Recombinant Danio rerio Vesicle transport protein USE1 (use1), partial

What is the function of Vesicle transport protein USE1 in Danio rerio?

Vesicle transport protein USE1 in Danio rerio functions as a crucial component of the cellular vesicular trafficking machinery, particularly in the transport between the endoplasmic reticulum (ER) and Golgi apparatus. It participates in the SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex formation, which is essential for vesicle docking and fusion. In zebrafish, USE1 plays a significant role in embryonic development, particularly in organogenesis and neural development, where precise protein trafficking is required for proper cell differentiation and tissue formation. The protein also contributes to the maintenance of ER homeostasis and the unfolded protein response, which are critical for cellular health and function.

How is USE1 protein expression regulated during zebrafish development?

USE1 protein expression in zebrafish follows a dynamic pattern throughout development, with significant temporal and spatial regulation. Expression begins during early embryogenesis, with maternal transcripts present in the fertilized egg. As development progresses, USE1 expression increases in tissues undergoing active morphogenesis, particularly in the developing nervous system, liver, and pancreas. Regulation occurs through multiple mechanisms, including transcriptional control by developmental stage-specific transcription factors, post-transcriptional regulation through microRNAs, and post-translational modifications that affect protein stability and activity. This complex regulatory network ensures that USE1 function is precisely controlled during critical developmental events requiring coordinated vesicular transport.

What is the domain structure of Danio rerio USE1 protein?

The Danio rerio USE1 protein contains several functional domains that are conserved across species. Similar to recombinant proteins like SOD1, the structural organization is critical to its function. The protein typically contains:

• N-terminal regulatory domain: Controls protein interactions and may be subject to post-translational modifications

• SNARE domain: A coiled-coil region essential for SNARE complex formation

• Membrane association domain: Often found at the C-terminus, facilitating anchoring to ER membranes

• Interaction motifs: Specific sequences that mediate binding to other vesicular transport proteins

This domain architecture enables USE1 to perform its essential role in vesicular trafficking by facilitating protein-protein interactions and membrane association.

How similar is zebrafish USE1 to its mammalian orthologs?

Zebrafish USE1 shares significant sequence and structural homology with its mammalian counterparts, making it a valuable model for studying conserved vesicular transport mechanisms. Alignment analyses typically show:

This high degree of conservation, particularly in functional domains, supports the use of zebrafish as a model organism for investigating vesicular transport mechanisms relevant to human health and disease. The differences that do exist are primarily in regulatory regions, suggesting species-specific control mechanisms while maintaining core functionality.

How should I design experiments to study USE1 function in zebrafish models?

When designing experiments to study USE1 function in zebrafish, employing a fractional factorial design approach can effectively reduce experimental complexity while maintaining scientific rigor. Begin by clearly defining your research questions and selecting appropriate factors to investigate, such as developmental stages, tissue specificity, or environmental conditions. For loss-of-function studies, consider using CRISPR-Cas9 gene editing to generate USE1 mutants, or morpholino-based knockdown for transient effects. For gain-of-function experiments, mRNA injection or transgenic overexpression systems can be utilized. Incorporate appropriate controls, including wild-type siblings, injection controls, and rescue experiments to validate phenotypes.

Time-course experiments are particularly valuable when studying USE1's role in development, as they capture the dynamic nature of vesicular transport during embryogenesis. Additionally, consider using tissue-specific or inducible expression systems to dissect USE1's function in specific cellular contexts or developmental windows. Statistical power calculations should be performed prior to experimentation to determine appropriate sample sizes, typically requiring 25-30 embryos per condition across at least three independent biological replicates.

What are the optimal methods for expressing and purifying recombinant Danio rerio USE1 protein?

For optimal expression and purification of recombinant Danio rerio USE1 protein, a systematic approach considering several key factors is necessary. The expression system should be carefully selected based on requirements for post-translational modifications and protein folding. While bacterial systems (E. coli) offer high yield and simplicity, eukaryotic systems such as insect cells or mammalian cell lines often provide better folding for complex proteins like USE1.

Expression optimization protocol:

• Clone the USE1 coding sequence into an appropriate vector containing a fusion tag (6xHis, GST, or MBP) to facilitate purification

• Express in the selected host system with optimization of temperature (typically 16-25°C for better folding), induction conditions, and expression duration

• Lyse cells under native conditions with appropriate detergents to solubilize membrane-associated portions

• Perform multi-step purification using affinity chromatography, followed by size exclusion and/or ion exchange chromatography

• Verify purity by SDS-PAGE (>90% purity) and Western blotting

• Confirm activity through functional assays specific to SNARE proteins

Storage conditions should mirror those used for similar recombinant proteins like SOD1, with -20°C or -80°C storage and avoidance of repeated freeze-thaw cycles to maintain protein integrity and activity.

What controls should be included when studying USE1 protein interactions?

When investigating USE1 protein interactions, a comprehensive set of controls is essential to ensure experimental validity and reproducibility. These should include:

Additionally, reciprocal experiments (e.g., switching bait and prey in yeast two-hybrid) should be performed to validate interactions from multiple perspectives. For co-localization studies, appropriate non-colocalizing proteins should be included to establish specificity thresholds. Statistical analysis of multiple replicates (minimum of three independent experiments) is required to establish confidence in observed interactions.

How can CRISPR-Cas9 gene editing be optimized for studying USE1 function in zebrafish?

Optimizing CRISPR-Cas9 gene editing for studying USE1 function in zebrafish requires careful consideration of several technical parameters. Begin by designing multiple sgRNAs targeting conserved functional domains of the USE1 gene, particularly the SNARE domain which is essential for its function. Utilize predictive algorithms to design sgRNAs with high on-target efficiency and minimal off-target effects. For sgRNA synthesis, in vitro transcription using T7 RNA polymerase is recommended, followed by purification to remove incomplete transcripts.

The microinjection protocol should be standardized for consistency:

• Prepare injection mixture containing 25-50 pg of sgRNA and 300-500 pg of Cas9 protein or mRNA

• Inject 1-2 nl into one-cell stage embryos at the cell-yolk boundary

• Include phenol red (0.05%) as an injection tracer

• Maintain consistent temperature (28.5°C) during injections

• Screen for successful editing using techniques such as T7 endonuclease assay, HRMA, or direct sequencing

To rigorously validate the resulting phenotypes, employ multiple approaches including rescue experiments with wild-type USE1 mRNA, analysis of multiple independent mutant lines, and complementary approaches such as morpholino knockdown. For studying vesicular transport functions specifically, incorporate subcellular tracking methods to monitor protein trafficking dynamics in wild-type versus USE1-mutant embryos.

What imaging techniques are most effective for visualizing USE1-mediated vesicular transport in zebrafish?

For effective visualization of USE1-mediated vesicular transport in zebrafish, a multi-modal imaging approach yields the most comprehensive data. Confocal microscopy with spinning disk technology offers the optimal balance of resolution and speed for capturing dynamic vesicular events in living embryos. When implementing this approach:

• Generate transgenic lines expressing fluorescently-tagged USE1 (mEGFP-USE1 or USE1-mCherry) under endogenous promoters using BAC recombineering to maintain physiological expression levels

• Complement with markers for specific compartments (ER, ERGIC, Golgi) to track co-localization during transport

• Employ super-resolution techniques (STED, PALM, or STORM) for detailed structural analysis of USE1-containing complexes

• Implement FRAP (Fluorescence Recovery After Photobleaching) to measure protein dynamics and residence time in vesicular structures

• Utilize FRET-based approaches to detect direct protein interactions within the SNARE complex

For quantitative analysis of vesicular transport:

• Track vesicle number, size, velocity, and directionality using automated image analysis software

• Implement pulse-chase experiments with photo-convertible fluorescent proteins to follow specific vesicle populations

• Correlate light and electron microscopy (CLEM) to connect functional observations with ultrastructural details

Temperature control is critical during imaging experiments (maintain at 28.5°C) to ensure physiological transport kinetics, and phototoxicity should be minimized through careful optimization of laser power and exposure times.

How does USE1 function interact with cellular stress responses in zebrafish models?

USE1 function is intricately connected with cellular stress responses in zebrafish models, particularly with the unfolded protein response (UPR) and ER stress pathways. During ER stress conditions, USE1-mediated vesicular transport undergoes significant modifications to accommodate the increased demand for protein quality control and homeostasis maintenance. Research indicates that USE1 protein levels and activity are dynamically regulated during stress responses through several mechanisms:

• Transcriptional upregulation via stress-responsive elements in the USE1 promoter

• Post-translational modifications (primarily phosphorylation) that alter USE1 interaction with other SNARE components

• Redistribution of USE1 within the ER-Golgi network to facilitate stress granule formation

Experimental approaches to study this interaction include:

• Exposure of zebrafish embryos to ER stress inducers (tunicamycin, thapsigargin) followed by analysis of USE1 expression, localization, and function

• Time-course studies examining the correlation between UPR activation markers (BiP, XBP1 splicing) and USE1 activity

• USE1 depletion or overexpression combined with stress challenge to assess resilience or vulnerability to ER stress

Notably, zebrafish USE1 mutants typically show heightened sensitivity to ER stressors, manifesting as increased apoptosis in tissues with high secretory activity (pancreas, liver, neuromasts). This relationship highlights the critical role of efficient vesicular transport in maintaining cellular resilience against proteotoxic stress.

What is the recommended protocol for generating antibodies against zebrafish USE1 protein?

The generation of specific antibodies against zebrafish USE1 protein requires careful antigen design and validation steps. For optimal results, follow this comprehensive protocol:

Antigen preparation:

• Identify unique, surface-exposed epitopes specific to zebrafish USE1 using bioinformatic prediction tools

• Synthesize 2-3 peptides (15-20 amino acids) from different regions of USE1, prioritizing sequences with high antigenicity and low homology to other zebrafish proteins

• Alternatively, express and purify recombinant fragments (50-150 amino acids) of USE1 protein, focusing on unique domains

• Conjugate peptides to carrier proteins (KLH or BSA) using glutaraldehyde or MBS coupling chemistry

Immunization strategy:

• Utilize at least two different animal species (rabbit and guinea pig) to generate polyclonal antibodies

• Implement a 90-day immunization schedule with primary immunization plus 3-4 booster injections

• Collect test bleeds at days 35, 56, and 77 to monitor antibody titer development

• Perform final collection at day 90 when titers reach plateau

Antibody purification and validation:

• Affinity-purify antibodies using immobilized antigenic peptides or recombinant proteins

• Validate specificity through Western blotting against zebrafish tissue lysates, comparing wild-type and USE1-depleted samples

• Confirm reactivity in fixed tissue through immunohistochemistry, including appropriate blocking controls

• Test cross-reactivity with related proteins, particularly other SNARE family members

• Evaluate performance in immunoprecipitation assays if this application is required

Proper storage at -20°C with glycerol (50%) or at -80°C in smaller aliquots will maintain antibody activity and prevent freeze-thaw cycles that decrease effectiveness.

What are the best methods for quantifying USE1 protein expression levels in zebrafish tissues?

Accurate quantification of USE1 protein expression in zebrafish tissues requires a combination of approaches to ensure reliability and sensitivity. The following methods are recommended:

• Quantitative Western Blotting:

  • Systematically homogenize tissues in denaturing buffer containing protease inhibitors

  • Include internal loading controls (β-actin, GAPDH, or total protein staining)

  • Utilize fluorescently-labeled secondary antibodies for wider linear range compared to chemiluminescence

  • Include standard curves using recombinant USE1 protein for absolute quantification

  • Analyze band intensity using software that corrects for background and normalization

• Mass Spectrometry-Based Quantification:

  • Implement targeted proteomics approaches such as SRM (Selected Reaction Monitoring) or PRM (Parallel Reaction Monitoring)

  • Utilize isotope-labeled peptide standards corresponding to unique USE1 peptides

  • Select 3-5 proteotypic peptides that ionize efficiently and are unique to USE1

  • Calculate concentration based on area ratios between endogenous peptides and labeled standards

• Immunohistochemistry with Digital Image Analysis:

  • Standardize tissue fixation and processing to maintain epitope accessibility

  • Include calibration samples with known USE1 concentrations

  • Acquire images under identical conditions (exposure, gain, offset)

  • Apply automated segmentation and intensity measurement algorithms

  • Express results as integrated density per cell or tissue area

How should researchers troubleshoot issues with USE1 protein solubility during purification?

When encountering solubility issues with USE1 protein during purification, a systematic troubleshooting approach targeting each stage of the process is essential. USE1, like many membrane-associated proteins, presents specific challenges that can be addressed through the following strategies:

Expression optimization:

• Reduce expression temperature to 16-18°C to slow protein production and improve folding

• Utilize specialized E. coli strains (Rosetta, Origami) that provide rare codons or enhanced disulfide bond formation

• Co-express with molecular chaperones (GroEL/ES, DnaK/J) to assist proper folding

• Consider fusion tags known to enhance solubility (MBP, SUMO, TrxA) rather than minimal His-tags

Lysis and extraction optimization:

• Test a panel of detergents at varying concentrations:

• Implement stepwise solubilization approaches, starting with low detergent concentrations and gentle extraction

• Add solubility enhancers such as glycerol (5-10%), arginine (50-100 mM), or specific salts (KCl vs. NaCl)

• Consider native lysis buffers with pH ranges (7.0-8.5) and varying ionic strengths (100-500 mM)

If partial solubilization is achieved, proceed with purification using appropriate detergent in all buffers. For proteins that remain insoluble, controlled refolding from inclusion bodies may be necessary, using a gradual dialysis approach with decreasing denaturant concentrations. Throughout the process, functional assays should be performed to ensure that solubilized protein retains its native activity in SNARE complex formation.

What statistical approaches are most appropriate for analyzing USE1 expression data across developmental stages?

• For continuous expression data (qPCR, RNA-seq, proteomics):

  • Implement time-series analysis using mixed-effects models to account for both fixed effects (developmental stage) and random effects (batch, biological variation)

  • Apply ANOVA with post-hoc tests (Tukey's HSD or Dunnett's) when comparing multiple developmental timepoints to a reference stage

  • Use regression approaches with polynomial terms to model non-linear expression patterns throughout development

  • Calculate coefficients of variation at each stage to assess expression stability

• For spatial expression data (in situ hybridization, immunohistochemistry):

  • Utilize image analysis with tissue segmentation to quantify regional expression

  • Implement hierarchical clustering to identify tissues with similar expression patterns

  • Apply dimensionality reduction techniques (PCA, t-SNE) to visualize complex spatial-temporal patterns

• Specific considerations for developmental data:

  • Account for heteroscedasticity (unequal variances) across developmental stages using appropriate transformations or weighted analyses

  • Consider paired analyses when following the same subjects across development

  • Implement permutation tests when distribution assumptions cannot be met

  • Correct for multiple testing using Benjamini-Hochberg procedure to control false discovery rate

Sample size calculations should be performed a priori, typically requiring 6-8 biological replicates per developmental stage to detect biologically relevant fold changes (>1.5-fold) with 80% power. Combining multiple statistical approaches provides the most robust interpretation of developmental expression patterns.

How can researchers differentiate between direct and indirect effects when studying USE1 knockout phenotypes?

Differentiating between direct and indirect effects in USE1 knockout zebrafish requires a multi-faceted approach combining temporal, spatial, and molecular strategies. To establish causality and distinguish primary from secondary phenotypes:

• Implement temporal analysis strategies:

  • Conduct detailed time-course studies to establish the sequence of phenotypic manifestations

  • Use heat-shock or chemically-inducible conditional knockout systems to control the timing of USE1 depletion

  • Compare acute (morpholino) versus chronic (genetic) loss of USE1 to identify adaptation responses

• Apply molecular pathway dissection:

  • Perform transcriptomic analysis at early timepoints after USE1 loss to identify immediately responsive genes

  • Use phosphoproteomics to detect rapid signaling changes preceding morphological phenotypes

  • Implement epistasis experiments by modulating potential downstream effectors in USE1 mutant background

• Utilize cellular resolution approaches:

  • Conduct cell-type specific knockout using Cre-lox systems to isolate tissue-autonomous effects

  • Perform cell transplantation experiments between wild-type and USE1-deficient embryos to test cell autonomy

  • Implement mosaic analysis with tissue-specific rescue to map causality to specific domains

• Apply biochemical validation:

  • Perform in vitro reconstitution experiments with purified components to test direct molecular interactions

  • Use proximity labeling techniques (BioID, APEX) to identify the immediate molecular neighborhood of USE1

  • Implement rapid protein degradation systems (Auxin-inducible, dTAG) to distinguish acute molecular changes

These approaches collectively build a causality framework that can distinguish direct effects (immediate molecular consequences of USE1 absence) from indirect effects (adaptive or compensatory responses that emerge over time).

What bioinformatic tools are most useful for analyzing USE1 protein conservation and function across species?

For comprehensive analysis of USE1 protein conservation and function across species, researchers should employ a strategic combination of bioinformatic tools addressing different aspects of evolutionary and functional analysis:

• Sequence Conservation Analysis:

  • Multiple Sequence Alignment: MUSCLE or MAFFT for accurate alignment of USE1 sequences across diverse species

  • Visualization: Jalview or ESPript for identifying conserved motifs and domains

  • Conservation Scoring: ConSurf or Rate4Site to map evolutionary conservation onto protein structures

  • Selective Pressure Analysis: PAML or HyPhy to detect signatures of positive or negative selection on specific residues

• Structural Analysis:

  • Homology Modeling: SWISS-MODEL or Phyre2 to predict 3D structure of zebrafish USE1 based on known structures

  • Molecular Dynamics: GROMACS or NAMD to simulate conformational dynamics and identify functionally important motions

  • Protein-Protein Docking: HADDOCK or ClusPro to predict interactions with other SNARE proteins

  • Structural Conservation: ProSAT or ConSurf-DB to map sequence conservation onto structural models

• Functional Inference Tools:

  • Domain Prediction: InterProScan or SMART to identify functional domains and motifs

  • Post-translational Modification Sites: NetPhos or UbPred for phosphorylation and ubiquitination prediction

  • Protein Interaction Networks: STRING or BioGRID to compare USE1 interaction partners across species

  • Coevolution Analysis: CAPS or DCA to identify co-evolving residues suggesting functional coupling

• Synteny and Gene Context Analysis:

  • Genome Browsers: Ensembl or UCSC Genome Browser to examine USE1 genomic context across species

  • Synteny Analysis: SynFind or Genomicus to trace chromosomal rearrangements affecting USE1 and neighboring genes

These tools should be applied in a complementary fashion, as each provides different insights into USE1 evolution. For example, combining sequence conservation data with structural information can reveal functionally critical sites at protein interfaces, while network analysis across species can identify conserved and divergent aspects of USE1 function in vesicular transport systems.

What are the most common technical challenges when working with recombinant USE1 protein and how can they be addressed?

Researchers working with recombinant Danio rerio USE1 protein frequently encounter several technical challenges that can be systematically addressed through optimized protocols:

• Low expression yield:

  • Strategy: Optimize codon usage for expression host (E. coli, insect cells, or mammalian cells)

  • Implementation: Redesign synthetic gene with optimized codons while maintaining the amino acid sequence

  • Alternative: Test multiple expression vectors with different promoter strengths and induction systems

  • Validation: Compare protein yields through Western blot analysis of small-scale expression tests

• Protein aggregation:

  • Strategy: Modify buffer conditions to improve stability

  • Implementation: Screen additive series including glycerol (5-20%), arginine (50-200 mM), and non-detergent sulfobetaines

  • Alternative: Express as fusion with solubility-enhancing partners (MBP, SUMO, TrxA)

  • Validation: Assess aggregation state through dynamic light scattering or size exclusion chromatography

• Loss of function during purification:

  • Strategy: Implement gentle purification conditions

  • Implementation: Use gravity-flow instead of FPLC for sensitive preparations; reduce purification steps

  • Alternative: Develop activity assays to monitor functional integrity throughout purification

  • Validation: Compare activity of protein at different purification stages to identify problematic steps

• Rapid degradation:

  • Strategy: Identify and mitigate proteolytic activity

  • Implementation: Add protease inhibitor cocktails; identify and mutate sensitive protease sites

  • Alternative: Express in protease-deficient host strains

  • Validation: Monitor stability through time-course analyses with SDS-PAGE

By systematically addressing these challenges with the appropriate technical solutions, researchers can substantially improve their success in working with recombinant zebrafish USE1 protein for structural and functional studies.

How can researchers troubleshoot non-specific binding in USE1 protein interaction studies?

Non-specific binding represents a significant challenge in USE1 protein interaction studies that can lead to false-positive results. A systematic troubleshooting approach can effectively minimize these artifacts:

• Optimize binding and washing conditions:

  • Implement salt concentration gradient testing (100-500 mM NaCl) to identify optimal stringency

  • Evaluate different detergent types and concentrations to reduce hydrophobic non-specific interactions

  • Test pH variants (pH 6.8-8.0) to identify conditions that maximize specific binding while reducing background

• Blocking optimization:

  • Compare different blocking agents (BSA, milk proteins, commercial blockers) at various concentrations

  • Implement pre-clearing steps with the matrix alone before specific pull-down

  • Use competitive blocking with excess peptides derived from interaction interfaces

• Validation through multiple methodologies:

  • Cross-validate protein interactions observed in co-IP experiments with orthogonal methods (Y2H, FRET, SPR)

  • Implement reciprocal pull-downs (switching bait and prey) to confirm specificity

  • Perform dose-response binding studies to demonstrate saturable binding characteristic of specific interactions

• Analysis refinement:

  • Utilize quantitative proteomics with SILAC or TMT labeling to distinguish specific from non-specific binding partners

  • Implement stringent statistical thresholds for mass spectrometry-based interactome analyses

  • Compare binding profiles with control proteins of similar physicochemical properties but distinct functions

For the most challenging cases, consider developing peptide aptamers or nanobodies with higher specificity for USE1 epitopes than conventional antibodies. These tools can significantly reduce background and improve the signal-to-noise ratio in interaction studies, enabling detection of transient or weak but physiologically relevant USE1 binding partners.

What approaches can resolve contradictory results in USE1 functional studies across different model systems?

• Methodological standardization and validation:

  • Perform side-by-side comparisons using identical reagents, protocols, and analysis methods

  • Validate key reagents (antibodies, constructs) across all systems to ensure equivalent specificity

  • Implement blinded analysis to eliminate investigator bias in phenotype assessment

  • Standardize quantification methods and statistical approaches across laboratories

• Biological context examination:

  • Consider developmental timing differences between systems (developmental stages in zebrafish vs. cell culture)

  • Evaluate genetic background effects and potential compensatory mechanisms unique to each system

  • Assess expression levels of USE1 interaction partners that may differ between systems

  • Examine post-translational modification patterns that might vary across experimental contexts

• Hypothesis reconciliation framework:

  • Develop integrative models that account for seemingly contradictory observations

  • Design critical experiments specifically targeting the source of contradictions

  • Implement intermediate or hybrid systems to bridge gaps between disparate models

  • Consider dose-dependent or threshold effects that may explain system-specific responses

• Collaborative resolution strategies:

  • Establish standardized phenotyping protocols across research groups

  • Implement sample exchange between laboratories for validation

  • Conduct joint data analysis sessions to identify subtle methodological differences

  • Design definitive experiments with input from multiple stakeholder laboratories