Recombinant Danio rerio NEDD4 family-interacting protein 1-like (ndfip1l)

How does ndfip1l expression and function compare to its orthologs in other species?

Research indicates significant functional conservation of Ndfip1 proteins across vertebrate species, though with some species-specific adaptations. In mammals, Ndfip1 has been shown to function as an adaptor protein that facilitates the interaction between NEDD4 family ubiquitin ligases and their substrates .

The role of Ndfip1 in neuronal function appears conserved, as studies in rats have shown that Ndfip1 expression correlates with learning ability. Fast learners in water maze tasks demonstrate decreased Ndfip1 mRNA and protein expression compared to slow learners . Similarly, spatial training in rats decreases Ndfip1 mRNA and protein expression levels .

In mice, genetic studies using conditional heterozygous (cHet) mice for Ndfip1 have shown enhanced spatial memory performance compared to controls, further supporting the protein's conserved role in cognitive function .

While zebrafish ndfip1l shares these fundamental functions, its specific expression patterns during embryonic development may differ from mammals. The zebrafish model offers unique advantages for studying ndfip1l function due to the transparent nature of embryos and rapid development, allowing for in vivo visualization of protein dynamics .

What experimental approaches are most effective for studying ndfip1l expression and function in zebrafish?

When designing experiments to study ndfip1l in zebrafish, researchers should consider the following methodological approaches:

Gene expression analysis:

• qRT-PCR for quantifying ndfip1l mRNA levels at different developmental stages

• In situ hybridization to visualize spatial expression patterns in embryos

• RNA-seq for genome-wide expression analysis in the context of ndfip1l manipulation

Protein detection methods:

• Western blotting using antibodies specific to zebrafish ndfip1l

• Immunohistochemistry for spatial localization in tissue sections

• Live imaging using fluorescently tagged ndfip1l in transgenic lines

Functional studies:

• Morpholino-mediated knockdown targeting ndfip1l

• CRISPR/Cas9 genome editing to generate knockout or knock-in lines

• Transgenic overexpression of wild-type or mutant forms

Protein interaction studies:

• Yeast two-hybrid screening as demonstrated for other zebrafish proteins

• Co-immunoprecipitation assays to validate interactions

• Proximity ligation assays for in situ detection of protein-protein interactions

For developmental studies, utilizing zebrafish embryos at specific time points (e.g., 48 hpf, 72 hpf, and 96 hpf) allows for systematic analysis of ndfip1l function throughout early development .

How should researchers design genetic manipulation experiments for ndfip1l in zebrafish?

When designing genetic manipulation experiments for ndfip1l in zebrafish, follow these methodological guidelines:

Knockout strategy:

• Design CRISPR/Cas9 guide RNAs targeting conserved exons of ndfip1l

• Inject a mixture of sgRNA and Cas9 mRNA/protein into one-cell stage embryos

• Validate editing efficiency through sequencing

• Establish stable lines through outcrossing and genotyping

Conditional knockout approach:
Similar to the strategy used for Ndfip1 in mice , researchers can:

• Insert loxP sites flanking critical exons (e.g., Exon 3) of ndfip1l

• Cross with tissue-specific Cre-expressing lines

• Validate conditional deletion through tissue-specific PCR

Knockdown strategy:

• Design morpholinos targeting the translation start site or splice junctions

• Inject morpholinos at the 1-4 cell stage

• Include appropriate controls (standard control morpholino)

• Validate knockdown efficiency by Western blot

Genotyping protocol:

• PCR amplification using specific primers flanking the target region

• Expected products: wild-type band at expected size, mutant bands showing deletions or insertions

• Run on 2% agarose gel for detection of small indels

Essential controls:

• Include wild-type siblings as controls

• Use rescue experiments with mRNA co-injection to confirm specificity

• Consider generating multiple independent mutant lines to confirm phenotypes

What approaches are effective for studying ndfip1l protein-protein interactions?

Based on research methodologies applied to similar proteins, the following approaches are recommended for studying ndfip1l protein-protein interactions:

Yeast two-hybrid screening:

• Use zebrafish ndfip1l as bait protein in the pGBKT7 vector

• Create a cDNA library from zebrafish embryos at relevant developmental stages (48-96 hpf)

• Transform bait and prey plasmids into yeast

• Select positive colonies on selection media and validate through sequencing

This approach has been successfully used to identify protein interactions in zebrafish, yielding libraries with titers of approximately 1.12 × 10^7 CFU/mL and recombination rates of 100% .

Co-immunoprecipitation:

• Express tagged versions of ndfip1l in zebrafish embryos or cell lines

• Immunoprecipitate with tag-specific antibodies

• Analyze co-precipitating proteins by Western blot or mass spectrometry

• Validate interactions through reciprocal co-immunoprecipitation

Proximity-based methods:

• BioID or TurboID fusion with ndfip1l to identify proximal proteins

• FRET or BRET assays for direct interaction analysis

• Split-GFP complementation to visualize interactions in vivo

Based on studies of mammalian Ndfip1, potential interaction partners to investigate include Nedd4, Nedd4-2, and Itch ubiquitin ligases, as these have been shown to be recruited by Ndfip1 into exosomes .

How does ndfip1l contribute to exosome biogenesis and function in zebrafish?

Based on studies of Ndfip1 in other systems, ndfip1l likely plays a significant role in exosome biology in zebrafish. Research has shown that Ndfip1:

• Is detectable in exosomes secreted from transfected cells and primary neurons

• Increases exosome secretion compared to control conditions

• Recruits Nedd4 family proteins (Nedd4, Nedd4-2, and Itch) into exosomes, which are normally absent from these vesicles

To investigate ndfip1l's role in exosome biology in zebrafish, researchers should consider:

Methodological approach:

• Express tagged ndfip1l in zebrafish embryos or derived cell lines

• Isolate exosomes through differential ultracentrifugation or size exclusion chromatography

• Characterize exosome content with and without ndfip1l manipulation

• Analyze exosome size distribution and number using nanoparticle tracking analysis

• Perform proteomics on isolated exosomes to identify cargo proteins

Exosomes are small vesicles (50-90 nm diameter) originating from late endosomes and multivesicular bodies (MVBs) that provide a rapid means of shedding obsolete proteins and facilitating cell-to-cell communication . The ndfip1l protein likely influences which proteins are sorted into these vesicles through its ability to interact with ubiquitin ligases.

Since exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress , studying ndfip1l's role in this process could reveal mechanisms of cellular response to stress in zebrafish.

How can researchers leverage the zebrafish model to understand ndfip1l's role in protein ubiquitination pathways?

Zebrafish offer unique advantages for studying ndfip1l's role in ubiquitination pathways through their transparency and rapid development. Based on the predicted functions of ndfip1l in protein ubiquitination , researchers can:

Experimental approach for studying ubiquitination:

• Generate transgenic zebrafish expressing fluorescently tagged ubiquitin

• Manipulate ndfip1l expression through knockdown or overexpression

• Visualize ubiquitination patterns in vivo through confocal microscopy

• Isolate specific tissues for biochemical analysis of ubiquitinated proteins

Biochemical assays:

• Ubiquitination assays using zebrafish embryo lysates with wild-type or manipulated ndfip1l

• Mass spectrometry to identify differentially ubiquitinated proteins

• Analysis of ubiquitin chain topology (K48 vs. K63 linkages) to distinguish between degradative and non-degradative ubiquitination

Target protein analysis:
Based on mammalian studies, potential targets to investigate include:

• Beclin 1, which has been studied in the context of Ndfip1-mediated ubiquitination

• PTEN, another known substrate mentioned in the research

From the literature, we know that siRNA-mediated knockdown of Ndfip1 has been used to study its effects on Beclin 1 and PTEN ubiquitination in mammalian cells . Similar approaches could be adapted for zebrafish studies, potentially using cell lines derived from zebrafish or direct manipulation in embryos.

What are the optimal conditions for storage and handling of recombinant ndfip1l protein?

Based on the product information for recombinant Danio rerio ndfip1l, researchers should follow these storage and handling recommendations:

Storage conditions:

• Store at -20°C for standard use

• For extended storage, conserve at -20°C or -80°C

• Avoid repeated freezing and thawing

• For working aliquots, store at 4°C for up to one week

Buffer composition:

• Tris-based buffer with 50% glycerol, optimized for protein stability

• The specific buffer composition should be maintained when diluting the protein

Handling precautions:

• Thaw aliquots on ice

• Avoid vigorous vortexing which may cause protein denaturation

• Centrifuge briefly after thawing to collect contents

• Use low-protein binding tubes for storage and dilution

The recombinant protein is typically provided at a quantity of 50 μg, though other quantities may be available through special request . When designing experiments using this protein, researchers should carefully calculate the appropriate concentration based on their specific assay requirements.

How can researchers address potential challenges in zebrafish ndfip1l knockout studies?

Based on experimental approaches in zebrafish and studies of Ndfip1 in other model systems, researchers may encounter these challenges when creating and analyzing ndfip1l knockout models:

Common challenges and solutions:

Genotyping considerations:
For reliable genotyping of ndfip1l mutants, design primers that can distinguish between wild-type and mutant alleles. Based on similar approaches for Ndfip1 in mice, the PCR parameters should include:

• 95°C for 30 s

• 60°C for 30 s

• 72°C for 10 s for 36 cycles

• Final elongation at 72°C for 90 s

• Analysis on 2% agarose gel

Phenotypic assessment:
When analyzing ndfip1l knockout phenotypes, consider examining:

• Developmental timing and morphology

• Ubiquitination patterns in relevant tissues

• Exosome production and content

• Neuronal development and cognitive function

• Protein trafficking in the secretory pathway

What considerations should researchers take into account when analyzing contradictory data about ndfip1l function?

When confronted with contradictory findings regarding ndfip1l function, researchers should apply these methodological approaches:

Systematic analysis framework:

• Evaluate experimental design differences between studies

  • Model systems used (cell lines vs. whole organisms)

  • Developmental timing of manipulations

  • Methods of gene/protein manipulation (knockout, knockdown, overexpression)

  • Assay sensitivities and specificities

• Consider context-dependent functions

  • Tissue-specific effects

  • Developmental stage-specific roles

  • Stress or stimulus-dependent activities

• Assess potential technical confounders

  • Antibody specificity issues

  • Off-target effects of genetic manipulations

  • Interference from compensatory mechanisms

Reconciliation strategies:

• Direct comparison experiments that reproduce contradictory conditions side-by-side

• Collaboration with labs reporting different results

• Meta-analysis of multiple datasets

• Development of more sensitive or specific assays

Example scenario:
If one study shows ndfip1l promotes exosome secretion while another suggests inhibition, consider that:

• Effects may differ based on cell type or developmental stage

• Baseline levels of Nedd4 family proteins might influence outcomes

• Acute vs. chronic manipulation may yield different results

• Specific cargo proteins might be differently affected

This approach is supported by research on Ndfip1 showing its diverse functions in protein trafficking, ubiquitination, and exosome secretion that may manifest differently depending on cellular context .

How can insights from zebrafish ndfip1l research be applied to understanding human NDFIP1 function?

Zebrafish ndfip1l research offers valuable translational insights that can enhance our understanding of human NDFIP1 function through these methodological approaches:

Comparative analysis framework:

• Sequence homology analysis between zebrafish ndfip1l and human NDFIP1

• Structural modeling to identify conserved functional domains

• Cross-species rescue experiments (human NDFIP1 expression in zebrafish ndfip1l mutants)

• Parallel manipulation of orthologous pathways

The zebrafish model offers specific advantages for translational research:

• Rapid development allows for high-throughput screening

• Transparency enables in vivo visualization of protein dynamics

• Conservation of core molecular pathways with mammals

• Feasibility of generating genetic models at lower cost and time investment

Specific translational applications:

• Neurological disorders - Based on Ndfip1's role in learning and memory , zebrafish models could help understand cognitive disorders

• Protein trafficking diseases - Given ndfip1l's predicted role in protein transport , findings could inform understanding of trafficking disorders

• Stress response mechanisms - Ndfip1's involvement in exosome-mediated protein removal during stress suggests applications in stress-related pathologies

The zebrafish larva has been validated as an attractive translational vertebrate screening model that bridges the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates , making it particularly valuable for studying ndfip1l/NDFIP1 function in a physiologically relevant context.

What experimental design principles should be followed when using ndfip1l research for drug discovery applications?

When leveraging ndfip1l research in zebrafish for drug discovery applications, researchers should follow these experimental design principles:

Screening framework design:

• Define clear molecular or phenotypic endpoints based on ndfip1l function

• Establish quantifiable readouts (e.g., fluorescent reporters for ubiquitination or exosome secretion)

• Develop appropriate positive and negative controls

• Optimize assay conditions for reproducibility and statistical power

• Consider both target-based and phenotypic screening approaches

Key experimental design elements:

Pharmacological considerations:

• Assess compound solubility, stability, and toxicity in zebrafish

• Determine optimal administration routes (bath exposure vs. injection)

• Evaluate ADME properties in the zebrafish system

• Consider potential off-target effects

For in vivo imaging of drug effects, confocal microscopy approaches as described for zebrafish studies can be employed, using:

• Sequential line scanning

• Excitation wavelengths of 488 and 561 nm

• Emission wavelengths of 500-540 and 570-620 nm

• Analysis with appropriate software such as OMERO and Fiji

By following these principles, researchers can develop robust drug discovery platforms targeting ndfip1l-related pathways while ensuring scientific rigor and translational relevance.