native activity can be challenging. Functional assays should include:

  • LC3B conversion detection by Western blotting

  • Co-localization studies with mitochondrial markers

  • Interaction analyses with known binding partners

• Antibody limitations: Due to limited availability of zebrafish-specific antibodies, researchers might need to:

  • Use cross-reactive antibodies from related species

  • Generate custom antibodies against DrFundc1

  • Employ epitope-tagged versions of the protein for detection

How can researchers distinguish between direct and indirect effects of DrFundc1 manipulation on developmental phenotypes?

When studying the effects of DrFundc1 manipulation on zebrafish development, distinguishing between direct and indirect effects requires sophisticated experimental approaches:

• Temporal-specific manipulations: Using inducible expression or degradation systems to alter DrFundc1 levels at specific developmental stages can help pinpoint when the protein's function is critical.

• Tissue-specific manipulations: Employing tissue-specific promoters to drive knockdown or overexpression can help determine if particular tissues are responsible for the observed phenotypes.

• Rescue experiments with domain mutants: Creating a series of DrFundc1 constructs with mutations in specific domains (e.g., the LIR motif or transmembrane regions) can identify which molecular functions are essential for normal development .

• Epistasis experiments: Combining DrFundc1 manipulation with modulation of downstream effectors can establish functional relationships within signaling pathways.

• Multi-omics approaches: Integrating transcriptomics, proteomics, and metabolomics data following DrFundc1 manipulation can reveal primary and secondary effects.

The existing research demonstrates that co-injection of wild-type DrFundc1 mRNA can rescue the developmental defects caused by DrFundc1 knockdown, confirming specificity . Further experimental designs building on this approach can provide deeper insights into direct versus indirect effects.

What are promising research avenues for understanding DrFundc1's role in disease models?

Given the critical role of DrFundc1 in development and mitochondrial quality control, several promising research directions emerge for disease modeling:

• Neurodevelopmental disorders: The high expression of DrFundc1 in brain tissue and its impact on neural gene expression suggest potential roles in neurodevelopmental conditions. Zebrafish models could be developed to study how DrFundc1 variants affect neural development and function .

• Mitochondrial diseases: As a mitophagy regulator, DrFundc1 may influence pathologies related to mitochondrial dysfunction. Research could explore how DrFundc1 manipulation affects models of mitochondrial disease in zebrafish.

• Hypoxia-related conditions: The differential regulation of FUNDC1 under hypoxic conditions in mammals suggests potential roles in hypoxia-related pathologies. Zebrafish models of stroke, cardiac ischemia, or tumor hypoxia could be valuable contexts to study DrFundc1 function .

• Regenerative medicine applications: Zebrafish are renowned for their regenerative capabilities. Investigating how DrFundc1-mediated mitophagy contributes to tissue regeneration could provide insights relevant to regenerative medicine.

Methodologically, these studies would benefit from:

• CRISPR/Cas9-mediated generation of DrFundc1 mutant lines

• High-resolution in vivo imaging of mitochondrial dynamics

• Integration with emerging spatial transcriptomics and proteomics approaches

• Drug screening platforms to identify modulators of DrFundc1 function

How might the evolutionary conservation of Fundc1 inform comparative studies across model organisms?

The remarkable evolutionary conservation of FUN14 domain-containing proteins across archaea, bacteria, and eukaryotes provides a rich foundation for comparative studies . This evolutionary perspective offers several research opportunities:

• Functional conservation testing: Expressing Fundc1 orthologs from different species in zebrafish or mammalian models can determine the degree of functional conservation across evolutionary distance.

• Domain-function relationships: Creating chimeric proteins combining domains from Fundc1 orthologs of different species can help identify which structural elements confer species-specific functions.

• Regulatory evolution: Comparing the transcriptional and post-translational regulation of Fundc1 across species can reveal how regulatory mechanisms evolved alongside protein structure.

• Adaptation to different oxygen environments: Different species have evolved in environments with varying oxygen availability. Studying how Fundc1 function differs between hypoxia-tolerant and hypoxia-sensitive species could reveal adaptive mechanisms.

The methodological approach should include:

• Rigorous phylogenetic analysis to identify true orthologs

• Structural biology techniques to compare protein folding and interaction surfaces

• Interactome mapping across species to identify conserved and divergent protein interactions

• Functional complementation experiments in various model systems

This evolutionary perspective can provide unique insights into both the fundamental biology of Fundc1 and its potential applications in biomedical research.