Recombinant Danio rerio Transmembrane protein 55B-A (tmem55ba)

What is Transmembrane protein 55B-A (tmem55ba) in zebrafish?

Transmembrane protein 55B-A (tmem55ba) is a full-length protein encoded by the tmem55ba gene in Danio rerio (zebrafish). It functions as a Type I phosphatidylinositol 4,5-bisphosphate 4-phosphatase-A (also known as PtdIns-4,5-P2 4-Ptase I-A) with an enzyme classification number EC 3.1.3.-. The protein consists of 270 amino acids and is involved in phospholipid metabolism pathways that are critical for cellular signaling .

What is the molecular structure of zebrafish tmem55ba?

The zebrafish tmem55ba protein has a complete amino acid sequence beginning with MADGERSPLLSDLGDGALGS and continuing through the full 270 amino acid residues. The protein contains multiple conserved domains including zinc finger motifs that are characteristic of phosphatase enzymes. Its tertiary structure includes transmembrane regions consistent with its localization in cellular membranes. The protein is cataloged in UniProt under accession number Q32PR0 and has a molecular weight consistent with its full-length sequence .

What are the optimal storage conditions for recombinant tmem55ba protein?

For optimal preservation of recombinant tmem55ba protein activity, store at -20°C in the short term. For extended storage periods, maintain at -20°C or preferably -80°C to prevent degradation. The protein is typically supplied in a storage buffer consisting of Tris-based solution with 50% glycerol, which has been optimized to maintain stability. Repeated freeze-thaw cycles should be strictly avoided as they significantly reduce protein activity. For ongoing experiments, working aliquots can be stored at 4°C for up to one week without significant loss of function .

What expression systems are most effective for producing recombinant tmem55ba?

Based on commercial production methods, E. coli expression systems have proven effective for producing recombinant zebrafish tmem55ba protein. The bacterial expression system allows for His-tag incorporation, facilitating downstream purification processes. For studies requiring post-translational modifications that more closely resemble in vivo conditions, eukaryotic expression systems such as insect cells (Sf9 or Sf21) or mammalian cell lines (HEK293 or CHO cells) may be more appropriate, though these systems typically yield lower protein quantities with higher production costs. Selection of the expression system should align with specific experimental requirements regarding protein yield, purity, and functional authenticity .

What purification methods are recommended for recombinant tmem55ba?

Purification of His-tagged recombinant tmem55ba is most efficiently achieved through immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices. For higher purity requirements, a two-step purification protocol is recommended: IMAC followed by size exclusion chromatography to remove protein aggregates and contaminants of different molecular weights. Maintaining all purification steps at 4°C and including protease inhibitors in all buffers is crucial to prevent degradation. The purified protein should be immediately transferred to the glycerol-containing storage buffer to ensure stability. Validation of purity should be performed using SDS-PAGE and Western blotting with antibodies specific to tmem55ba or the His-tag .

What is the enzymatic activity of tmem55ba and how can it be measured?

Tmem55ba functions as a Type I phosphatidylinositol 4,5-bisphosphate 4-phosphatase (EC 3.1.3.-), catalyzing the removal of a phosphate group from the 4-position of phosphatidylinositol 4,5-bisphosphate (PIP2). Its enzymatic activity can be quantified using:

• Malachite Green Phosphate Assay: Measures inorganic phosphate released during the reaction

• HPLC Analysis: Separates and quantifies phosphoinositide species before and after enzyme treatment

• Radiolabeled Substrate Method: Uses 32P-labeled PIP2 to track phosphate removal

• Fluorescence-based Assays: Employs fluorescently-labeled phosphoinositides

The standard assay conditions include buffer at pH 6.8-7.2, presence of divalent cations (typically Mg2+ or Mn2+), and temperature of 28-30°C (optimal for zebrafish proteins). Activity is typically expressed as nmol phosphate released per minute per mg of protein .

How does tmem55ba contribute to phospholipid signaling pathways?

Tmem55ba plays a crucial role in phosphoinositide metabolism by hydrolyzing the 4-position phosphate from PIP2, thereby regulating membrane phospholipid composition. This enzymatic activity affects:

• Membrane Dynamics: By modifying PIP2 levels, tmem55ba influences membrane curvature and fluidity

• Signaling Cascades: PIP2 serves as a precursor for secondary messengers like IP3 and DAG

• Protein Localization: Many proteins contain PIP2-binding domains that determine their subcellular localization

• Vesicular Trafficking: Phosphoinositide balance regulates endocytosis and exocytosis

In zebrafish development, this regulation likely influences cell migration, differentiation, and tissue morphogenesis. Research suggests that perturbation of tmem55ba activity could disrupt these processes, potentially affecting embryonic development and organogenesis, particularly in neural tissues .

What is the expression pattern of tmem55ba during zebrafish development?

The tmem55ba gene (zgc:123304) shows dynamic expression patterns throughout zebrafish development. While the search results don't provide specific expression data for tmem55ba, research methodologies to determine its expression pattern would include:

• In situ hybridization: To visualize spatial distribution of tmem55ba mRNA in whole embryos at different developmental stages

• qRT-PCR: To quantify temporal expression changes during development

• Transgenic reporter lines: Creating tmem55ba:GFP lines to monitor expression in live embryos

• Single-cell RNA sequencing: To identify cell types expressing tmem55ba at high resolution

Based on related proteins, expression would likely be detected in developing neural tissues, digestive organs, and possibly hematopoietic tissues. The expression pattern may vary between embryonic, larval, and adult stages, suggesting stage-specific functional roles .

How can CRISPR-Cas9 be used to study tmem55ba function in zebrafish?

CRISPR-Cas9 gene editing provides a powerful approach for studying tmem55ba function in zebrafish through these methodological steps:

• Target site selection: Design guide RNAs targeting exonic regions of tmem55ba, preferably early exons to ensure functional disruption

• Knockout generation: Inject Cas9 protein with guide RNAs into single-cell embryos to create mosaic F0 fish

• Founder identification: Screen F0 adults for germline transmission of mutations using fin clip genotyping

• Stable line establishment: Outcross founders with wild-type fish and identify heterozygous F1 carriers

• Phenotypic analysis: Examine homozygous mutants for:

  • Developmental abnormalities

  • Cellular phosphoinositide levels

  • Membrane trafficking defects

  • Neural development and behavior

For more subtle manipulations, CRISPR knock-in approaches can introduce point mutations to specifically disrupt the phosphatase catalytic domain while preserving other protein functions. Phenotypic rescue experiments using wild-type mRNA injection into mutant embryos provide confirmation of phenotype specificity .

What zebrafish disease models might benefit from tmem55ba research?

Based on the known functions of phosphoinositide-modifying enzymes, tmem55ba research could contribute to several zebrafish disease models:

• Neurological disorders: As phosphoinositide signaling is crucial for neural development and function, tmem55ba studies may inform models of neurodevelopmental disorders

• Immune response regulation: Research examining TiLV infection in zebrafish demonstrates the importance of proper immune regulation, which may involve phosphoinositide signaling pathways possibly mediated by tmem55ba

• Metabolic disorders: Disruption of membrane lipid composition can affect cellular metabolism and may contribute to metabolic disease models

• Cancer models: Phosphoinositide signaling is frequently dysregulated in cancer, making tmem55ba potentially relevant to zebrafish cancer models

Methodological approaches would include creating tmem55ba mutant or transgenic lines, exposing them to disease-inducing conditions, and analyzing phenotypic outcomes through histology, functional assays, and molecular profiling .

How can phosphoproteomic analysis be applied to study tmem55ba signaling networks?

Phosphoproteomic analysis offers a powerful approach to elucidate tmem55ba-dependent signaling networks in zebrafish through:

• Sample preparation protocol:

  • Generate tmem55ba knockout or overexpression zebrafish models

  • Extract proteins from specific tissues or whole larvae

  • Enrich for phosphopeptides using TiO2 or IMAC (Immobilized Metal Affinity Chromatography)

• Mass spectrometry analysis:

  • Perform LC-MS/MS analysis using high-resolution instruments

  • Implement data-dependent or data-independent acquisition strategies

  • Use label-free or isotope labeling approaches (SILAC, TMT) for quantification

• Data analysis pipeline:

  • Identify phosphopeptides using database search algorithms

  • Quantify phosphorylation changes between experimental groups

  • Perform motif analysis to identify kinase-substrate relationships

  • Construct signaling pathway networks using enrichment analysis

This methodology would identify proteins with altered phosphorylation status in response to tmem55ba perturbation, potentially revealing novel components of phosphoinositide signaling networks and unexpected connections to other cellular pathways .

What approaches can be used to visualize tmem55ba-mediated phosphoinositide dynamics in live zebrafish?

Visualizing tmem55ba-mediated phosphoinositide dynamics in live zebrafish requires sophisticated imaging techniques:

• Genetically encoded phosphoinositide biosensors:

  • Generate transgenic zebrafish expressing PIP2-binding domains (e.g., PH domain from PLCδ1) fused to fluorescent proteins

  • Use ratiometric sensors that change FRET efficiency upon binding to specific phosphoinositides

  • Create tissue-specific or inducible expression systems for targeted analysis

• Advanced microscopy methods:

  • Employ spinning disk confocal microscopy for rapid acquisition with minimal phototoxicity

  • Use light-sheet microscopy for whole-embryo imaging with cellular resolution

  • Implement super-resolution techniques (STED, PALM) for subcellular localization studies

• Experimental design:

  • Compare wild-type vs. tmem55ba mutant or morphant embryos

  • Perform time-lapse imaging during critical developmental processes

  • Combine with optogenetic tools to acutely manipulate tmem55ba activity

This approach would provide unprecedented insights into the spatiotemporal dynamics of phosphoinositide metabolism in a vertebrate model organism, revealing how tmem55ba regulates these critical signaling lipids during development and in response to physiological stimuli .

How can single-cell transcriptomics enhance our understanding of tmem55ba function?

Single-cell transcriptomics provides a high-resolution approach to understanding tmem55ba function through these methodological steps:

• Sample preparation protocol:

  • Dissociate zebrafish embryos or specific tissues into single-cell suspensions

  • Perform fluorescence-activated cell sorting (FACS) if targeting specific cell populations

  • Prepare single-cell libraries using platforms like 10x Genomics Chromium or Drop-seq

• Experimental design strategies:

  • Compare wild-type to tmem55ba mutant/knockdown embryos at key developmental stages

  • Analyze cell type-specific responses to tmem55ba perturbation

  • Perform temporal analysis to capture dynamic gene expression changes

• Analytical approaches:

  • Identify cell clusters using dimensionality reduction and clustering algorithms

  • Perform differential expression analysis between experimental conditions

  • Conduct trajectory analysis to map developmental progressions

  • Integrate with spatial transcriptomics for positional context

• Validation methods:

  • Confirm key findings using in situ hybridization or immunohistochemistry

  • Functional validation of identified genes through targeted knockdown

This comprehensive approach would reveal cell types most affected by tmem55ba perturbation, identify compensatory mechanisms, and potentially discover novel functions through unexpected gene expression changes in specific cell populations .

What are the key differences between tmem55ba and tmem55bb in zebrafish?

Zebrafish possess two paralogous genes, tmem55ba and tmem55bb, resulting from the teleost-specific genome duplication. The key differences between these proteins include:

These differences suggest potential subfunctionalization, where each paralog may have adopted specialized roles in specific tissues or developmental contexts. Methodological approaches to study these differences include comparative expression analysis using in situ hybridization, parallel knockdown studies, and biochemical characterization of substrate specificities .

How do zebrafish phosphoinositide phosphatases compare with mammalian counterparts?

Zebrafish phosphoinositide phosphatases, including tmem55ba, share important similarities and differences with their mammalian counterparts:

• Structural conservation:

  • Core catalytic domains show high sequence conservation

  • Regulatory domains may exhibit greater divergence

  • Zebrafish often possess additional paralogs due to genome duplication

• Substrate specificity comparison:

  • Similar substrate recognition for key phosphoinositides

  • Potentially different kinetic parameters (Km, Vmax) reflecting adaptation to different cellular environments

  • Temperature optima aligned with organism physiology (28°C for zebrafish vs. 37°C for mammals)

• Developmental roles:

  • Conservation of fundamental signaling pathways

  • Species-specific adaptations for unique developmental processes

• Experimental considerations:

  • Lower incubation temperature required for zebrafish protein assays

  • Different optimal buffer conditions may be necessary

  • Cross-reactivity of antibodies should be carefully validated

This comparative information is essential for translating findings between zebrafish models and mammalian systems, particularly when using zebrafish to model human diseases involving phosphoinositide signaling .

What functional redundancy exists in the phosphoinositide phosphatase family in zebrafish?

The zebrafish genome encodes multiple phosphoinositide phosphatases with potentially overlapping functions, creating a complex network of functional redundancy:

• Paralog compensation mechanisms:

  • tmem55ba and tmem55bb likely provide partial functional redundancy

  • Knockdown of one paralog may trigger upregulation of the other

  • Complete functional assessment requires double knockout models

• Cross-family redundancy:

  • Different phosphatase families may target the same phosphoinositide position

  • Functional overlap can mask phenotypes in single gene perturbations

  • Comprehensive analysis requires combinatorial gene targeting approaches

• Methodological approaches to study redundancy:

  • Generate combinatorial mutants targeting multiple family members

  • Perform quantitative phosphoinositide profiling in various mutant backgrounds

  • Use graded knockdown approaches to reveal dosage-sensitive phenotypes

  • Conduct rescue experiments with paralogs to assess functional equivalence

Understanding this redundancy is crucial for interpreting knockout phenotypes, as the absence of obvious defects may reflect compensatory mechanisms rather than lack of function. This necessitates sophisticated genetic approaches and careful biochemical characterization to fully elucidate the roles of individual phosphatases like tmem55ba within the broader network .

What are the current limitations in studying tmem55ba function in zebrafish?

Research on tmem55ba in zebrafish faces several methodological and conceptual challenges:

• Technical limitations:

  • Limited availability of specific antibodies for zebrafish tmem55ba

  • Challenges in direct measurement of enzymatic activity in vivo

  • Complex redundancy with tmem55bb requiring sophisticated genetic approaches

• Knowledge gaps:

  • Incomplete understanding of tissue-specific expression patterns

  • Limited characterization of developmental roles

  • Uncertain relationship between phosphatase activity and physiological outcomes

• Experimental constraints:

  • Difficulty in visualizing phosphoinositide dynamics with sufficient spatiotemporal resolution

  • Challenges in distinguishing primary effects from compensatory responses

  • Limited cellular models derived from zebrafish for in vitro studies

Addressing these limitations will require development of new tools, including zebrafish-specific antibodies, improved phosphoinositide sensors, and refined genetic approaches for paralog-specific manipulation .

How might high-throughput screening approaches be optimized for tmem55ba inhibitor discovery?

Developing a high-throughput screening platform for tmem55ba inhibitors requires a carefully designed methodological approach:

• Assay development strategy:

  • Express and purify recombinant zebrafish tmem55ba with high activity

  • Optimize a fluorescence-based or colorimetric phosphatase assay

  • Validate assay performance metrics (Z', signal-to-background ratio, variability)

  • Miniaturize to 384- or 1536-well format for higher throughput

• Compound library selection:

  • Include diverse chemical scaffolds to maximize chemical space coverage

  • Incorporate known phosphatase inhibitors as positive controls

  • Consider focused libraries targeting phosphoinositide-binding pockets

• Screening cascade design:

  • Primary screen at single concentration (10-20 μM typical)

  • Confirmation of hits in dose-response format

  • Counter-screening against related phosphatases to assess selectivity

  • Cellular assays in zebrafish-derived cell lines

  • In vivo validation in zebrafish embryos

• Data analysis approach:

  • Implement machine learning algorithms to identify structure-activity relationships

  • Cluster hits by chemical scaffold and mechanism of action

  • Prioritize compounds with favorable physicochemical properties

This comprehensive approach would facilitate discovery of chemical probes to further dissect tmem55ba function and potentially lead to therapeutically relevant compounds for human orthologs .

What emerging technologies might advance our understanding of tmem55ba biology?

Several cutting-edge technologies hold promise for transforming our understanding of tmem55ba biology in zebrafish:

• CRISPR-based technologies:

  • Base editing for precise introduction of point mutations

  • Prime editing for targeted nucleotide substitutions without double-strand breaks

  • CRISPR interference/activation for reversible modulation of gene expression

  • CRISPR screening in zebrafish for genetic interaction mapping

• Advanced imaging approaches:

  • Expansion microscopy for improved subcellular resolution

  • Lattice light-sheet microscopy for high-speed volumetric imaging

  • Correlative light and electron microscopy for ultrastructural context

  • Intravital microscopy for studying tmem55ba dynamics in adult zebrafish

• Multi-omics integration:

  • Spatial transcriptomics to map gene expression changes in anatomical context

  • Lipidomics to comprehensively profile phosphoinositide alterations

  • Proteomics to identify interaction networks and post-translational modifications

  • Metabolomics to capture downstream consequences of altered phosphoinositide signaling

• Computational advances:

  • AlphaFold2-based structural prediction of tmem55ba and its interactions

  • Molecular dynamics simulations of enzyme-substrate interactions

  • Network analysis tools to integrate multi-omics datasets

These technologies, especially when applied in combination, have the potential to resolve current knowledge gaps and provide unprecedented insights into tmem55ba function in development, physiology, and disease contexts .